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Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic drugs WL Blalock1, C Weinstein-Oppenheimer1,2, F Chang1, PE Hoyle1, X-Y Wang3, PA Algate4, RA Franklin1,5, SM Oberhaus1,5, LS Steelman1 and JA McCubrey1,5

1Department of Microbiology and Immunology, 5Leo Jenkins Cancer Center, East Carolina University School of Medicine Greenville, NC, USA; 2Escuela de Quı´mica y Farmacia, Facultad de Medicina, Universidad de Valparaiso, Valparaiso, Chile; 3Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, MN, USA; and 4Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

Over the past decade, there has been an exponential increase growth factor), Flt-L (the ligand for the flt2/3 receptor), erythro- in our knowledge of how cytokines regulate signal transduc- poietin (EPO), and others affect the growth and differentiation tion, cell cycle progression, differentiation and apoptosis. Research has focused on different biochemical and genetic of these early hematopoietic precursor cells into cells of the 1–4 aspects of these processes. Initially, cytokines were identified myeloid, lymphoid and erythroid lineages (Table 1). This by clonogenic assays and purified by biochemical techniques. review will concentrate on IL-3 since much of the knowledge This soon led to the molecular cloning of the genes encoding of how cytokines affect cell growth, signal transduction, and the cytokines and their cognate receptors. Determining the apoptosis has been elucidated from research with IL-3-depen- structure and regulation of these genes in normal and malignant hematopoietic cells has furthered our understanding of dent cell lines. Some other cytokines (eg GM-CSF and IL-5) neoplastic transformation. Furthermore, this has allowed the exert their effects through similar mechanisms. design of modified cytokines which are able to stimulate mul- IL-3 was initially defined by its ability to induce the enzyme tiple receptors and be more effective in stimulating the repopu- 20-␣-hydroxysteroid dehydrogenase in cultures of splenic lation of hematopoietic cells after myelosuppressive chemo- lymphocytes from nude mice.5 However, it soon became therapy. The mechanisms by which cytokines transduce their regulatory signals have been evaluated by identifying the apparent that IL-3 was being studied by a number of investi- involvement of specific protein kinase cascades and their gators under a variety of aliases. It was called persisting cell- downstream transcription factor targets. The effects of cyto- stimulating factor (PSF),6 mast cell growth factor (MCGF),7 kines on cell cycle regulatory molecules, which either promote hematopoietic cell growth factor (HCGF),8 histamine-produc- or arrest cell cycle progression, have been more recently exam- ing cell-stimulating factor,9 multi-colony stimulating factor ined. In addition, the mechanisms by which cytokines regulate 10 5 apoptotic proteins, which mediate survival vs death, are being (multi-CSF), Thy-1-inducing factor, and burst promoting 11 elucidated. Identification and characterization of these com- activity (BPA). All of these growth stimulatory activities were plex, interconnected pathways has expanded our knowledge of subsequently identified as the same protein and renamed IL-3. leukemogenesis substantially. This information has the poten- IL-3 was shown to act on both myeloid and lymphoid lin- tial to guide the development of therapeutic drugs designed to eages by in vitro studies. In vivo administration of pharmaco- target key intermediates in these pathways and effectively treat patients with leukemias and lymphomas. This review focuses logical doses of recombinant IL-3 to mice resulted in the on the current understanding of how hematopoietic cytokines increased production of red blood cells, leukocytes and plate- such as IL-3, as well as its cognate receptor, are expressed and lets.12 Moreover, over-expression of the IL-3 gene in hemato- the mechanisms by which they transmit their growth regulatory poietic progenitors via retroviral transduction of bone marrow signals. The effects of aberrant regulation of these molecules cells resulted in a non-neoplastic, myeloproliferative syn- on signal transduction, cell cycle regulatory and apoptotic 13 pathways in transformed hematopoietic cells are discussed. drome in vivo. Finally, anti-neoplastic drugs that target crucial constituents in To further understand the role of IL-3 in vivo, transgenic these pathways are evaluated. mice expressing antisense IL-3 RNA were created. These Keywords: cytokines; signal transduction; anti-neoplastic drugs; mice, which exhibited decreased serum levels of IL-3, oncogenes; apoptosis developed either a B cell lymphoproliferative syndrome or a neurological dysfunction.14 In more recent studies with IL-3- deficient mice, a role for IL-3 in contact hypersensitivity was Cytokines and hematopoiesis observed. IL-3 was determined to be necessary for efficient priming of hapten-specific contact hypersensitivity.15 Cytokines stimulate cell cycle progression, proliferation, and IL-3 transgenic mice have also been used to examine the differentiation, as well as inhibit apoptosis of hematopoietic 1–3 effects of constitutive IL-3 expression on development and cells. Peripheral blood cells are generated from self-renew- neoplasia. Recent evidence indicative of neurological dys- able, pluripotential hematopoietic stem cells in the bone function in IL-3-transgenic mice supports the idea that this marrow. Cytokines such as interleukin-3 (IL-3), hematopoietic cytokine performs key roles in the central ner- granulocyte/macrophage colony stimulating factor (GM-CSF), vous system as well.14–19 The pleiotropic roles of this cytokine stem cell factor (SCF, or steel factor, c-Kit-L, macrophage must be considered when therapies are developed based upon alteration of IL-3 expression, down-regulation of cognate Correspondence: JA McCubrey, Department of Microbiology and receptor expression and function, or destruction of IL-3 recep- Immunology, East Carolina University School of Medicine, Brody tor (IL-3R)-positive cells by chimeric IL-3-toxin molecules. Building 5N98C, Greenville, NC 27858, USA; Fax: 252-816-3104 Received 2 March 1999; accepted 14 May 1999 Review WL Blalock et al 1110 Table 1 Abbreviations of cytokines, growth factors, their receptors and cellular targets

Abbreviation Full name Target cells or function

EGF Epidermal growth factor Affects the growth of many cells epo Erythropoietin Erythroid cells flt-2/flt-3L Ligand for the flt2/flt3 receptor Early hematopoietic progenitor cells G-CSF Granulocyte-colony stimulating Granulocyte/monocyte progenitor cells factor GM-CSF Granulocyte/macrophage- Many early hematopoietic precursor cells of the myeloid lineage, neutrophilic colony stimulating factor granulocytes, monocyte–macrophages, eosinophils; has the ability to activate macrophages gp130 Glycoprotein 130 Signal transducing receptor protein shared among IL-6, LIF, oncoM and other cytokines IL-3 Interleukin-3 Many early hematopoietic precursor cells of both myeloid and lymphoid lineages IL-3R Interleukin-3 receptor Present on many early hematopoietic cells IL-4 Interleukin 4 B cells, mast cells, some T cells IL-5 Interleukin 5 B cells, eosinophils LIF Leukemia inhibitory factor Homology with oncoM, inhibits proliferation of tumor cell lines, affects a broad range of cells M-CSF Macrophage-colony stimulating Granulocyte–monocyte progenitor, receptor is the product of the c-fms proto- factor oncogene oncoM Oncostatin M 28-kDa protein with homology to LIF, inhibits proliferation of tumor cell lines, affects a broad range of cells, expression induced by Jak/STAT pathway SCF Stem cell factor, also known as Many early hematopoietic cells, receptor is the product of the c-kit proto-oncogene, c-kit ligand stimulates proliferation of myeloid, erythroid and lymphoid progenitors TGF-␤ Transforming growth factor-beta Often inhibits cell proliferation, wide tissue distribution TPO Thrombopoietin Growth and differentiation factor for megakaryocytes, receptor is the c-mpl proto- oncogene

Recombinant/chimeric cytokines and therapy tration of daniplestim and G-CSF results in higher numbers of short-term and long-term clonogenic cells compared to A variety of engineered recombinant cytokines are currently sequential administration of daniplestim and G-CSF.29 In the under investigation for use in hematopoietic reconstitution of rhesus myelosuppression model, daniplestim accelerated patients undergoing myelosuppressive chemotherapy hematopoietic reconstitution in radiation-induced cytopenia. (Table 2).20–47 A fusion molecule containing IL-3 and GM-CSF Daniplestim significantly reduced the nadir of neutropenia (PIXY 321) developed by Immunex activates both the IL-3 and and the duration of thromocytopenia in animals with radi- GM-CSF receptors.22–25 ation-induced myelosuppression.27 Taken together, these data GD Searle Pharmaceuticals has developed a structurally and suggested that daniplestim may be clinically useful in patients functionally distinct IL-3 receptor agonist (daniplestim) which with chemotherapy-induced myelosuppression and support binds to the IL-3 receptor with higher affinity than native IL- the utility of combination therapy. 3 but has reduced inflammatory effects.26–29 Daniplestim is a Some of the chimeric cytokines developed by Searle truncated form of hIL-3 but has complete biological activity. include the myelopoietin (MPO) family (IL-3 and G-CSF Daniplestim binds to the IL-3 receptor ␣/␤ complex with 20- receptor agonists), the promegapoietin (PMP) family (IL-3 and fold greater affinity compared to native IL-3.26 Daniplestim TPO receptor agonists) and the progenipoietin (ProGP) family induced cell proliferation with 10-fold greater potency than (Flt3 and G-CSF receptor agonists). These chimeric molecules native IL-3 in the human acute myelogenous leukemia were developed based on the concept that a single molecule AML193 cell line. In colony-forming unit (CFU) assays using with bifunctional activities could provide synergy, that is an human bone marrow CD34+ cells, daniplestim demonstrated enhancement in activity that is greater than the addition of 22-fold greater potency than native IL-3.27 molecules with monofunctional activities.33,34 Additional The in vivo utility of daniplestim was evident in the rhesus enhancement in activity was achieved through circular per- mobilization and myelosuppression models.28,29 In the rhesus mutation of the molecules such that the termini of the protein mobilization model, the combination of daniplestim and G- sequence were ‘linked’ and new termini were generated by CSF mobilized higher and sustained levels of stem cells in the ‘breaking’ the sequence at a new location. Such a process peripheral blood. These studies demonstrated that coadminis- could relieve conformational constraints, add flexibility, result

Table 2 Abbreviations of recombinant/chimeric cytokines and drugs and their sources

Name Brief description Source

Daniplestim Modified IL-3 with higher affinity binding to the IL-3 receptor than unmodified IL-3 Monsanto/Searle Leridistim IL-3/G-CSF chimera Monsanto/Searle PIXY-321 Fusion cytokine which binds both the IL-3 and GM-CSF receptors Immunex Progenipoietin Flt-3L/G-CSF chimera Monsanto/Searle Promegapoietin IL-3/TPO chimera Monsanto/Searle Review WL Blalock et al 1111 in different conformational constraints and introduce a new ases ␣ and ␤ (I-␬K␣ and I-␬K␤).55 These kinases phosphorylate structure–activity relationship.33,34 In addition, permutation of I-␬B on serine residues which results in degradation of the a molecule with two functional entities could impart an protein and allows NF-␬B to enter the nucleus and transactiv- advantage with respect to the relative positioning of the two ate gene expression. The I-␬ kinases are activated by NF-␬B molecules as they bind to their respective receptors. inducing kinase (NIK) and the mitogen-activated protein kin- In CFU assays, MPOs demonstrate greater potency than the ase kinase kinase-1 (MEKK1).55 coaddition of native IL-3 and G-CSF.33 In rhesus myelosuppr- 15-Deoxyspergualin (DSG), an immunosuppressive drug ession model, MPOs have been shown to be more effective in currently in phase I/II clinical trials may represent one repopulating specific hematopoietic cell compartments than a approach to inhibiting NF-␬B activation. DSG inhibits the combination of native IL-3 and G-CSF.36,37 In addition, MPOs localization of heat shock protein 70 (Hsp 70) to the nucleus effectively mobilize CD34+ and clonogenic cells in rhesus in response to heat stress, as well as the activation of NF- monkeys.36–38 In phase I/II clinical trials in breast cancer or ␬B, through its interaction with Hsp70.56 Another approach lymphoma patients, MPOs are well-tolerated mobilizing to inhibiting NF-␬B activation involves introducing adenoviral agents.39–41 vectors which overexpress I-␬B.57 This gene therapeutic PMPs support the differentiation of progenitors along the approach may prove beneficial in the suppression of tumor megakaryocytic lineage and induces expansion of CD41+ cells growth. in vitro.42–44 In rhesus myelosuppression model, PMPs sig- Increased levels of intracellular Ca2+ following TCR aggre- nificantly improve the platelet nadir and accelerates platelet gation allows calmodulin to activate calcineurin, a recovery.45 In CFU assays, ProGPs demonstrate greater serine/threonine phosphatase.53 Activated calcineurin dephos- potency than coaddition of native Flt3 ligand and G-CSF sug- phorylates the cytoplasmic (c) form of the transcription factor, gesting a cooperativity of hematopoietic activities in a chim- NF-ATc (nuclear factor of activated T cells) enabling NF-AT eric molecule.46,47 In mice and rhesus monkeys, ProGPs to translocate to the nucleus (n). This results in the transactiv- mobilize substantial numbers of hematopoietic stem cells into ation of cytokine gene expression, including IL-3 and GM- the peripheral blood.37,48 In rhesus myelosuppression model, CSF whose promoters contain NF-AT binding sites.59–74 The ProGPs also support multilineage hematopoietic reconsti- immunosuppressive drugs cyclosporin A (CsA) and FK506 tution.37 Interestingly, ProGPs have recently been shown to mediate their activity by inhibiting calcineurin activation, mobilize a large number of functional dendritic cells suggest- thereby preventing the dephosphorylation of NF-ATc58,74 ing that it may also be effective in this area of immunother- (Table 4). An illustration of the regulation of IL-3 gene apy.46,47 In summary, these chimeric cytokines represent a expression is presented in Figure 1. Similar mechanisms novel line of biotechnology, which shows significant promise mediate the expression of IL-2, GM-CSF and other T cell- in the treatment of certain leukemia patients. derived cytokines. In addition to stimulating proliferation and differentiation of There are additional pathways by which activated PKC can hematopoietic cells, cytokines such as IL-3 also promote cell stimulate cytokine gene expression. PKC can activate the Ras survival. IL-3-dependent cells undergo apoptosis after with- pathway by inactivating the GTPase activating protein (GAP), drawal of IL-3 for a prolonged period of time (12 to 48 h a negative regulator of Ras.61,68–70 Ras is a member of a large depending upon the cell type and species of origin).48,49 How- multi-gene family, which encodes small GTP-binding proteins ever, addition of IL-3 to IL-3-deprived cells can prevent that serve as molecular switches. Inactivation of GAP stimu- apoptosis in a significant proportion of these cells.48,49 These lates Ras activity, which results in an enhancement of acti- anti-apoptotic functions of IL-3 and other important cytokines vator-protein-1 (AP-1) binding activity.61,68–70 AP-1 can then are critical concerns in therapeutic cytokine intervention, stimulate cytokine gene expression, including IL-3 (Figure 2a). especially with patients having certain leukemias and minimal Interestingly, the neurofibromatosis-1 (NF1) gene, a tumor residual disease. suppressor frequently lost in juvenile chronic myelogenous leukemia (CML), is functionally related to GAP.77,78 NF1 likely serves to block Ras activation, thus its loss leads to constitutive Regulation of IL-3 expression Ras activation and contributes to the generation of CML. Ras is frequently targeted by anti-neoplastic drugs including farnesyl Most hematopoietic cells do not usually synthesize the 26- transferase (FT) inhibitors (see below). Addition of a farnesyl kDa IL-3 protein. In those cells that do express the IL-3 gene, group is necessary for Ras localization to the cytoplasmic the gene is normally under stringent controls.50–76 In periph- membrane. Drugs, which block Ras farnesylation, are cur- eral blood, activated T cells, natural killer cells, mast and rently being developed by pharmaceutical companies for some megakaryocytic cells can synthesize IL-3.50–53 For opti- therapeutic use (eg Janssen, Merck).79 mal IL-3 expression, T cells must be activated via the T cell receptor (TCR)/CD3 pathway, or by agents that mimic this pathway, eg the combination of PMA and calcium ionoph- Transcriptional regulation of IL-3 expression ores.50–53 When a T cell is activated, aggregation of the TCR/CD3 complex occurs (Table 3). Receptor aggregation is The cis-acting elements of the human IL-3 promoter include followed by a complex series of biochemical events leading two activation regions separated by an inhibitory region.59–74 to the activation of protein kinase C (PKC) and a rise in the These genetic elements lie within a region that extends to concentration of intracellular Ca2+.50–68 Activated PKC can ෂ300 bp upstream of the transcription start site (Figure 2a). phosphorylate and inactivate the repressor protein, inhibitor Inhibitory elements in the IL-3 promoter which suppress IL-3 ␬B (I-␬B), thus permitting I␬B to disassociate from nuclear fac- transcription include a NIP (nuclear inhibitory protein) bind- tor-␬B (NF-␬B).54 This allows NF-␬B to assume an active form ing sequence located between bp −271 to −250.59 Figure 2 (Figure 1) which subsequently enters the nucleus and trans- depicts some of the transcription factor binding sites involved activates cytokine gene expression. In addition, there is a in the regulation of IL-3 expression. complex of proteins which phosphorylates I-␬B, the I-␬B kin- Sequence motifs common to many cytokine promoters, Review WL Blalock et al 1112 Table 3 Abbreviations of signal transducing molecules and a brief description

Abbreviation Full name Brief description

␤c IL-3 receptor ␤ common chain ␤ subunit of IL-3, IL-5 and GM-CSF receptor chains ␤IL-3 IL-3 receptor ␤IL3 chain ␤ subunit of IL-3 receptor; present in mice, not present in humans 14-3-3 Chaperonins Approximately 250 aa proteins, coordinate signal transduction pathway by binding a speciﬁc motif (FSXSXP or RXY/FXSXP, S is phosphorylated) present in certain proteins, binds Raf, Bad, PKC⑀, PKC␦, BCR and other proteins containing the interaction domain, members of the 14-3-3 gene family include ␤/␣, ⑀, ␩, ␴, ␶, ␨ and ␦ isoforms which exist as homo- and heterodimers A-Raf Serine threonine kinase, acts downstream of Ras and PKC, upstream of MEK; often involved in the transmission of signals from mitogens and growth factors to the nucleus; member of the Raf gene family Abl Abelson proto-oncogene Tryosine kinase, product of the c-abl proto-oncogene, involved in chromosome translocations in chronic myelogenous leukemia (see BCR-ABL) AP-1 Activator Protein-1 Complex of Fos and Jun family transcription factors, binds the promoter regions of many genes, including cytokine and immediate–early genes Act-1 Transcription factor which binds the regulatory region of the IL-3 and certain other cytokine genes Akt 480 aa product of the c-akt proto-oncogene; serine/threonine kinase, activated by PI3K which results in the translocation of Akt to the plasma membrane, can phosphorylate the Bad proapoptotic protein as well as caspase-9; also known as PKB and RAC␤ APS Adaptor protein containing plekstrin homology and SH2 domains which inhibits the Jak-STAT pathway through interactions with c-cbl B-Raf Serine threonine kinase acts downstream of Ras and PKC, and upstream of MEK, often involved in the transmission of signals from mitogens and growth factors to the nucleus, member of the Raf family BCR Breakpoint cluster region Region of the bcr locus in which chromosomal translocation occurs with the c-abl gene in CML; BCR gene encodes a serine/threonine kinase which can phosphorylate the BCR-ABL protein and inhibit its activity BCR-ABL Tyrosine kinase; similar to Abl, product of the BCR-ABL chromosomal translocation, present in Ͼ90% of patients with chronic myelogenous leukemia (CML) c-Cbl Signal transducing adaptor molecule; product of the c-cbl proto-oncogene; may serve to inhibit cytokine signaling through interactions with the APS adaptor protein CD3 Signal transducing molecules associated with T cell receptor (TCR) complex, CD3/TCR consists of CD3 ␥, ␦, ⑀, ␨ and ␩ subunits and TCR subunits C/EBP CAAT/enhancer binding protein Transcription factor which binds the promoter region of many genes, including some genes encoding signal transduction molecules c-Erb-B Receptor for EGF and transforming growth factor-alpha (TGF-␣), which has an intrinsic tyrosine kinase activity; product of the c-erb-b proto-oncogene c-Fms Product of the c-fms proto-oncogene, receptor for M-CSF with intrinsic tyrosine kinase activity c-Fps Tyrosine kinase; product of the c-fps proto-oncogene c-Fos c-Fos transcription factor Product of the c-fos proto-oncogene; member of a gene family which includes Fos-B, Fra-1 and Fra-2; forms heterodimers with c-jun members which bind Ap-1 sites present in the promoter regions of many genes c-Jun c-Jun transcription factor Product of the c-jun proto-oncogene; member of a gene family which includes Jun-B and Jun-D; forms homodimers or heterodimers with c-fos family members and binds AP-1 sites present in the promoter regions of many genes c-Myc With Max and Mad, they are all part of a multi-gene family of transcription factors implicated in both cell growth and induction of apoptosis, c-Myc is a product of the c- myc proto-oncogene; often rearranged in Burkitt’s lymphoma and ampliﬁed in other types of tumors, often induced after growth factor and mitogen stimulation of growth factor-deprived cells; heterodimerizes with Max proteins, (Mad also heterodimerizes with Max to inhibit Myc/Max heterodimers), the above described Mad is not the same as the Drosophila Mad which is related to the Smads that are involved in TGF-␤ signal transduction Cis Cytokine inducible sequence Member of a family of Jak/STAT inhibitors, the protein encoded by the cis gene and related family member binds either Jak or cytokine receptors to inhibit their functions, Cis proteins serve to limit the response of cells to cytokines CREB Cyclic AMP response element Transcription factor which binds the promoter regions of many genes including binding protein cytokine genes CRK-L An adaptor protein with a single SH2 and two SH3 domains DB-1 Transcription factor which binds the regulatory region of the IL-3 and other cytokine genes EGF-R Epidermal growth factor Product of the c-erb-b proto-oncogene, receptor for EGF, TGF-␣, (see c-Erb-B) receptor EGR-1 Early growth response-1 Transcription factor which binds the regulatory region of the IL-3 and other cytokine genes ERG-2 Early growth response-2 Transcription factor which binds the regulatory region of the IL-3 and other cytokine genes Review WL Blalock et al 1113 Table 3 Continued

Elf-1 Ets-like transcription factor Transcription factor which binds the regulatory regions of many genes including cytokine genes, member of the ETS gene family ERK1, ERK2 Extracellular regulated kinases Also known as p42MAPK and p44MAPK, serine/threonine kinases; act downstream of 1 and 2 MEK1 and upstream of p90Rsk often their activity is induced by growth factors Ets Ets transcription factor Product of the c-ets oncogene; binds the promoter region of many genes; member of the Ets gene family GAP GTPase-activating protein Enhances removal of phosphate from GTP by Ras thereby inactivating Ras GRB-2 growth factor receptor bound Adaptor protein involved in coupling the signal from membrane receptors to other protein-2 signaling proteins, contains one SH2 and two SH3 domains Hck Hematopoietic cell kinase Scr family tyrosine kinase expressed in hematopoietic cells HCP Hematopoietic cell phosphatase See SHP-1 HER2 Heregulin growth factor receptor Membrane receptor tyrosine kinase expressed on mammary epidermal cells; often implicated in breast cancer; drugs targeted to this molecule (eg herceptin) are used to treat certain breast cancer patients I␬B Inhibitory ␬ B protein Binds NF-␬B in the cytoplasm and prevents NF␬B from entering the nucleus, when it is phosphorylated, it disassociates from NF-␬B protein which can then enter the nucleus and activate gene expression; product of the Bcl-3 locus I-␬K␣,I-␬K␤ I-␬B kinases ␣ and ␤ Phosphorylate I-␬B on serines 32 and 36 which results in inactivation and subsequent degradation of I-␬B and activation of NF-␬B JAB Jak/STAT inhibitor; member of Cis family of Jak inhibitors Jak Janus associated kinase, Family of tyrosine kinases which are activated by ligand binding to hematopoietin (‘Just another kinase’) receptors; can phosphorylate STAT transcription factors, transmit signals from cytokine receptors to the nucleus; can also transduce effects by phosphorylating other signaling molecules JIP1 Non-enzymatic scaffolding protein which anchors JNK, MLK and MKK4 kinase proteins in a signaling complex JNK c-Jun N-terminal kinase Serine/threonine kinase (380 to 460 aa), phosphorylates the c-Jun transcription factor, member of gene family which includes JNK1␣2, JNK1␤1, JNK1␤2, JNK1␣1, JNK2␤1, JNK3␣1 and JNK3␣2, also known as SAPK, substrates include c-Jun, JunD, Elk-1, AFT-2, can be activated by ␥-irradiation, UV JNKK c-Jun N-terminal kinase kinase Tyrosine/threonine kinase which phosphorylates JNK KSR Kinase suppressor of ras Serine/threonine kinase; acts upstream of Raf; can phosphorylate and activate Raf Lck Lymphocyte cell kinase Src family kinase; expressed predominately in T cells; interacts with TCR and CD3 complex Mad Mothers against Drosophila Mad binds to and is required for the activation of an enhancer within the decapentaplegic vestigial wing-patterning gene. Mad also binds to decapentaplegic (dpp)-response elements in other genes. Smads, which function in TGF-␤ mediated signal transduction are related MEK-1 Mitogen activated extracellular Dual-specificity kinase (serine/threonine, tyrosine); acts downstream of Raf and regulated kinase 1 upstream of MAPK (ERK1 and ERK2); target of the drug PD98059 MKK4 Map kinase kinase-4 Dual tyrosine and serine/threonine kinase; phosphorylates p38MAPK and JNK MEKK7 Map kinase kinase-7 Dual tyrosine and serine/threonine kinase; phosphorylates JNK MEKK1 MEK1 kinase Dual tyrosine and serine/threonine kinase which can phosphorylate MEKs MKK3/MKK6 Map kinase kinase 3/6 Dual tyrosine and serine/threonine kinase; phosphorylates p38MAPK MKP-1 Map kinase phosphatase-1 Phosphatase which removes the phosphate from activated MAPK; member of a gene family of phosphatases which have different but sometimes overlapping specificities MLK Serine/threonine kinase involved in the JNK-induced pathway; involved in oocyte maturation Mos Serine/threonine kinase encoded by the c-mos proto-oncogene; has been shown to activate the MEKs MP1 Non-enzymatic scaffolding protein which anchors MEK and MAP kinase proteins mSos Mammalian homologue of son Coupling molecule involved in transferring signal from activated receptor(s) to Ras or of seven-less a nucleotide exchange factor like Vav NF-AT Nuclear factor of activated T Transcription factor complex; binds the promoter regions of many cytokine genes, cells sensitive to calcineurin; the target of cyclosporin and FK506 NF-IL3-A Nuclear factor IL-3-A Transcription factor complex which binds the IL-3 regulatory region; comprised of NF- ␬B and NF-AT like proteins NF1 Neurofibromatosis 1 Tumor suppressor gene, functionally related to GAP NF-␬B Nuclear factor ␬ (light Transcription factor originally shown to bind the enhancer region of the immunoglobulin chain) B cells immunoglobulin ␬ light chain; regulates the expression of many genes; member of the NF-␬B (c-rel) gene family, product of the c-rel proto-oncogene, naturally inhibited by I- ␬B, also inhibited by the immunosuppressive drug 15-Deoxyspergualin NIK NF-␬B inducing kinase Mediates activation of NF-␬B by TNF-␣ and IL-1, activated by TRAF2, phosphorylates I␬K Oct-1 Octamer consensus sequence-1 Transcription factor which binds the regulatory region of the IL-3 and other cytokine genes Review WL Blalock et al 1114 Table 3 Continued

p38Mapk Serine/threonine kinase, member of MAPK family, phosphorylated on threonine and tyrosine residues by MEK p70S6 kinase p70 ribosomal S6 kinase 70-kDa serine/threonine kinase thought to act downstream of PI3K p90Rsk p90 ribosomal 6 kinase 90-kDa serine/threonine kinase, acts downstream of ERK1 and ERK2 PI3 Kinase Phosphatidylinositol 3 kinase kinase which will phosphorylate 3′ position of the inositol ring, may lead to activation of AKT/PKB Pim-1 Serine/threonine kinase; expressed primarily in hematopoietic cells, can closely cooperate with c-Myc in lymphomagenesis, may contribute to cell transformation by inhibiting apoptosis, also can interact with c-Myc to stimulate apoptosis PKC Protein kinase C Serine/threonine protein kinase; member of a family which consists of multiple family

members including: ␣, ␤1, ␤2 (derived by alternative splicing) ␥, ␦, ⑀, ␩, ␮, ␪, and ␰ PTB Protein tyrosine binding domain Motif present in certain signaling proteins; capable of binding to NPXY motif Rac Ras-related protein proposed to play a role in the JNK pathway Raf-1 Serine/threonine kinase; product of the c-raf proto-oncogene; acts downstream of Ras and PKC, and upstream of MEK; often involved in the transmission of signals from mitogens and growth factors to the nucleus; member of a multigene family which includes A- Raf, B-Raf and Raf-1 Ras Family of GDP/GTP exchange proteins; includes Ha-Ras, Ki-Ras, N-Ras, ras gene are frequently mutated in human cancer Rho Ras-related protein involved in signal transduction SAPK Stress-activated protein kinase Serine/threonine protein kinase, also known as JNK SEK1 Stress/extracellular-regulated Dual speciﬁcity serine/threonine and tyrosine protein kinase, also known as JNKK, MKK4 kinase-1 and MEK4, activates SAPK/JNK in response to mitogenic factors or environmental stress SH1 Src homology region 1 A protein domain related in sequence to kinase domain present in c-Src tyrosine kinase SH2 Src homology region 2 A protein domain related to phosphotyrosine binding domain of Src SH3 Src homology region 3 A protein domain related to a proline rich binding domain found in Src Shc Adapter protein which serves as a substrate for tyrosine kinase activity and can serve as a docking site for proteins with SH2 domains, related to IRS-1 and IRS-2 SHP-1 SH2 containing hematopoietic Removes phosphates from certain activated cytokine receptors and other molecules, cell phosphatase-1 SHP-1 gene is mutated in motheaten mice, also know as SHP-1, SHPTP1, and PTP1C protein tyrosine phosphatase SHP-2 SH2 containing hematopoietic Phosphatase; previously called Syp/SHPTP2 cell phosphatase-2 SHIP SH2 containing inositol Phosphorylated upon M-CSF receptor (c-Fms) stimulation; found in complex with Shc, phosphate Grb2, SOS and c-Fms; involved in phospholipid signaling, phosphatase catalytic activity 5-phosphatase removes 5-phosphate from PI3,4,5 P, the product of PI3K activity, purported to have negative proliferative effects Smad Family of mammalian homologues of Drosophilia MAD gene; activity varies with cell type; transcription factor complex activated by TGF␤ SOCS Proteins which are Jak/STAT inhibitors Src Product of the c-src proto-oncogene, tyrosine kinase, acts upstream of Raf, and downstream of membrane receptors SRE Serum response element Present in the promoter regions of many immediate-early genes, including c-fos STAT Signal transducers and Transcription factors which couple signals from Jak kinases to the nucleus activators of transcription Syk Tyrosine kinase involved in lymphocyte antigen receptor signaling; related to Zap 70 TCR T cell receptor Antigen recognition complex present on T cells Tec Family of tyrosine kinases which includes BTK, ITK, EMT and Tec TEL-ABL Encoded by the chromosomal translocation between tel and c-abl on chromosomes 12 (TEL) and 9 (Abl) TEL-JAK Encoded by the chromosome translocation between TEL and JAK2 on chromosomes 12 (TEL) and 9 (JAK2) Tyk2 Member of the Jak family of tyrosine kinases Vav Product of the c-vav proto-oncogene; GDP/GTP exchange protein

including CK (cytokine)-1 and CK-2/GC elements, are found ively and can enhance the transcriptional activity of the IL-3 in the IL-3 promoter. Although speciﬁc binding of transcrip- promoter when trans-activated by Tax.71 tion factors to the CK regions has been observed, these Two regions of the IL-3 promoter play important positive sequences appear to be dispensable for the activity of the IL- regulatory roles in the response of T cells to mitogens. One 3 promoter.61–64 A CT/GC-rich region located between bp −76 region is called Act-1 and is located between −175 to to −47 confers a basal transcriptional activity to the IL-3 pro- −135 bp.62,63 The 5′ part of this region binds a mitogen- moter and responds to trans-activation by the human T cell inducible, T cell-speciﬁc, octamer-1-associated protein (Oct- leukemia virus type I-encoded Tax protein.64,66 Three tran- 1). Nuclear factor-IL3A (NF-IL-3A) binds in the middle, while scription factors, EGR1, EGR2 and DB1 have been shown to the 3′ Act-1 sequence contains a consensus binding site for a bind to this region. The binding of EGR1 and EGR2 to these cAMP responsive element binding protein (CREB). The role of sites increases IL-3 promoter activity when the cell is acti- the Act-1 region is to coordinate the functions of several cis- vated.70 In contrast to EGR1 and EGR2, DB1 binds constitut- acting transcription factors, which leads to a maximal effect Review WL Blalock et al 1115

Figure 1 Activation of IL-3 expression. The effects of diacyglycerol (DAG) and Ca2+ on PKC activation and the subsequent activation of calmodulin, calcineurin, NF-␬B and NF-AT. NF-␬B can also be activated by NIK. I-␬B can be inhibited by phosphorylation by PKC, I-␬K␣ and I-␬K␤. The activated transcription factors are indicated in yellow. Once activated, NF-␬B and NF-AT enter the nucleus and stimulate IL-3 and GM-CSF expression. Also shown in this diagram are the sites of inhibition by the immunosuppressive drugs CsA, FK506 and DSG.

Table 4 Drugs which inhibit signal transduction and their sources

Name Brief description Source

AG192 Tyrphostin, Jak inhibitor Agouron AG6450 Tyrphostin, Jak inhibitor Agouron Bryostatin PKC inducer, derived from starfish, sea urchins Cyclosporin Immunosuppressive drug which interferes with calcineurin and Novartis cytokine gene expression D609 Phosphatidylcholine-specific phospholipase C inhibitor DSG 15-Deoxyspergualin is an immunosupressive drug which inhibits the localization of the heat shock protein 70 (Hsp 70) to the nucleus in response to heat shock, it will inhibit the activation of NF-␬B Erbstatin Tyrosine kinase inhibitor, inhibits Abl tyrosine kinase FK506 Immunosuppressive drug which interferes with calcineurin and Merck cytokine gene expression FT inhibitors Farnesyl transferase inhibitor, prevents localization of Ras to the cell Merck, Janssen, Schering membrane Plough, Parke Davis Genistein Tyrosine kinase inhibitor Herbimycin Tyrosine kinase inhibitor Okadaic acid Inhibits protein phosphatase 2A PD 98059 MEK1 inhibitor Parke Davis PMA Phorbol myristate acetate; activates PKC; protein tumor promoter, prolonged stimulation results in down regulation of PKC Rapamycin Immunosuppressive drug which inhibits p70S6 Wortmannin Inhibits PI3 kinase Review WL Blalock et al 1116 In addition to the cis-acting elements 5′ to the IL-3 transcription start site, there is another set of cis-acting elements found in the region between the IL-3 and GM-CSF genes which are sensitive to the immunosuppressive drugs CsA and FK-506 (Figure 2b).74 This region contains four NF-AT sites which are bound by transcription factors upon mitogen activation. The binding of the IL-3 promoter region by proto-oncogene products, (eg c-Fos, c-Jun, NF-␬B, EGR and Ets-related transcription factors) suggests that abnormal expression of these oncoproteins may result in autocrine transformation and lead to leukemia. In many transformed cells, the pathways controlling the activities of these transcription factors are dysregulated.78,80–83 For example, constitutive activation of the Ras/Raf/MEK/ERK (extracellular regulated kinase) cascade can alter the activity of transcription factors, to induce autocrine growth factor synthesis.82,83 In addition, certain chromosomal translocations have been linked to aberrant IL-3 expression. In certain human B cell lymphomas, chromosomal translocations between the immunoglobulin heavy chain (IgH) locus on chromosome 14 and the IL-3 gene on chromosome 5 [t(5;14)(q31;q32)] were detected.84,85 The IgH enhancer, a strong tissue specific enhancer involved in many chromosomal translocations in hematopoietic cells (eg Burkitt’s lymphoma, follicular B cell lymphomas involving Bcl-2) induced the transcriptional activation of the IL-3 gene. Genomic DNA demethylation is also believed to influence the propagation of specific T cell cytokine profiles. The extent of methylation of certain cytokine genes such as IL-3 and interferon-␥ (IFN-␥) may contribute to distinct patterns of cytokine gene expression in T cell clones. Demethylation of the Figure 2 Transcription regulation of IL-3 expression. (a) The effects IL-3 promoter was shown to be confined to specific CpG sites of activated Ras, PKC, KSR and Src family kinases on the Raf/MEK/ERK within the same clones.76 signal transduction pathway and IL-3 expression. Ras activation can be induced by external stimuli and inhibited by NF1 or GAP. Ras can further transmit the signal to Raf, MEK, ERK and p90Rsk which can result in the activation of the AP-1 and CREB transcription factors Post-transcriptional regulation of IL-3 gene expression (shown in yellow) which bind the promoter region of the IL-3 and GM-CSF genes. Raf can also be activated by KSR and Src family tyro- IL-3 mRNAs are very unstable and decay within one-half to sine kinases. Other transcription factors (eg c-Jun, Elf-1) are activated 1 h after their synthesis.86–104 This property appears to be criti- by other kinases such as JNK and p38 that in turn are activated by cal for their normal function, since the degradation of IL-3 MKK4, MEKK1 and MKK3/6 and SEK. The NF-␬B and NF-AT as well as the Oct-1 and CREB transcription proteins bind to the ACT-1 mRNA, as well as other cytokine mRNAs, is stringently con- 86–104 ′ region. Possible control mechanisms for NF-␬B, I-␬B and NF-AT (NIK, trolled. An AU-rich element (ARE), found within the 3 I␬K and calcineurin) were presented in Figure 1. Also shown in this untranslated region (UTR) of IL-3 and other cytokine mRNAs, picture are the negative effects of the NIP protein which binds the is involved in the regulation of IL-3 mRNA stability88–92 NIP region and suppresses transcription. In addition, there are CK1 (Figure 3). These evolutionarily conserved ARE sequences and CK2 transcription factors, which bind to the CK1 and CK2 serve to tightly regulate cytokine expression, a critical function regions, as well as the Tax, EGR1, EGR2 and DB1 transcription factors, which bind to the CT/GC region. (b) This panel depicts the bind- due to the potent growth stimulatory and anti-apoptotic effects ing of NF-AT molecules to the intergenic region between the IL-3 and of cytokines. GM-CSF genes. The binding of these proteins to the intergenic region Electrophoretic mobility shift assays (EMSAs) and UV- influences the chromatin configuration of this gene cluster. crosslinking experiments have identified the proteins that bind to cytokine AREs.91 These included proteins with apparent upon IL-3 transcription. The other important transcriptional molecular weights of 36, 40, 43, 46, 55, 57, 68 and 95 kDa. regulatory region is located between bp −315 to −274 and The adenine/uridine binding protein (AUF1, also known as contains AP-1 and Elf-1 binding sites. The c-Jun and c-Fos heterogeneous nuclear ribonuclear protein D [hnRNP D]) was heterodimer (AP-1) binds to the AP-1 site52 while the Elf-1 site shown to bind to the IL-3 ARE, by an EMSA followed by is bound by an Ets-related transcription factor, Elf-1.60,70 immunoprecipitation of IL-3 ARE-binding proteins with a spe- Tissue specific expression of the IL-3 gene may result from cific ␣AUF1 antibody.91 interactions between the Act-1 and this latter region.70–73 All three isoforms of hnRNP D, which exhibited apparent There are many kinases which regulate the activity of the molecular weights of 40, 43 and 46 kDa, bind to the IL-3 transcription factors that bind the IL-3 promoter region. Signal ARE.91 hnRNP C also binds to the IL-3 ARE region.91 Calcium transduction cascades originating from extracellular signals ionophore treatment prevents/reverses binding of these pro- often regulate these kinases (MEKK1, MKK4, JNK, MKK3/6, teins to the IL-3 ARE and results in stabilized IL-3 mRNA91 p38MAPK, p90Rsk, and others). It may be possible to control (Figure3b). The affinities of the hnRNP D proteins for their either directly or indirectly the activities of these important RNA substrates in another study are also negatively correlated regulatory molecules in transformed cells.56,61,69,75,78 with mRNA stability.98 Review WL Blalock et al 1117

Figure 3 Post transcriptional regulation of IL-3 expression. (a) The wild-type IL-3 ARE is shown which binds the indicated proteins. The binding of these proteins results in mRNA with a short mRNA half-life. p36 and p95 are the only proteins that were demonstrated by northwestern analysis to bind directly to the IL-3 gene.91 p95 is depicted as a larger sphere due to artistic constraints. The exact sequences where p36 and p95 bind the IL-3 UTR are not known, nor is the stoichiometry of binding. (b) Calcium ionophores disrupt the binding of RNA- binding proteins to the IL-3 ARE resulting in conditional stabilization of IL-3 mRNA. (c) The RNA-binding proteins are prevented from binding the truncated IL- 3 gene in tumorigenic autocrine transformed cells which contain an IAP provirus inserted into the IL-3 ARE.86–91 The prevention of binding of these proteins results in the continuous stabilization of IL-3 mRNA. The sizes of the coding and non-coding IL-3 sequences are not drawn to scale.

CsA and FK506 decrease IL-3 production by certain mast These studies indicated that the IAP transposition stabilized cells via mRNA destabilization, as well as affecting NF-ATc IL-3 mRNA directly. Furthermore, the remaining two AUUUA activation.103 The immunosuppressive drug rapamycin, which motifs could not efficiently destabilize IL-3 mRNA, and hence has a different biochemical target than CsA, also destabilizes the transfected cells were autocrine-transformed and tumorig- IL-3 mRNA is certain autocrine transformed cells.104 Rapamy- enic. Site-directed mutagenesis studies indicated that destabil- cin in primarily thought to affect p70 ribosomal S6 kinase ization of IL-3 mRNA requires a clustering of either the three (p70S6K) phosphorylation, which subsequently modulates the 5′ or the distal three 3Ј AUUUA motifs present in the IL-3 efficiency of protein translation (see below). The mechanisms ARE. However, the cluster of the three 3′ AUUUA motifs was by which these drugs prevent the binding of proteins to the a stronger destabilizer.92 IL-3 ARE are unknown. The drugs may alter the phosphoryl- In order to determine how the IAP transposition altered the ation states of ARE-binding proteins, which prevent them from binding of proteins to the IL-3 ARE, EMSAs were performed. interacting with the IL-3 ARE. Proteins specifically bound to the wild-type IL-3 mRNA ARE region whereas no protein binding was detected to the RNA which had only two AUUUA motifs, or to an artificial RNA Biological consequences of IL-3 ARE disruption probe which did not contain any AUUUA motifs91 (Figure 3c). Thus certain IAP transpositions disrupt IL-3 AREs and prevent We have characterized autocrine-transformed cells which the binding of proteins to this region. These mutations result secrete IL-3 and have an intracisternal type A particle (IAP) in autocrine growth stimulation leading to malignant trans- transposed into the IL-3 ARE.87 In these autocrine-transformed formation. FL-IL3-R cells, only two AUUUA motifs adjacent to the IL-3 gene remain intact due to the IAP transposition. IL-3 mRNA isolated from these cells has a much longer half-life (t. = 16 h) Therapy directed at cytokine gene expression than wild-type IL-3 mRNA (t. = 0.5 to 1 h). Moreover, the IL- 3-secreting hematopoietic cells expressing the mutated IL-3 As discussed, the regulation of cytokine gene expression can mRNAs were tumorigenic upon injection into immuno- occur by both transcriptional and post-transcriptional mech- compromised nude mice.84–89 anisms, and dysregulation of either mechanism can result in Chimeric IL-3 gene constructs containing portions of the the autocrine transformation of hematopoietic cells. Identifi- wild-type and IAP-disrupted IL-3 genes were made, trans- cation or synthesis of drugs that alter either one or both of fected into IL-3-dependent parental FL5.12 cells and exam- these regulatory mechanisms could be used to control cyto- ined for their ability to abrogate cytokine dependency.86–88 kine synthesis. Specific drugs could be designed to either Review WL Blalock et al 1118 inhibit cytokine gene expression when it is deleterious (eg Both IL-3 receptor ␣ and ␤ chains belong to the cytokine inflammatory and autoimmune responses) or stimulate cyto- receptor superfamily, all members of which are characterized kine gene expression when it is advantageous (eg to repopu- by a conserved region homologous to the fibronectin type-III late hematopoietic precursor cells after myelo-ablative chem- domain. This sequence motif consists of a 200 amino acid otherapeutic treatments or other conditions which induce extracellular domain and a conserved WSXWS amino acid hematopoietic cell stress). sequence at the C-terminus.129 In addition, there is a set of It may also be possible, under certain circumstances, to conserved cysteine residues in this extracellular domain as block the growth of certain autocrine tumors with monoclonal well. Mutations in the IL-3R␤ chain gene in cytokine-depen- antibodies directed to the growth promoting cytokine.105 dent cultured cells can result in factor-independent growth However, this immunotherapy strategy will only be successful and the inhibition of apoptosis.131,132 when it can neutralize the activity of both intracellular cytokines, whose production by the cells may be sufficient to promote proliferation by interaction with nascent receptors not Functions of the IL-3 receptor chains as determined by yet inserted into the membrane, as well as extracellular cyto- knockout and transgenic mice kines.106 This immunotherapy technique may have to be coupled with therapies designed to inhibit the effects of cyto- To investigate further the biological functions of IL-3 and its kines and cytokine gene expression (cytokine antagon- cognate receptor, knockout mice lacking either one or the ists,107,108 anti-sense RNA,109–112 ribozymes,109–112 and see other of the two IL-3R␤ genes were generated.133–136 Since ␤ ␤ below). they had a functional c chain, mice lacking the IL3 chain Another promising approach to inhibiting leukemic cell were phenotypically normal as they could respond to IL-3.133– 136 ␤ growth is to treat cells with modified toxic cytokines which However, mice lacking the c chain exhibited a pulmonary bind the respective receptors. Thus there is specificity in the alveolar proteinosis-like disease134–136 with a phenotype targeting of the toxins to the particular cells which express resembling the deficiency of the GM-CSF and IL-5 genes.134– 138 ␤ the given receptor. Toxic recombinant cytokines have been In these mutant mice, IL-3 could still act through the IL3 generated by ligating modified Diphtheria toxin or Pseudo- chain, whereas the receptors for GM-CSF and IL-5 were non- ␤ monas exotoxin sequences to cytokine genes. These chimeric functional as they lacked the c chain. To investigate further proteins have shown promise in their ability to kill certain the effects of GM-CSF, IL-3 and IL-5 deficiencies, double- ␤ tumors by binding the receptors and then initiating cell knockout mice lacking both c and IL-3 genes were cre- lysis.113–120 Although this approach is designed to kill tumors ated.139 These mice had neither a functional IL-3 cytokine, expressing the receptors for a particular cytokine, it could be nor functional receptors that could respond to GM-CSF or IL- ␤ easily modified to treat autocrine tumors which express a 5. Surprisingly, these mice had a phenotype similar to c- given cytokine as well as the cognate receptor. knockout mice as no further effects on hematopoiesis were An overview of potential mechanisms to inhibit the growth observed.139 These results indicated the presence of func- of hematopoietic tumors is presented in Figure 4. Although tionally overlapping hematopoietic growth factors in mice. this review focuses on the therapy of hematopoietic tumors, Even in the absence of functional IL-3, GM-CSF and IL-5, we have also provide information concerning the therapies of there appeared to be other cytokines, which could compen- other types of tumors as they may serve as general models for sate for these deficiencies so that ‘normal’ hematopoiesis the treatment and cure of diverse types of cancer. could take place, at least in a laboratory setting. Recently, the effects of GM-CSF over-expression in GM- ␤ 140 CSF-transgenic, c-knockout mice were examined. In these The IL-3 receptor: a cytokine receptor gene family member mice which over-expressed the GM-CSF gene, the low-affinity GM-CSF␣ receptor chain did not induce the same tissue ␤ IL-3 exerts its biological activity by binding to its cognate pathologies that were induced by the c chain in GM-CSF- receptor (IL-3R), which is found on the surface of appropriate transgenic mice.140 These results supported the theory that the target cells.121–129 The IL-3R is comprised of a unique ␣ chain, effects of cytokines are mediated exclusively by high-affinity which associates with IL-3,123,124 and a ␤ chain, which is receptors.140 The phenotypes of cytokine and receptor essential for signal transduction.122 Two contiguous residues transgenic/knockout mice are summarized in Table 5. on human IL-3, D21 and E22, which is conserved in the IL- 3, GM-CSF and IL-5 cytokine family, interact with the IL-3 ␣ ␤ 130 ␣ ␣ ␤ receptor and chains and are crucial for IL-3 activity. Lack of mutations in the IL-3R , GM-CSFR and c chain The IL-3 and GM-CSF genes are located in the 5q region of genes in human hematopoietic cells chromosome 5, which has been associated with hematopoietic malignancies.1–3,122 Likewise, the IL-3R␣ and GM-CSFR␣ Mutations have not been frequently detected in the IL-3 and ␣ ␤ chain genes are closely linked in the X-Y pseudosomal region GM-CSF receptor and c chain genes in cells isolated from of the sex chromosomes.126 It is highly likely that both the IL- patients with hematopoietic disorders and leukemias.141–143 3 and GM-CSF cytokines as well as the IL-3R␣ and GM- These results may indicate that mutations in these genes are CSFR␣ chain genes arose by tandem duplications of original not well tolerated as they may readily lead to life-threatening cytokine and cytokine receptor genes, respectively. conditions. In contrast, mutations in other cytokine or growth The murine IL-3R has two types of ␤ chains; one uniquely factor receptor genes have been documented (c-Fms, c-Kit, ␤ 144–164 binds IL-3 ( IL3), whereas the other is shared with the receptors Flt-2 and G-CSFR). ␤ 122,125 ␤ ␤ ␤ for GM-CSF and IL-5 ( c). The murine IL3 and c genes In contrast, truncated c mRNAs and proteins have been are closely linked and highly conserved in sequence suggest- observed in certain leukemias.165 The function(s) of these ing they also probably arose due to tandem duplication.1–3,122 truncated proteins is not clear, but they may act as naturally In contrast, the human IL-3R has only one ␤ chain, the ␤ com- occurring dominant-negative mutations which prevent signal ␤ 122,123 mon chain ( c) (Figure 5). transduction through the IL-3/GM-CSF/IL-5 receptors, thereby Review WL Blalock et al 1119

Figure 4 Potential methods to inhibit leukemic cell growth. This diagram depicts how the growth of tumor cells can be arrested/suppressed by different mechanisms. The points where certain inhibitors act in these pathways are indicated. Many of these approaches have arisen in the past 25 years due to the discovery of monoclonal antibodies, the cloning of cytokines and their cognate receptors, the identification of signal transduction, cell cycle and apoptotic pathways, and the realization that viruses can be used to deliver modified genes. Although chimeric IL- 3-toxin molecules have not been described in the literature yet, they are currently being evaluated for their ability to eliminate the growth of certain malignant hematopoietic cells. limiting the response of these cytokines after a particular Some of the Cis family of proteins can bind this region and phase of cell differentiation. prevent Jak binding.176,185 The Src and phosphatidylinositol-3 kinases (PI3K) can also bind this region.178 In addition, the Vav GDP/GTP exchange protein binds this region which can Functional regions of the receptor ␣ and ␤ chains result in the association of Vav with the Tec protein tyrosine kinase through interactions of Tec’s Src homology 2 region ␣ ␤ 182,183,186,187 The regions in the IL-3/GM-CSF/IL-5 and c chains respon- (SH2) domains. The membrane proximal region is sible for interaction with signal transduction molecules, regu- also required for the induction of c-myc, cis, pim-1 and lation of gene expression, DNA synthesis, proliferation, differ- oncostatin M (onco M) gene transcription.122,175–177,179,183,19 entiation and prevention of apoptosis have been mapped by Moreover, this membrane proximal region is also sufficient to the creation of a series of truncation and substitution promote DNA synthesis. 122,132,166–195 ␤ mutants (Figure 6). Cysteines 86, 91 and 96 in the Although the distal region of the c chain contains residues ␤ extracellular portions of the c chain are required for recep- which are associated with growth inhibition, this region of the 189,190 ␤ tor dimerization. c chain mostly appears to function in the promotion of ␤ 167 The cytoplasmic portion of the c chain has been divided viability. This region contains binding sites for the Shc pro- into membrane proximal and distal domains, although there tein and the SH2-containing hematopoietic cell phosphatase is some ambiguity regarding the border region of these two 1 and 2 (SHP also known as HCP).172 The distal region is also ␤ domains. The membrane proximal region of the receptor c necessary for the transcriptional induction of the c-fos and c- chain is associated with Janus kinase-2 (Jak2) and signal trans- jun genes and is important in Raf and p70S6K activation, ducers and activators of transcription (STAT) activation.174,175 which can result in the prevention of apoptosis.44,122,170,177,192 Review WL Blalock et al 1120 diverse proteins involved in the regulation of cell growth and differentiation. The ␣ chains of the IL-3, GM-CSF, and IL-5 receptors are less well characterized. However, they all contain three extracellular domains, a transmembrane domain and a short intracellular region.193,195 The proximal intracellular region of the IL-3R␣ chain is necessary for activation of STAT5 and proliferation while the distal region is linked to cell survival. In addition, a group of three proline residues in the cytoplasmic domain of the GM-CSFR␣ chain has been associated with proliferation and differentiation.193–195 Likewise, the GM-CSFR␣ chain plays a direct role in the activation of the Jak/STAT pathway as well as stabilization of the receptor complex.195 In summary, we now know some of the important regulatory regions of the IL-3 and GM-CSF receptors. The current dilemma is how this information can be used to design therapies to treat particular leukemias.

Modulation of tumor growth by antibodies to cytokine/growth factor receptors

Immunotherapeutic techniques designed to target cytokine/growth factor receptors are beginning to show promise in the clinic.196–204 An anti-IL-3␣ chain monoclonal antibody, which functions as a speciﬁc IL-3 receptor antagonist, has been isolated, and treatment of certain terminal cancer patients with modiﬁed antibodies is currently in clinical trials.204 Strategies have been designed to block the activation of receptors which stimulate cell growth in certain types of ␤ 197–201 Figure 5 Sharing of the c chain between the IL-3, GM-CSF and cancer (eg breast, brain, prostate, head and neck). For ␤ IL-5 receptors. The c chain is shared between the IL-3R, GM-CSFR ␣ example, the epidermal growth factor receptor (EGF-R) and and IL-5R. The chains of the different receptors are distinct. Thus the closely related HER2 growth factor receptor have been the ␣ chains differentiate the abilities of these three cytokines to bind their different receptors. implicated in the etiology of breast, kidney, prostate, head and neck, and brain tumors. Therapies based upon the cytolytic properties of monoclonal antibodies to the EGF-R and HER2 ␤ are currently in clinical trials (Genetech; Imclone Systems).105 In addition, the distal region of the murine c receptor contains the binding sites for the Src-family kinases Hck, Lyn and In some cases tumors, after treatment with these monoclonal Syk.191 Thus, there appears to be different regions where the antibodies, have been reduced by more than 50% in test 105 Src and PI3 kinases (proximal region) vs Hck, Lyn and Syk patients. Likewise, monoclonal antibodies conjugated to ␤ bacterial toxins or radioisotopes have been created to kill kinases (distal region) bind the c chain. This distal region is also associated with macrophage differentiation.194 In sum- tumor cells and are currently being investigated in clinical ␤ trials.105,202,203 mary the c chain serves as a target for the binding of many

Table 5 Phenotypes of IL-3/GM-CSF and receptor transgenic mice1

Affected gene Phenotype

IL-3 transgenic Macrophage/microglial-mediated primary demyelination and motor diseases in the central nervous system, neuronal death prevented13,16–19 IL-3 antisense Below-normal serum levels of IL-3, B cell lymphoproliferative syndrome, neurological dysfunction14 IL-3 knockout Impaired hypersensitivity reactions (IL-3 required for efﬁcient priming of hapten-speciﬁc contact hypersensitivity)15 GM-CSF transgenic Elevated serum GM-CSF, abnormal peritoneal cell population, eye destruction, tissue lesions, death from wasting and paralysis, abnormal production of TNF-␣ and IFN-␥140 GM-CSF knock-out Pulmonary alveolar proteinosis-like disease, macrophage hypofunction137–139 IL-3R␣ Not reported in literature IL-3R␤ Lung pathology and impaired immune response, pulmonary alveolar proteinosis-like disease, macrophage hypofunction133–135 GM-CSFR␣ Not reported in literature Review WL Blalock et al 1121

Figure 6 Important regulatory regions of IL-3 receptor. By creation of a series of truncation and substitution mutants, many regions of the ␤ c chain involved in speciﬁc biochemical functions have been identiﬁed. There are some discrepancies as to where the different Src family ␤ ␣ kinases bind the cytoplasmic regions of the c chain. Certain regions of the IL-3R chain have been shown to be necessary for ligand binding, Jak activation and proliferation.

Cytokine-induced signal transduction tional induction of proto-oncogenes such as c-myc and c-fos.213–222 The intracellular signal trandsducing machinery, which controls cell growth, represents another logical area under inten- sive investigation to exploit in the therapy of leukemia. The The Jak/STAT pathway of cytokine-induced signal ultimate goals of these studies are the development of specific transduction compounds which will modulate key intermediates in signal transduction pathways. An overview of the growth pathways Upon binding of IL-3 to the IL-3 receptor, the IL-3 receptor ␣ induced by IL-3 is presented in Figure 7. and ␤ chains heterodimerize, and the entire receptor oligo- Neither the ␣ nor the ␤ chains of the IL-3R has any obvious merizes with other IL-3 receptors.223–225 The association homology to known signaling molecules, such as kinases, of Jak2 with the cytoplasmic membrane-proximal region of ␤ phosphatases, nucleotide binding proteins or src homology the c chain allows for the subsequent oligomerization, (SH)-containing proteins. However, as mentioned earlier, the phosphorylation and activation of Jak2, upon IL-3 receptor ␤ 225 c-chain of the cytokine receptor functions in the activation aggregation. of signal transduction pathways by recruiting the necessary Members of the Janus tyrosine kinase family which kinases.122,129,205–228 An immediate response of cells upon IL- includes Jak1, Jak2, Jak3, and Tyk2, were initially identified 3 activation is the tyrosine phosphorylation of Jak and STAT either by low stringency hybridization with cDNA libraries, proteins,122,207,225 and the activation of Ras, Raf, MEK, and followed by molecular cloning or by PCR amplification MAPK (mitogen-activated protein kinases ERK1/2 (MAP kinase approaches.207,225–227 A unique characteristic of Jak family is a generic name referring to a group of three serine-threonine members is that they contain two tyrosine kinase domains, a MAP kinases (ERK, p38 and JNK)).208–213 Subsequently, these carboxy terminal catalytic domain and a more amino terminal effects are transduced to the nucleus resulting in the transcrip- pseudo-kinase domain.225–228 Jak2 has been demonstrated to Review WL Blalock et al 1122

Figure 7 Overview of effects of IL-3 on cell growth. This diagram depicts the different effects which IL-3 has on cell growth and the prevention of apoptosis. IL-3 can stimulate Jak kinases, which activate gene expression through STAT proteins. Some of the genes that are induced by STAT stimulate proliferation, whereas others serve to inhibit the Jak/STAT signal transduction pathway. IL-3 can also induce anti-apoptotic pathways by stimulating the Ras, cAMP, or PI3K pathways, which can result in the phosphorylation of the pro-apoptotic Bad protein. Also shown are the negative effects of phosphatases, which can dampen IL-3 mediated signal transduction.

be the molecule responsible for some of the immediate cell leukemia,233 as well as v-Abl,234 BCR-ABL,235 and v- responses of IL-3 stimulation and is required for mitogen- Src236,237 mediated transformation of various hematopoietic esis.174,207,225 cells. Mutant Jak proteins have also been observed in certain Jak kinases are sufficient but not required for STAT acti- patients with immunodeficiencies.238–245 Thus, modulation of vation by tyrosine kinase receptors such as the c-Kit, flt2/3 Jak and STAT activities may be a method of therapeutic inter- and M-CSFR (c-Fms) receptors.174,225 In contrast, Jak activity vention in HTLV-I-induced leukemias, chronic myelogenous is necessary for STAT activation by non-tyrosine kinase recep- leukemia and immunodeficiency, as well as other diseases. tors such as the IL-3 receptor.174–225 Experimental data suggest that the SH2 domains of the STAT proteins dictate which Phenotypes of Jak and STAT knock-out mice receptor/kinase complexes they will form.228 IL-3 receptor binding leads to the activation of Jak2 and the recruitment To determine the biological functions of the different Jak pro- and subsequent tyrosine phosphorylation of STAT5 (Y694 of teins, mice were created which lack Jak1, Jak2 or Jak3.245–248 STAT5a and Y699 of STAT 5b).225 Tyrosine phosphorylation Jak-1-deficient mice are runted at birth and died shortly there- of STATs leads to their activation and dimerization.225 These after.245 Deficiencies were seen in the ability of Jak1-knockout STAT dimers then translocate to the nucleus where they act mice to respond to cytokines which signaled through class as transcription factors by binding regulatory sites within the II receptors (interferon-␥) (INF-␥), gp130 subunit containing promoter region of immediate–early genes such as c-myc, ␤- receptors (IL-6R, IL-11R, Onco-R, LIF-R, CNTF-R) or the IL- 176,225 ␥ casein and onco M (see below) as well as feedback 2R c receptor subunit (IL-2R, IL-4R, IL-9R, IL-13R, IL-15R). inhibitors of the JAK-STAT pathway (eg cis).176 Although STAT Phenotypes of Jak and STAT knockout mice are summarized activation requires tyrosine phosphorylation by Jak kinases or in Table 6. tyrosine kinase receptors, STAT translocation to the nucleus A Jak2 deficiency created an embryonic lethal mutation, is enhanced by threonine phosphorylation as a result of namely due to the absence of erythropoiesis.246 During devel- Raf/MEK/ERK activation.229–232 The consequences of acti- opment of these mouse embryos, there was a pronounced vation of this pathway will be discussed later. absence of red blood cells in the fetal liver. The loss of Constitutive activation of members of the Jak-STAT pathway erythropoiesis resulted from the inability of these mice to has been associated with the onset of HTLV-induced adult T- respond to IL-3, GM-CSF, INF␥, EPO and TPO. Review WL Blalock et al 1123 Table 6 Phenotypes of Jak and STAT knockout mice

Gene knocked out Phenotype

Jak1 Runted at birth, early mortality, inability to respond to class II receptors, and receptors that utilize IL- 2R␥ or gp130245 Jak2 Death at embryonic stage, absence of erythropoiesis, inability to respond to IL-3, GM-CSF, IFN-␥, EPO and TPO246 Jak3 Altered T cell responses, lack of response to IL-2, IL-4, IL-7, reduced levels of IL-2 secreted, low levels of CD8, increased T cell apoptosis, B cell differentiation blocked at pre-B cell stage247,248 Tyk Not reported in literature STAT1 Increased susceptibility to microbial infection, inability to respond to INF-␣ and INF-␥249 STAT2 Not reported in literature STAT3 Lethal253 STAT4 Defects in IL-12 response250 STAT5 Hematopoietic cells show slower growth in response to IL-3, GM-CSF and IL-5, reduced expression of Cis, A1 protein251,252 STAT6 Decreased response to IL-4 and 1L-13, weaker pulmonary responses to allergens. Increased activity of p27Kip-1254–256

Jak3 knockout mice displayed an altered T cell response the peribronchial region as well as an absence of eosinophilia. phenotype as well as abnormal lymphoid tissue organization These mice also had impaired MHC class II expression, and and a lack of response to the cytokines IL-2, IL-4 and IL- lymphocytes from these mice failed to proliferate, secrete IgE 7.247,248 These mice possessed T cells, which appeared consti- or develop a Th2 phenotype. Moreover, the loss of STAT6 tutively active but were non-responsive to mitogenic stimuli. resulted in increased activity of the cell cycle inhibitor p27Kip1. The loss of Jak3 apparently had little effect on the immediate– In summary, studies of these various knockout mice have indi- early response of T cell activation, but Jak3-deficient mice cated that it is imperative to have a properly functioning Jak- failed to respond to IL-2 and did not proliferate in response STAT pathway for immune responses, lymphocyte develop- to this cytokine. These mice also had additional abnormalities ment and lymphocytic tissue formation. in T cell development including abnormally small thymuses and a low CD8+ cell count in the periphery, as well as increased T cell apoptosis. Although T cell development pro- Inhibitors of Jak and STAT proteins duced a mature non-responsive T cell population in the periphery of Jak3-deficient mice, B cell development was com- Recently, a family of proteins, which inhibits the activation, pletely blocked at the pre-B stage of development. and/or function of Jaks and STATs was described. This family Functional loss of the Jak tyrosine kinase family members is referred to as the Cis family of proteins and consists of Cis, equates with the loss of STAT activity in response to non-tyro- Soc1, Soc2, and Soc3 as well as other proteins.176,257–261 Some sine kinase receptor activation and may very well have of these proteins inhibit multiple Jak and STAT proteins, additional consequences as other targets of the Jak kinases whereas others only suppress a specific Jak or STAT pro- have been discovered. The function of a number of these tein.176,257–261 In addition, expression of Socs proteins appears STATs has been examined in STAT-deficient mice.249–256 to be tissue specific. These inhibitors can function either by Absence of STAT1 has been linked to increased suscepti- binding the Jak protein and blocking activity or by binding the bility to certain viruses as well as other microbial pathogens cytokine receptors thereby preventing Jak binding and acti- resulting from the inability to respond to INF␣ and INF␥.249 vation. Moreover, there are naturally occurring STAT isoforms, While a STAT1-deficiency shows a mild phenotype, STAT3 which can function as dominant-negative mutants.262 Thus, it loss is lethal, as this STAT family member appears to be neces- may be possible to target the Jak-STAT pathway in certain sary for embryonic development.253 STAT3 is activated by a cells by the introduction of vectors which over-express these variety of cytokines including GM-CSF, IL-6 and EGF. natural inhibitory proteins. Finally, certain drugs such as the IL-12 can mediate the differentiation of T helper cells to tyrphostins AG198 and AG6450 specifically inhibit Jak kin- either Th1 or Th2 type cells. An IL-12-stimulated divergence ases, as well as the growth of lympoblastic leukemia.263,264 requires STAT4.250 Mice with an STAT4-deficient phenotype Thus, there exist both natural and chemical means to inhibit lose the ability to respond to IL-12 resulting in a more Th2- Jak and STAT activities. characteristic development. Growth promoting cytokines of hematopoietic precursors such as IL-3, GM-CSF and IL-5 were shown to activate Additional receptor-associated signal transduction pathways STAT5a as part of their response to receptor binding.251 The lymphoid cells of STAT5a-deficient mice appeared quite nor- There are other signal transduction pathways besides the Jak- mal with the exception that they grew much slower than nor- STAT pathway, which are associated with the IL-3 receptor mal lymphoid cells. In addition, the loss of STAT5a resulted and serve to transmit additional regulatory signals in hemato- in reduced expression of Cis and the Bcl-2- like protein, poietic cells. Upon IL-3 stimulation, the adapter molecule Shc A1.251–253 is rapidly phosphorylated and associates with the phosphoryl- ␤ 183,265,266 Mice deficient in STAT6 displayed a decreased response to ated c chain. Shc contains two domains that are IL-4 and IL-13, which most likely resulted in their reduced capable of interacting with tyrosine-phosphorylated proteins: hyperreactivity to allergens in the airway passages.254–256 an N-terminal phosphotyrosine-binding (PTB) domain and a STAT6-deficient mice exhibited decreased inflammation in C-terminal SH2 domain.181,265–267 The PTB domain is respon- Review WL Blalock et al 1124 sible for the physical association of the Shc protein to the phorylates S6 in vitro and enhances protein translation of cer- ␤ 181 282 receptor c chain. Phosphorylated Shc protein binds to tain mRNAs. The inhibitors, Wortmannin and rapamycin, another adapter protein, Grb2 (growth-factor-receptor-bound can suppress the activities of PI3K and p70S6K, respectively. protein-2), which in turn associates with the GTP exchange Alternatively, p70S6K can be activated by PI3K-independent factor, mSos (mammalian son of sevenless homologue), to means as well. Activated Akt can further transduce the signal activate Ras.266,267 IL-3 stimulation also results in tyrosine to other targets (eg glycogen synthase-3 and Tec family phosphorylation of an SH2 containing inositol phosphatase kinases) and mediate anti-apoptotic functions by phosphoryl- (SHIP) which forms a complex with Shc, Grb2 and Sos and ating the pro-apoptotic Bad protein (see below).280,282–285 In may act to regulate this pathway.268 Phosphorylation of SHIP contrast to p70S6K and Akt, NF-␬B can lead to gene

does not appear to be necessary for its IP3-phosphatase expression that under certain circumstances promotes growth activity; rather, it may be involved in the binding of proteins as well as prevents apoptosis.286,287 necessary for targeting SHIP to the correct subcellular compo- The protein tyrosine kinases responsible for the phosphoryl- nents where its catalytic activity is necessary. Indeed, phos- ation of Shc and PI3K proteins have been suggested, but not phorylated SHIP is found predominately in the membrane exclusively identiﬁed. The kinase which phosphorylates Shc fraction of cells.268 A diagram of IL-3 mediated signal trans- is proposed to be Jak2228,266 while the kinase responsible for duction is presented in Figure 8. phosphorylation of PI3K may be a member of the Src tyrosine The stimulation of appropriate target cells by IL-3 also leads kinase family, which includes Fgr, Fyn, Hck, Lyn, Src, Syk, to the rapid activation of PI3K.269 PI3K is a heterodimeric pro- Tec and Yes in hematopoietic cells.186–188,191,237,269,274 In tein consisting of an 85 kDa regulatory subunit, which con- addition, Ras, as well as other Rac and Rho family proteins, tains SH2, and SH3 domains, and a 110 kDa catalytic sub- can activate or enhance PI3K activity.286,288 unit.269–276 IL-3 stimulation leads to the phosphorylation and The Vav protein is yet another signaling molecule activated activation of an 80-kDa adapter protein which associates with by IL-3/GM-CSF stimulation.182,289–291 Vav contains a single ␤ 289–291 the receptor c chain and with the SH2 domain of the p85 SH2 domain and two SH3 domains. The SH2 domain subunit.179,273 The p85 subunit is then phosphorylated which mediates the interaction of Vav with Jak2, which has been subsequently leads to the activation of the p110 catalytic sub- proposed to be responsible for the phosphorylation and acti- unit that in turn activates the downstream targets p70 S6 kin- vation of Vav.182 Once phosphorylated, Vav can interact with ase (p70S6K), protein kinase B (PKB), also known as Akt, and the Tec protein kinase through Tec’s SH2 domains. Tec can NF-␬B.277–281 then bind PI3K and initiate additional signal transduction cas- The p70S6K is a S6 ribosomal protein kinase that phos- cades. In addition, PKC can also activate Vav leading to

Figure 8 IL-3 mediated signal transduction. IL-3 mediates activation of the Jak/STAT and Ras/Raf/MEK/ERK signal transduction pathways. IL- 3 can also affect apoptosis by inducing the PI3K pathway. Also shown are the activation of PKC and KSR, which can also activate the Raf pathway. Inactivated proteins are depicted in purple whereas the activated forms are depicted in green. ER, endoplasmic reticulum. Review WL Blalock et al 1125 Ras/Raf activation.290 Several pharmaceutical companies have control gene expression. Many of the members of this path- focused on modifying PKC activity as a potential therapeutic way, eg Ras, Raf, MEK, as well as additional downstream tar- ␬ treatment. Many PKC inhibitors (G0,GF) as well as activators, gets, eg c-Fos, c-Jun, Egr-1, Ets and NF- B are proto-onco- including bryostatin, are being used in clinical trials with genes. This pathway is often aberrantly regulated in patients who suffer from certain types of cancer.292–294 Pro- transformed hematopoietic cells. Thus elucidation of the regu- longed activation of PKC by inducers like bryostatin results in lation of this pathway may help in the development of drugs PKC downregulation. Thus activation of PKC by such differen- which will aid in the treatment of various leukemias. tiation-inducing compounds as byrostatin may be a viable Ras is a small monomeric GTP-binding protein whose GTP- therapeutic approach. bound form can associate with its downstream target, which These signal transduction pathways are also under the regu- in some cases is Raf.310–323 Because Ras is ubiquitously lation of phosphatases.295–305 p145 SHIP is purported to be a expressed and often mutated in human cancer, Ras was one of growth inhibitory regulator which down-regulates PI3K and the first oncogenes identified as a potential chemotherapeutic Ras signaling activities. SHIP contains a conserved SH2 target by pharmaceutical companies. For Ras to be functional, domain and an inositol polyphosphate-5-phosphatase it must be farnesylated by the enzyme farnesyl transferase (FT) domain.268,273,288,295,297–302 Targeted disruption of SHIP has which attaches a 15-chain fatty acid to Ras. This modification suggested an important role of this molecule in controlling allows Ras to be tethered to the plasma membrane. Compa- cytokine signaling. In addition to Shc protein association with nies, including Janssen Pharmaceutica, Schering Plough, SHIP, IL-3 induces the transient association of SHIP with Merck and Parke-Davis, either plan to perform clinical trials another phosphatase, SHP2.268,288,295–302 The intacellular lev- or have clinical trials in progress with FT inhibitors (Figure 9, els of this complex may influence whether a cell proliferates Refs 79, 105, 317–322). There is a large family of Ras-related or undergoes apoptosis. SHIP knock-out mice have increased proteins, including Rho and Rac.317 The roles of these Ras- numbers of granulocyte–macrophage progenitors, possibly as related proteins in the growth and transformation of hemato- a consequence of hyper-responsiveness to stimulation with IL- poietic cells remains undefined, but they may serve as poten- 3 and other cytokines.302 tial targets for the FT inhibitors as well. In addition to the signal transducing molecules, binding Ras frequently passes its mitogenic signal on to the Raf pro- ␤ sites are also present on the receptor c chain for SHP-1, a teins, a family of three serine/threonine kinases (Raf-1, A-Raf tyrosine phosphatase bearing two SH2 domains.299,301,303 and B-Raf) which contain binding sites for interaction with SHP-1 is also known as HCP, SH-PTP1 and PTP1C. SHP-1 Ras.310–316 Activated Ras will induce the translocation of Raf ␤ 173 310–312 associates with the c chain via its N-terminal SH2 domain. from the cytosol to the plasma membrane. Thus, SHP-1 is preferentially expressed in hematopoietic cells303 mutations, which alter Ras activity, may also perturb the and negatively regulates the growth stimulation induced by actions of Raf and the downstream cascade. The raf gene is hematopoietic growth factors.173 SHP-1 activity is an essential highly conserved evolutionarily as homologues have been component for controlling the events of cell activation described in Drosophilia and C. elegans.324–326 Loss of raf induced by hematopoietic growth factors as evidenced by a gene expression leads to severe developmental consequences mutation at the SHP-1 gene, which is responsible for the for the organism.317–329 Evidence suggests that this pathway is moth-eaten phenotype in mice.304 Moth-eaten mice have sev- intimately associated with the control of apoptotic machinery ere immundeficiency and autoimmune syndromes. SHP-1 in myelo-monocytic cells.49 negatively regulates erythropoiesis based upon the observations that erythroid cells from these mice are hypersensitive to Epo.304 Clearly cytokines induce phosphatases which also play critical roles in regulating signal transduction. Expression of Raf proteins

The individual functions of the three different Raf proteins in Inhibitors of kinases induced by IL-3 signal transduction, cell cycle progression and the regulation of apoptosis are not fully understood because most biochemi- Many compounds have been described which are tyrosine- cal studies to elucidate Raf function have been performed with kinase inhibitors (Refs 305–309, Table 4, Figure 9). Sugen, Raf-1.325–391 However, since some, but not all, Raf knock-out Zeneca, Oncogene Sciences/Pfizer and other companies are mutants are lethal, the three Raf proteins likely have unique currently using tyrosine kinase inhibitors in clinical trials with non-overlapping functions which do not entirely compensate terminal cancer patients who have gliomas and various solid for one another.327,329 tumors.80,105 Based on the structures of the regulatory and The A-Raf protein is expressed in epididymis, urogenital catalytic sites of these and other kinases it should be possible, and ovary tissues, and hematopoietic cells.327,343,379,388 Mice in the near future, to isolate compounds, using rational drug lacking A-Raf contain defects in the development of the gas- design, which will inhibit a specific tyrosine kinase. The sites trointestinal track.327 In terms of its ability to phosphorylate its of action for certain inhibitors to key enzymes involved in IL- downstream substrate, MEK1, A-Raf has a lower activity than 3-mediated signal transduction are presented in Figure 9. B-Raf or Raf-1 (500- and 10-fold lower, respectively).324,375,377 B-Raf is expressed at high levels in neuronal cells and certain hematopoietic cell lines.378,380–387 Knockout mutations of The Ras/Raf/MEK/ERK signal transduction pathway B-Raf are embryonic-lethal in mice. These mice fail to develop certain key neurological tissues.329 The Ras/Raf/MEK/ERK cascade is perhaps one of the best-stud- Raf-1 and A-Raf are more phylogenetically related to each ied signal transduction pathways. It is centrally involved in other than to B-Raf.324–328 The Raf-1 protein is more ubiqui- the transmission of mitogenic and anti-apoptotic signals in a tously expressed and detected in almost every tissue exam- variety of hematopoietic cells as it couples information initiat- ined.324,328,343 Raf-1 knockout mutants have also been stated ing from membrane receptors to transcription factors which to be embryonic-lethal.329 Review WL Blalock et al 1126 oteins (in black). Deletion or mutation Inhibitors of cytokine signaling molecules. Sites of intervention of pharmaceutically derived chemical drugs (in yellow) and naturally derived pr Figure 9 of the genes encoding the proteins shown in black can contribute to hematological defects and in some cases malignant transformation. Review WL Blalock et al 1127 Structure and activation of Raf proteins Phosphorylation of the tyrosine residues 340 and 341 are critical for the ability of the Raf-1 protein to transform NIH- The Raf proteins consist of three different functional domains; 3T3 cells.336,337,354,377 Moreover, mutation of these residues to CR1, CR2, and CR3. The CR1 region has the binding site for aspartic acid, which gives the Raf-1 regulatory amino acids a an activated Ras protein.313–316,323 The CR2 region negatively conformation resembling phosphorylated residues, converts regulates the Raf kinase domain (CR3) which is located in the the Raf-1 protein into an ‘active’ configuration. This mutation carboxy terminus of Raf.313–316,323 Raf-1 has recently been leads to constitutive Raf-1 activation as well as oncogenic shown to dimerize, and certain drugs (eg coumerimycin) conversion.336,337,377 In contrast, changing these two residues which induce Raf-1 dimerization, stimulate kinase to phenylalanine (FF) which results in a Raf protein that can activity.340–342 not be phosphorylated at the activation domain leads to a Raf- Activation of the Raf-1 pathway is essential for growth fac- 1 protein that does not transform NIH-3T3 cells.336,337,377 tor-induced proliferation during hematopoiesis.330 The events Adjacent to the two tyrosine residues in Raf-1 are two serine that lead to activation of Raf-1 at the plasma membrane are residues338,339 which also have been shown to be important not fully understood, however Raf-1 activation often occurs in the regulation of Raf-activity.354 in the presence of GTP-Ras. Inactive Raf-1 proteins are present The analogous regulatory tyrosine residues in the wild-type in the cytosol bound to 14–3-3 chaperonin proteins. The 14– A-Raf protein are tyrosines 299 and 300.336,337,377 Mutation of 3-3 proteins may bind to a cysteine rich domain (CRD) present the tyrosine residues in A-Raf to aspartic acid increases the in Raf.331,332 Cytosolic Raf-1 is translocated to the plasma transforming ability of this protein whereas substitution to membrane through interactions with GTP-Ras. This occurs phenylalanine residues decreases the capacity of this protein between the Ras binding domain (RBD) on Raf-1 (aa 55 to to transform fibroblastic cells.377 We have extended these 131) and the switch region of GTP-Ras.316,336 Once Raf-1 is observations to hematopoietic cells and shown that site- localized to the plasma membrane, Ras can interact with the directed mutagenesis of the tyrosine residues in either Raf-1 Raf-CRD via the Ras switch-2 region.316,335 These interactions or A-Raf to phenylalanine decreases the ability of these Raf between Ras and the Raf-CRD serve to displace the 14–3-3 proteins to abrogate the cytokine dependency of human hem- proteins from Raf-1 and uncover the kinase domain, allowing atopoietic cells.378 While both Raf-1 and A-Raf contain tyro- the phosphorylation of two regulatory tyrosine residues (Y340 sine residues at these regulatory sites, they each have different and Y341 on Raf-1) by a Src-related protein-tyrosine kin- levels of kinase activity. Hence, there must be structural differ- ase.336–353 ences between these proteins which result in their varying Displacement of the 14–3-3 proteins from the Raf-CRD also abilities to transform cells. permits the dephosphorylation of two regulatory serine resi- The wild-type B-Raf protein contains aspartic acid residues dues on Raf-1 (S621 and S259). Once all of these protein at the corresponding regulatory positions (492 and modifications have occurred, Raf-1 is activated. At least par- 493).336,337,377 It has been postulated that these aspartic acid tial activation of Raf-1 can also be achieved through phos- residues confer a very strong kinase activity.336,337,377 phorylation by other membrane-associated kinases. There is Recently, it was shown that B-Raf could transform NIH-3T3 some evidence for direct activation of Raf-1 by certain PKC fibroblast cells more effciently than either Raf-1 or A- isotypes.338,344–347 The ␣, ␦, and ⑀ isoforms of PKC will lead Raf.336,337,377 However, when site-directed mutagenesis stud- to phosphorylation of Raf-1, however, only PKC ⑀ may func- ies were performed with B-Raf by introducing either tyrosine tionally activate Raf-1.338,346,347 Alternatively, different PKC (DD → YY) or phenylalanine (DD → FF) into these sites, sig- isoforms may stimulate autocrine growth factor loops that in nificant decreases in the transforming abilities of these turn activate Raf-1.82 There is evidence for cross talk mutants were not readily observed.377 These results indicate between the Jak/STAT and the Ras/Raf pathways. Activation that there are additional sites/domains in the B-Raf kinase, of Raf-1 by Jak is dependent upon recruitment of Raf-1 to the which control its high intrinsic kinase activity as well as its plasma membrane by Ras and occurs by phosphorylation of transforming capacity. Raf-1 at Y340 and Y341.231,233,237,339 Certain Raf proteins appear to promote cell cycle arrest. Recent studies have shown that hydrolysis of sphingomyelin High levels of B-Raf and Raf-1 induce p21Cip1 expression, generates ceramide which activates a serine/threonine protein which interferes with Cdk4, Cdk6 interactions with cyclin D kinase (CAPK).343–346 CAPK seems to correspond to a kinase preventing cell cycle progression.389–391 p21Cip1 binds suppressor of Ras (KSR) (see Figure 8) which was originally Cdk/cyclin and blocks the phosphorylation of proteins such identified in Drosophilia and C. Elegans.345 The KSR kinase as the retinoblastoma protein (Rb). We have shown that the phosphorylates Raf-1 on T261 leading to Raf activation. Thus, B-Raf oncoprotein was the least efficient Raf oncoprotein in many different kinases can serve to activate/inactivate Raf-1 abrogating the cytokine dependency of human hematopoietic by tyrosine and/or serine/threonine phosphorylation.339 cells.378,381 This may be a reflection of the enhanced capacity of the B-Raf oncoprotein to induce the expression of cell cycle inhibitory proteins. Proliferative and oncogenic effects induced by aberrant Raf It is conceivable that there are specific interactions between expression certain Raf and 14–3-3 family members.392–402 These interactions may modulate the activity of Raf proteins and regulate Removal of the amino-terminal regulatory domains of all three their ability to lead either directly or indirectly to the phos- Raf proteins results in constitutively active oncoproteins which phorylation of the pro-apoptotic Bad protein. Phosphorylation will transform fibroblastic NIH-3T3 cells and abrogate the of Bad, which leads to sequestering of Bad by 14–3-3, can cytokine dependency of certain hematopoietic cells.374–378 inhibit apoptosis (see below). Thus removal of the Ras binding domain results in activated In hematopoietic cells there may be a delicate balance Raf proteins. The three different Raf oncoproteins vary in their between inducing cell growth and inducing cell cycle arrest. ability to transform fibroblast cells and abrogate the cytokine A Raf oncoprotein with high kinase activity may actually be dependency of hematopoietic cells. the least efficient protein in terms of abrogating cytokine Review WL Blalock et al 1128 dependency. These studies indicate that results obtained with forms of Mos (v-Mos) transform fibroblasts via an MEK1-ERK- fibroblastic models may not always be relevant for other cell dependent pathway implicating Mos as a MEK kinase.411 Mos systems (eg hematopoietic cells). A diagram of the balancing preferentially phosphorylates MEK1 on serine 222.410,411 In effects of the different Raf oncoproteins on cell growth and contrast, MEK kinase-1 (MEKK1), which is associated with induction of cell cycle arrest proteins is presented in Figure stress activated pathways (SAPK) and whose activation is asso- 10. ciated with the induction of apoptosis, preferentially phosphorylates MEK1 on serine 218.372 However, this particular activation of MEK1 does not lead to significant ERK activation, Effects of Raf on downstream kinase activation and its function remains undetermined.368,371 Thus MEK1 may represents a common intermediate in pathways that exert anti- In the mitogen-activated Ras/Raf/MEK/ERK cascade, Raf acti- and pro-apoptotic effects.

vates the dual specific serine/threonine and tyrosine kinase Another potential means of activating MEK1 is via the PI3K MEK1, which in turn activates the MAP kinases ERK1 and pathway.413 The effector directly leading to MEK1 activation ERK2 (p42/p44).405–415 In cells expressing normal MEK1, the in this pathway has not been determined but does not appear 383,385,387 413 kinase appears as a 45-kDa protein. The amino ter- to be Raf-1. Active PI3K leads to the induction of ERK1 and minal end of the kinase has a negative regulatory domain, ERK2 and may be responsible for the prolonged ERK activity since deletion of these residues results in constitutive acti- observed in certain cytokine-stimulated cells.413 Other activation of MEK1, while the catalytic activity is localized to the vators of Raf, such as PKC, can also result in MEK1 and carboxy-terminus of the protein.403–405 Proline-rich sequences ERK activation.414,415 between kinase domains IX and X are required for Raf-1 bind- Use of dominant-negative mutants of upstream activators of ing and subsequent activation of MEK1.405 MEK1, as well as the MEK-specific inhibitor PD98059, Raf-1 activation of human MEK1 requires the phosphoryl- blocked MEK1 and ERK activation.372,373,413–415 The MEK1 ation of serine residues 218 and 222.406–409 Substitution of inhibitor, in turn blocked Ras and Raf mediated transformation either of these residues with aspartic or glutamic acid results and cytokine-stimulated growth.372,373 PD98059 and related in a 10- to 50-fold increase in MEK1 activity.403–406 When drugs that inhibit MEK1 activity may be useful in turning off both serines are replaced with aspartic acid or aspartic and this major pathway in rapidly proliferating malignant cells. glutamic acid (218 and 222), MEK1 activity was 400- to 6000- Recently a MEK1 binding partner, MP1, was identified and fold greater.403–409 These substitutions are believed to confer shown to enhance the enzymatic activation of the MAP kinase to the MEK1 protein a configuration which is constitutively cascade.416 MP1 is a nonenzymatic, scaffolding protein that active.406,408,409 When these MEK1 mutants were transfected serves to anchor both the MEK and ERK proteins facilitating into NIH3T3 cells, constitutive activation of p42/p44 ERKs the activation of both proteins. Raf has not been demonstrated occurred as well as foci formation.405–409 This suggests ERK to be present in this scaffolding complex. It was suggested that activation through Raf requires MEK1. In support of this MP1 functions as an adapter to enhance the efficiency of the hypothesis, PD98059, a MEK1 inhibitor developed by Parke- Ras/Raf/MEK/ERK cascade. A functionally similar protein (JIP- Davis, prevents ERK activation by activated Raf con- 1) has been identified which binds another MAP kinase family structs.372,373 member, JNK.417 The JIP-1 protein also serves as a scaffolding In addition to the three Raf kinases, several other kinases protein to bind the MLK and MKK7, upstream activators of influence MEK activity. One such kinase is the oocyte- JNK, in a complex with JNK. Thus, these signal transduction expressed proto-oncogene, Mos.410,411 Constitutively active cascades also contain other matrix proteins that serve to form

Figure 10 Different interactions of RAF, apoptotic and cell cycle regulatory proteins. This Figure depicts the different effects that the three different Raf proteins can have on cytokine dependency, cell cycle progression and prevention of apoptosis. Review WL Blalock et al 1129 scaffolding devices, which can enhance sequential activation some cells, signal through other molecules that will allow of downstream substrates. growth. As with Raf, constitutive activation of MEK1 has been associated with a variety of neoplasias including hepatocellular Regulation of transcription factors by the Raf/MEK/ERK carcinoma, renal cell carcinoma, breast cancer, squamous cascade cell carcinoma, AIDS-related Kaposi’s sarcoma, acute myelogenous leukemia, and chronic myelogenous leukemia.441,447–461 In addition, constitutive activation of other downstream mem- Ultimately, the signals generated by the Raf/MEK/ERK pathway bers of this pathway has been implicated in oncogenic pro- are transmitted to the nucleus where they lead to activation cesses such as invasion, metastases, angiogenesis, and radior- of various transcription factors necessary for the regulation of esistance.454,455,457,458 One factor involved in invasiveness of cell growth and differentiation.359–370 Raf-1 can activate the c- cancers is the urokinase-plasminogen activator gene whose Jun transcription factor.367 c-Jun and c-Fos heterodimers bind synthesis is increased following ERK activation.458,459 AP-1 driven elements which are contained in promoter Loss of a regulatory phosphatase, responsible for controlling regions of many cytokine and immediate–early genes. Raf this pathway has been associated with neoplasia as well.460,461 activity is also linked to activation of another AP-1-like site MKP-1 is a phosphatase that is activated by mitogenic signals by phosphorylation of the TAR, ATF3, and c-Jun transcription and calcium.460,461 This phosphatase serves to turn off acti- factors.367,369,370 This protein complex binds a response vated ERK in a negative feedback manner, and inhibits ERK- element and leads to cell survival. The removal of growth fac- stimulated DNA synthesis.460 Alterations in MKP-1 expression tors leads to formation of a JunD/ATF3 complex, which acts have been observed in prostate, colon and bladder cancer as a repressor of this response.367,370 where MKP-1 is over-expressed in the early stages but pro- Activated ERK can enter the nucleus where it can act as a gressively diminishes with higher histological grade and met- kinase and phosphorylate certain key regulatory proteins. For astases.460–462 Moreover, MKP-1 over-expression inhibits the example, activation of Elk-1 by ERK occurs in the nucleus differentiation of myoblasts.463 In v-Raf-transformed cells, where active Elk-1 binds the serum response element (SRE) MKP-1 expression appears to be suppressed.464 The sup- contained in the promoter regions of certain cytokine and pression has been speculated to occur via feedback regulation immediate–early genes (ie c-fos).361,362 In addition, ERK can of MKP-1 by v-Raf.464 MKP-1 might be a target for gene ther- also directly activate p90Rsk, an S6 kinase family member (see apy in aggressive hematopoietic tumors, which have lost Figures 9 and 11). p90Rsk activates the cyclic adenosine mono- MKP-1 expression. Inhibitors to MKP-1 or related phospha- phosphate response element (CRE) binding protein (CREB) tases have been isolated, suggesting that it may be possible to which binds CRE response elements in immediate–early gene modulate the activity of these proteins in certain leukemias promoters (see Figure 2).364,366,374 Thus, induction of the Raf (Figure 9).75 In contrast, the involvement of this and other pathway leads to activation of at least three transcription fac- phosphatases in hematopoietic malignancies is an area of tors that bind elements contained in the c-fos promoter (Elk- research in its infancy and requires further investigation. 1, CREB and AP-1).

Alternative signal transduction pathways involved in Involvement of Ras/Raf/MEK/ERK cascade in neoplasia cytokine-mediated growth and response to stress

Ras is one of the most frequently mutated oncogenes in In addition to the Jak-STAT and Raf/MEK/ERK pathways, sev- human malignancies.310,311 There are three Ras related genes, eral alternative signal transduction cascades are activated by Ha-Ras, Ki-Ras and N-Ras.310,311 Mutations in Ras are Ras-dependent and -independent mechanisms.324,465–471 observed in 10 to 50% of patients with myelodysplastic syn- These related pathways could interact with the Raf/MEK/ERK drome and acute myeologenous leukemia.418–422 These pathway (Figure 15). Ras-dependent activation of the MEK1 lesions often result from point mutations in three conserved kinase, MEKK1, a serine/threonine kinase, is responsible for codons, 12 (Ha-Ras, Ki-Ras), 13 (Ki-Ras), or 61 (Ha-Ras, N- activating three MAPK pathways.465–468 MEKK1 phosphoryl- Ras), which convert all three Ras proteins into constitutively ates and activates MEK1 and MEK2368,371 and the dual active proteins.418–422 Altered Ras activity often leads to alt- serine/threonine and tyrosine stress/extracellular regulated ered Raf activity. kinases (SEK).371,469–473 Activation of the SEKs (MKK4 and Deregulated Raf expression and activation as well as MKK3/MKK6) results in the activation of the SAPKs, JNK and mutated forms of the Raf oncogenes have been observed in p38MAPK.371,469–472 JNK is a 46-kDa MAP kinase responsible diverse neoplasias including hematopoietic, breast, cervical, for activating the AP-1 transcription factor component c-Jun renal, laryngeal, hepatocellular, small cell lung carcinomas, by phosphorylating its serine residues 63 and 73.472 p38MAPK and in lung biopsies recovered from cigarette smokers.423–442 is responsible for phosphorylating and activating certain mem- However, the role(s) of Raf in the initial transformation events bers of the AP-1 transcription factor family, the C/EBP tran- are not clear, since the biopsied cells were established scription factor family member, CHOP, and the MAPK-asso- tumors.441 Mechanisms responsible for the expression of acti- ciated protein kinase (MAPK-AP).469,470,475 Activation of vated Raf include point mutation, deletion, gene rearrange- MEKK1 preferentially leads to the activation of ment and gene amplification.375–385 JNK Ͼ p38MAPK Ͼ ERK1 and ERK2.469 On the other hand, Raf- In addition, there are also mutations that eliminate Raf-1 1 leads to the activation of ERK1 and ERK2 but does not result kinase activity. Site-directed mutations, which change the in activated JNK or p38MAPK.385 Although pharmaceutical lysine in the ATP-binding site of the catalytic domain, render companies have produced specific inhibitors to p38MAPK, the kinase inactive, and can serve as dominant-negative direct inhibitors to JNK and ERK have not yet been developed. mutations.443–446 Kinase-inactive mutants of Raf-1 inhibit Ha- Recent evidence suggests that a significant amount of cross

Ras-mediated cell transformation although Ha-Ras can, in talk occurs among the PI3K, MEKK/SEK/SAPK and Review WL Blalock et al

1130 480–495 Raf/MEK/ERK pathways. PI3K has been implicated in cell lines, which can serve as preliminary models for under- MEKK1 activation as well as MEK1/ERK activation,413,485,486 standing cell cycle regulation in hematopoietic cells. whereas oncogenic Raf-1 and MEK1 have also been reported The process of cell division, or the cell cycle, is complex, to activate p70S6K in a PI3K-independent way.494 Inhibitors of orderly and highly regulated in mammals. The eukaryotic the PI3K-p70S6K pathway, wortmannin and rapamycin, exert chromosomes must be replicated and condensed. The centri- their effects at different points in the pathway.488,490,491 Wort- oles must duplicate, separate and then migrate to the opposite mannin inhibits PI3K, while the immunosuppressive drug sides of the nucleus. Following centriole migration, the rapamycin inhibits p70S6K via an upstream target of rapamy- nuclear membrane is broken down and the mitotic spindle cin (TOR) (Figure 9). However, once cells have entered the apparatus is assembled. The condensed chromosomes are seg- cell cycle, inhibition of p70S6K does not stop prolifer- regated via the spindle network. Once the chromosomes are ation.482,483 Use of these inhibitors has documented the PI3K- segregated, the mitotic spindle complex is subsequently independent activation of p70S6K.495 Rapamycin has been broken down and the nuclear membrane is reassembled. The recognized for years as a potentially useful immunosuppres- chromosomes de-condense and then the cell membrane sive drug.58 Thus these inhibitors or derivatives of them may invaginates to complete cytokinesis. The cell cycle is divided be useful in the treatment of patients with certain leukemias. into stages with respect to these different processes. DNA replication/synthesis (S phase) and mitosis (M phase) must be properly coordinated to ensure constant maintenance of the Cytokines and cell cycle regulatory proteins genetic material. The model proposed for cell cycle pro-

gression is relatively simple. A cell in G0 (a quiescent resting We have addressed cytokines, their receptors and the control stage theoretically out of the cell cycle)/G1 (Gap1) receives of gene expression by downstream signal cascades which positive (mitogens and growth factors) and negative (contact regulate the activity of transcription factors. The next area to inhibition or cellular damage) stimuli. These signals are inte- consider for therapy is modulation of the cell cycle regulatory grated to decide whether or not the cell should enter the cell 496 proteins by cytokines, including IL-3. Much of the information cycle. The decision whether to progress from G1 into S for this section has been derived from studies with ﬁbroblastic phase is a position in the cell cycle termed the restriction (R) point.496 Coordination of cell cycle progression is achieved through the activity of the cyclins and their associated Cdks, as well as interactions with tumor suppressor genes (eg p53, p21Cip1) that regulate the activity of these complexes. These signaling and regulatory molecules comprise the cell cycle clock. In mammalian cells, three major Cdks are associated with

G1 progression. Cdk4 and Cdk6 bind to cyclins D1–3 and activate them in mid to late G1 stage of the cell cycle whereas Cdk2 is activated by binding cyclin E or cyclin A later in G1 497–519 (Figure 12). Cyclin E is expressed only in G1 phase and disappears once cells enter S phase. In contrast, cyclin A is

expressed later than cyclin E in the G1 phase and steadily 498,499 accumulates during the S and G2 phases. Cdk1 is associated with mitosis and is active at the G2/M transition. Although the levels of Cdks remain relatively constant throughout the cell cycle, the cyclins ‘cycle’ during pro-

gression through G1 to M. Ectopic over-expression of either cyclin D1 or cyclin E can promote G1 progression which leads to a shorter G1 phase, reduced cell size and decreased serum dependency.500–504 In contrast, abolishment of cyclin D1 or E/Cdk complex activity by neutralizing antibodies or antisense oligonucleotides effectively blocks the entry of the cells into 503,504 S phase. It is believed that in addition to G1 progression and S phase entry, cyclin A is also required for the normal transition from S phase to mitosis as it is needed for the activation of Cdk1.505,519 Loss of cyclin A prevents a round of DNA replication and disrupts key checkpoint events that couple mitotic initiation to the completion of DNA synthesis.506,507 Cdks are serine/threonine kinases which, when bound to their regulatory cyclin subunits, cyclins, can phosphorylate the retinoblastoma protein (pRb) and pRb-like proteins such as p130 and p106, collectively termed pocket proteins.510–514 Figure 11 Alternative signaling pathways. In addition to the The normal function of these pocket proteins is to negatively Ras/Raf/MEK/ERK pathway, there are other signal transduction path- control passage from G1 into S phase by sequestering E2F tran- ways which may cross-talk (interact) with each other. These pathways scription factors that are required for transcriptional activation can be induced by many different stimuli that induce cell stress. The 510–514 kinases, which are functionally similar, are at the same horizontal of genes responsible for the G1/S transition. The pocket positions in the pathway. Additional related molecules and other proteins are phosphorylated by Cdks, initially triggered by the names are shown on the left and right hand sides of the ﬁgure. cyclin D-Cdk4/6 complex503,517 and then accelerated by the Review WL Blalock et al 1131

Figure 12 Interactions between cyclins, kinases and inhibitory molecules. Potential interactions between cyclin, CDK and cell cycle inhibitory molecules are indicated. Functional interaction of Rb with E2F is indicated in the conversion from the inactive in purple to the active hyperphosphorylated form, which is indicated in green. The cyclin A/E/CDK4/p27 complex should read cyclin A/E/CDK2/p27. cyclin E-Cdk2 complex.502,504 E2F transcription factors are Recent experimental results suggest that instead of redun- then released, bind to specific promoter regions, and modu- dancy these two families of Cdk inhibitors actually work in late the gene expression. Target genes for activation by the concert to regulate cell cycle progression.541,543 transcription factor E2F include regulators of S-phase entry (eg B-myb, Cdk1, cyclin E and cyclin A), and other genes required for DNA replication (dihydrofolate reductase, DNA pol␣, and INK4 family thymidine kinase).520–528 The activity of the cell cycle machinery is regulated by the The Ink4 family contains four members, p16Ink4a, p15Ink4b, presence of cell cycle inhibitors that can arrest cell cycle pro- p18Ink4c, and p19Ink4d. Ink4 proteins, which consist of tandem gression at certain checkpoints during the cycle. Many of repeats of an ankyrin-like sequence, are able to specifically these inhibitors have earned the name ‘tumor suppressors’ bind Cdk4 and its close homologue, Cdk6.515,538–540 This because a loss of expression (or activity) will often result in direct binding can prevent the dimerization of Cdk4/6 with tumorigenesis. Thus, there is an expectedly high mutation fre- their cyclin partners, cyclin D1–3, and hence prevent kinase quency in the genes that code for these ‘tumor suppressors’. activity (Figure 12). p16Ink4a, the first Ink4 member to be The two most widely studied inhibitors are p53 and pRB. p53, cloned and characterized, was initially identified in SV40- a transcription factor, relies on stimuli from outside the cell transformed fibroblasts.542 The observed binding of this 16- cycle clock. Signals such as DNA damage, contact inhibition, kDa protein with Cdk4 and Cdk6 was specific since no bind- hypoxia, and viral infection may increase p53 protein lev- ing to cyclins or other Cdks was observed. In addition, p16Ink4a els.529 p53 is believed to be the most commonly mutated gene also bound to preformed and catalytically active cyclin in human cancer.530 p53 induction often leads to increased D:Cdk4 and cyclin D:Cdk6 complexes in insect Sf9 cells and expression of the p21 protein, Cip-1/Waf-1/SDI-1.531–533 In inhibited their activity.542 Ectopic overexpression of p16Ink4a 515 mammalian cells, two families of Cdk inhibitors are involved can induce G1 arrest in primary fibroblasts. This induction Ink4a in the regulation of G1 progression, the Ink4 family and the of G1 arrest by p16 is believed to be pRb-dependent since Cip/Kip family.534–542 Even though they both act on Cdks to overexpression of p16Ink4a prevents proliferation in pRb+ cells, inhibit their kinase activity, their structure, their spectrum of but not in pRb− cells.515,538,544 substrates, and the way they bind with Cdks are very different. Three other Ink4 gene family members, p15Ink4b, p18Ink4c, Review WL Blalock et al 1132 and p19Ink4d, were subsequently cloned and identified. All tion to inhibit the kinase activity of the complex.537 It was three possess the capacity to bind Cdk4/6, inhibit their kinase found that in normal fibroblast cells, Cdks normally form a activities and induce cell cycle arrest.538,545–547 p15Ink4b was quaternary complex with cyclin, the proliferating cell nuclear induced in epithelial cells specifically after transforming antigen (PCNA) and p21Cip1. However, when cells are prolifer- growth factor-␤ (TGF-␤) treatment, suggesting that it functions ating, p21Cip1-cyclin-Cdk-PCNA complexes retain their Cdk as an effector of TGF-␤-mediated cell cycle arrest.546 p18Ink4c kinase activity. When more p21Cip1 proteins were introduced displayed a common feature with p16Ink4a in that it was into the cells, the Cdk complexes lost their kinase induced by cell cycle arrest in a pRb-dependent pathway.538 activity.555,556 These results indicated that the concentrations It remains unclear as to how each Ink4 protein contributes to of p21Cip1 proteins are critical for the cell to undergo cell cycle Cdk4/Cdk6 regulation in vivo. However, the different progression, division or arrest. mutation frequencies of Ink4 genes in human cancers and The mechanism by which the Cdk kinase inhibitor p21Cip1 tumor cell lines and the divergent patterns of gene expression binds to Cdk/cyclin complexes is not yet clear. It is possible during cell cycle progression suggests they each have dis- that p21Cip1 can modify Cdk kinase activity in more subtle tinct functions. manners. The fact that p21Cip1 protein associates with a PCNA p19Ink4d is expressed ubiquitously in proliferating cultured molecule, a subunit of DNA polymerase-␦, implies that cells and in normal mouse tissues.540 More interestingly, in p21Cip1 may coordinate the regulation of cell cycle pro- accordance with its theoretical in vivo function, an oscillation gression and DNA replication and/or DNA repair. In vitro of p19Ink4d mRNA as well as protein levels occurs during the DNA replication studies have demonstrated that p21Cip1 can cell cycle in cultured macrophages and fibroblasts.540 The uni- inactivate DNA polymerase-␦ by binding PCNA with its car- versal p19Ink4d expression in cell lines and tissues and its per- boxy-terminus.557,558 However, although DNA polymerase-␦ iodic expression during the cell cycle suggests that p19Ink4d is also involved in DNA repair, p21Cip1 does not appear to may be the Cdk inhibitor that actually regulates cyclin D- affect the efficiency of DNA repair.559,560 dependent kinase activity as cells enter S phase.540 p21Cip1 may play important roles in the organism’s A high percentage of deletion or hypermethylation in the responses to cellular stress. Following DNA damage, human promoter regions of the p15Ink4b and p16Ink4a genes has been cells lacking p21Cip1 can still undergo cell cycle progression observed in human non-small cell lung carcinomas, malignant through S phase. This often leads to DNA replication in the gliomas, renal cell carcinomas, head and neck tumors, pros- absence of cell division. Uncoupled cell cycle progression tate tumors, bladder carcinomas, and leukemia and lym- can lead to nuclear abnormalities (ie aneuploidy) and ulti- phoma cells.548–550 In contrast, mutations or changes in the mately apoptosis of the cell.541 methylation status of the p18Ink4c and p19Ink4d genes have not p21Cip1 can be transcriptionally upregulated by the p53 pro- 548,551 been frequently detected in human cancer or cell lines. tein, and is a primary mediator of the p53-dependent G1 arrest Thus, only the p15Ink4b and p16Ink4a genes have been widely that occurs following DNA damage.532,553,561,566 It appears accepted as tumor suppressor genes. Members of the Ink4 that p53 can bind to the promoter region of p21Cip1 and family, the Cip/Kip family and additional cell cycle regulatory increase its expression.562 However, induction of p21Cip1 does proteins are listed in Table 7. not always require p53 expression as p53-independent pathways of p21Cip1 induction have also been described.390,391,565–570 Multiple Sp1 binding sites are con- Cip/Kip family tained in the proximal region of p21Cip1 promoter. These binding sites are believed responsible for the constitutive The Cip/Kip family consists of p21Cip1, p27Kip1, and p57Kip2. expression of p21Cip1. This promoter region also mediates the Cip/Kip proteins contain a homologous amino-terminal transcriptional activation of p21Cip1 by members of the Smad domain, which contains contiguous cyclin- and Cdk-binding family of proteins.567 Kip1 regions. These adjacent binding regions allow the Cip/Kip pro- p27 was first identified in cells arrested at G1 by TGF- teins to selectively bind cyclin/CDK complexes. Cell cycle ␤.571 Like p21Cip1, p27Kip1 affects many Cdks, and appears to components, which are targets for binding Cip/Kip proteins, bind to cyclin-Cdk complexes more efficiently than to Cdks include cyclin D-Cdk4/6 and cyclin E-Cdk2, which are acti- alone. p27Kip1 shares significant amino acid homology in its Cip1 Cip1 vated at the late G1 phase, and cyclin A-Cdk2, that is active amino-terminus with p21 ; however, unlike p21 ,no at the G1/S transition and throughout the S phase.533,552–581 PCNA binding domain has been identified in p27Kip1. Saos-2 p21Cip1 was discovered as a Cdk-interacting protein (Cip) cells, a human osteosarcoma cell line which does not express

which could bind and inhibit the activity of cyclin-cdk com- functional pRb and p53, arrested in G1 after transfection with plexes.532,533,535,552–565 Binding of the cyclins blocks Cdk a vector encoding p27Kip1.572 It was thereby suggested that Kip1 phosphorylation of pRB as well as other proteins that require p27 -mediated G1 arrest is pRb- and p53-independent. phosphorylation during cell cycle progression. p21Cip1 Since the quantity of mRNA encoding p27Kip1 remained the inhibited the activity of each member of the cyclin/Cdk family same after TGF-␤ treatment571 and the p27Kip1 protein levels Cip1 indicating that p21 was a universal inhibitor of did not change when cells exited G0 and progressed through cyclin/Cdk complexes.536 the cell cycle, the regulation of p27Kip1 may occur predomi- Surprisingly, p21Cip1 was also found to be a component of nantly at the post-transcriptional level.572 active cyclin/Cdk complexes. After this crucial discovery, it p57Kip2 is the most recent member of the Cip/Kip family to became apparent that p21/cyclin/Cdk complexes could alter- be identiﬁed.573 Structurally, it is more similar to p21Cip1 than nate between active and inactive states. Regulation of the p27Kip1 since it also contains a cyclin-Cdk binding domain at activity of these complexes is thought to be a function of the the N terminus and a PCNA binding domain at the C ter- stoichiometry of the p21Cip1 subunit within the cyclin/Cdk minus.575 The p57Kip2 protein can inhibit the kinase activities complex.537 One theory suggests that p21Cip1 acts as an of cyclin D-Cdk4/6, cyclin E/A-Cdk2, and cyclin E-Cdk3 com- assembly factor for the cyclin/Cdk complexes. However, plexes in vitro. Like p27Kip1, when transfected into mink lung when p21Cip1 is present at higher concentrations it may func- cells or into human Saos-2 cells, p57Kip2/cyclin/Cdk com- Review WL Blalock et al

571–573 1133 plexes induced G1 arrest. Control of cell cycle and sup- both Cdk2 and Cdk4 before the G1/S transition. Ectopic over- pression of cell transformation by p57Kip2 required both Cdk expression of Ras or its downstream molecules, such as ERK and PCNA inhibitory activity. Disruption of either function or Ets-2,584 can lead to the induction of cyclin D but has little leads to uncontrolled cell growth.575 or no effect on cyclin E or A.582,584–588 The regulation of cyclin D is thought to be the more critical and limiting step because of its rapid degradation. Its expression is dependent on con-

The interplay between Ink4 and Cip/Kip tinued growth factor stimulation until cells pass the G1 restriction point. Mutations at either Ink4a/Ink4b or Cip/Kip can result in malig- DNA sequencing analysis has revealed an AP-1-like nant transformation. These mutants present a dilemma in sequence in the promoter region of the human cyclin D1 understanding tumor progression. Why does a single knockout gene.584 This AP-1-like sequence is required for activation of mutation lead to cancer when there appears to be redun- the cyclin D promoter by c-Jun; a target gene of Ras mediated dancies present in the overall system? It remains a mystery signal transduction. Site-directed mutagenesis of this AP-1-like why two families of Cdk inhibitors are required for the proper sequence may abolish Ras-dependent activation of cyclin D control of cell cycle progression. However, more and more expression. Several AP-1-related proteins, including c-Jun, studies have indicated that cooperation occurs between these JunB, JunD, and c-Fos, bind to this AP-1-like sequence in the two sets of inhibitors of cell proliferation. One example is the cyclin D promoter region. Down-regulation of the Cdk inhibi- interplay between p15Ink4b and p27Kip1 in TGF-␤-treated epi- tor p27Kip1 was also observed in rapidly proliferating Ras trans- thelial cells. Even though p27Kip1 can bind cyclin D-Cdk4/6 formed cells.586 and cyclin A/E-Cdk2 complexes, the interaction of p27Kip1 with these two kinase complexes is very different. p27Kip1 can inhibit the activity of cyclin A/E-Cdk2 but not cyclin D- Antiproliferative effects of oncogenic Ras Cdk4/6.576 In rapidly proliferating cells, p27Kip1 is predominately bound with cyclin D-Cdk4/Cdk6572,576,577 without The effects of Ras on proliferation and tumorigenesis have inhibiting complex kinase activity. Cyclin D-Cdk4/6 serves as been well-documented in immortal cell lines.588–590 However, a reservoir to sequester free p27Kip1 from its target, cyclin A/E- antiproliferative responses of oncogenic Ras were observed in Cdk2. When cells were treated with TGF-␤, p15Ink4b was non-transformed fibroblasts, primary rat Schwann cells, rat induced and competitively bound to cyclin D-Cdk4/6. As a pheochromocytoma PC12 cells, NIH-3T3-L1 fibroblast cells, result, there was more free p27Kip1, which could bind to cyclin and primary fibroblasts of human and murine origins.591–596 It A/E-Cdk2 to inhibit its kinase activity.576,578 This resulted in a is believed that the cell proliferation arrest induced by oncog- combined inhibition of both cyclin D-Cdk and cyclin E/A- enic Ras was not an immediate outcome of Ras signaling, but

Cdk; hence the cells were arrested at the G1 phase. In vitro a late cellular response due to abnormal Ras activity. In the studies have shown that p15Ink4b can only prevent p27Kip1 fibroblasts that were transfected with oncogenic Ras, induc- binding to cyclin D-Cdk4/6 when it has access to cyclin D- tion of cell cycle inhibitors, p16Ink4a, p21Cip1, and p53 as well Cdk4/6 first. This was determined by examining the different as decreases in the levels of cyclin A and Cdk2 kinase activity subcellular locations of these two inhibitors. In normal proli- and hyperphosphorylated pRb, were observed. In p53− and ferating epithelial cells, p15Ink4b and Cdk4 are localized p16− mutant fibroblasts, proliferation was observed in the mostly in the cytoplasm, whereas p27Kip1 is present in the presence of oncogenic Ras. Thus p53 and p16Ink4a tumor sup- nucleus.579 pressor genes are essential for the cell cycle arrest mediated Recently, a negative feedback control of cyclin E-Cdk2 by Ras. complexes on the activity of p27Kip1 was observed.580 The expression of cyclin E-Cdk2 in murine fibroblasts can cause phosphorylation of p27Kip1 on T187, and an in vitro experi- Cell cycle arrest induced by Raf ment demonstrated that this phosphorylation event could be directly mediated by purified active cyclin E-Cdk2.580 It was Over-expression of activated Raf protein has also been asso- proposed that the interactions between cyclin-Cdk and Cdk ciated with cell cycle arrest in some cell lines, including rat inhibitor occur in two phases. First, binding of cyclin E-Cdk2 Schwann cells, mouse PC12 cells, human promyelocytic leu- with p27Kip1 can inhibit Cdk2 activity. Secondly, this binding kemia HL-60 cells, and small cell lung cancer (SCLC) cell can also result in p27Kip1 phosphorylation, reverse the cell lines.389–391,597–600 In contrast, T cell differentiation is acceler- cycle blockage caused by p27Kip1, and initiate a pathway lead- ated in transgenic mice expressing activated Raf in the thy- ing to elimination of p27Kip1 from the cells. Binding of ATP to mus.601 It is not clear why over-expression of the Raf gene the Cdks determines which state predominates. At low ATP can lead to such conflicting results, but recent reports have concentrations (less than 50 ␮M), p27Kip1 is primarily a Cdk suggested that it is the amount or activity of the Raf oncoprot- inhibitor, but when the ATP concentration increased to more ein that leads to the opposing outcomes.390,391 Depletion of than 1 mM, p27Kip1 is more likely to be a substrate.576–578 It is Raf-1 was shown to abrogate p21Cip1 induction after taxol not clear whether this kind of feedback regulation also exists treatment in a human prostate cancer cell line.597 Further- with other cyclin-Cdk complexes and Cdk inhibitors.576–578 more, recent studies involving conditionally active ⌬Raf:ER proteins have implicated the Raf family of kinases in cell cycle control.598–607 Raf-1 was determined to be sufficient for Links between the Ras/Raf/MEK/ERK pathway and cell p21Cip1 induction as long as the MEK/ERK pathway was left cycle proteins intact.598 Further study of Raf-mediated p21Cip1 induction has lead to a model that integrates proliferative signals with cell The Ras-activated signal transduction pathway provides a link cycle arrest. The effect of Raf activation seems to be determ- between IL-3 and stimulation of cell cycle machinery.582–588 ined either by the level or duration of Raf activity in the cell. Ras is required for cell cycle progression and activation of Upon stimulation, Raf delivers a proliferative signal that Review WL Blalock et al 1134 results in immediate–early gene activation and induction of in its cytoplasmic tail. A diagram of the hypothetical interac- the cyclins. However, prolonged signaling through the tions between IL-3 and TGF-␤ signal transduction pathways MEK/ERK proteins results in induction of p21Cip1 and cell is presented in Figure 13. cycle arrest. This dual activity of Raf might serve as a means In a model proposed by Derynck and Feng, ligand binding of feedback inhibition and could maintain checks and to the TGF-␤ heterodimeric receptor complex induces auto- balances on the generation of proliferative signals. The three phosphorylation of the receptor.615 The activated TGF-␤ different Raf proteins have varying levels of kinase acti- receptor then recruits Smad2 and Smad3 to the membrane vity.375–378,391 Thus one would expect differences in the bio- and phosphorylates them.618,619 The phosphorylated Smad2/3 logical effects of A-Raf, B-Raf, and Raf-1. This is supported by complex is subsequently released from the receptor complex the fact that A-Raf, a weaker kinase than either B-Raf or Raf- and associates with Smad4. The activated heterotrimeric Smad 1, is a poor inducer of p21Cip1 expression while B-Raf and complex is then translocated into the nucleus, where it can Raf-1 can readily induce p21Cip1 and cell cycle arrest in function as a transcriptional activator and induce the NIH3T3 fibroblasts.391 expression of its target genes. TGF-␤ treatment can lead to In NIH-3T3 cells transfected with the different Raf genes, CREB phosphorylation and increase the binding of CREB to the Raf proteins were able to upregulate the expression of the AP-1 or CRE element in the promoter region of the col- cyclin D1, cyclin E, Cdk2 and Cdk4 and downregulate the lagenase gene.620 Therefore, CREB has been suggested to expression of Cdk inhibitor p27Kip1.391,602–614 These changes associate with the Smad complex in the transcriptional acti-

induced cells to pass through G1 phase and enter S phase. vation of certain genes. In addition, the transcription factor However, in B-Raf and Raf-1 transfected cells, there was also CTF-1 has also been shown to respond to TGF-␤ and mediate a signiﬁcant induction of p21Cip1. Study of Raf-mediated cell the transcriptional induction of certain genes.621 The identiﬁ- cycle arrest should provide multiple targets for therapeutic cation of TGF-␤-responsive transcriptional complexes and intervention in malignancies involving a deregulated cell promoter sequences are currently under investigation. Some cycle. Cdk inhibitors, including p15Ink4b, p16Ink4a and p21Cip1 are considered possible target genes because they are induced after TGF-␤ treatment. TGF-␤ and cell cycle arrest

TGF-␤ can induce a strong growth inhibitory effect on many Identification of cell cycle regulatory molecules as cell types and also stimulate cell proliferation in therapeutic targets others.571,593,606–612 Although the links between IL-3 and TGF- ␤ have not been well documented or examined, we have Elevated levels of cyclins are found in fibroblastic and lymph- included this section on TGF-␤ because it is an important oid tumors. Cyclin D is over-expressed in 86% of breast carci- regulator in cell cycle progression and could have significant nomas, while the loss of p16Ink4a results in increased levels of therapeutic potential. The stimulatory effects of TGF-␤ are active cyclin D/Cdk4 and has been associated with malignant most likely indirect (ie through expression of other endogen- melanoma and thyroid cancer. Thus it is relevant to develop ous growth factors, such as platelet-derived growth factor, drugs which are cell cycle antagonists. Indeed, many anti-can- connective tissue growth factor, or from increased expression cer drugs are aimed at inhibiting specific phases of the cell of EGF receptors). In contrast, TGF-␤ induced growth arrest is cycle.622–633 more direct and is correlated with the maintenance of pRb in Vinblastin, pironetin and a demethyl derative, NK1098P, 609 ␤ its hypophosphorylated state. The effects of TGF- on G1 inhibit cell proliferation in M-phase (Table 7) whereas, the cyclins, Cdks, and Cdk inhibitors are well documented. Treat- drug RK682 inhibits cell cycle progression in the G1 to S tran- ment of epithelial cells with TGF-␤ can lead to inhibition of sition.623,626 In addition, Cdk inhibitors are also being tested Cdk2, Cdk4, cyclin A and cyclin E.610,611 In these cases, phos- in clinical trials by the pharmaceutical company Hoechst- phorylation of pRb was inhibited and the cell cycle was Marion-Roussell on patients with various tumors.105

arrested at G1 stage. Over-expression of Cdk4 can reverse the Another therapeutic target is the p53 protein. Some pharma- ␤ 611 ␤ G1 arrest induced by TGF- in epithelial cells. TGF- can ceutical companies such as Onyx are conducting clinical also induce the expression of Cdk inhibitors p15Ink4b and trials that exploit the fact that many tumors are p53-negative p21Cip1,546,579,612 and stimulate a redistribution of p27Cip1 with (p53−). They have designed an adenovirus vector, which does cyclin-Cdk complexes.613 TGF-␤ induces p15Ink4b to be bound not replicate in or lyse wild-type p53 cells but can replicate to Cdk4 and Cdk6 and thereby inhibits their functions. In in and kill p53− cells.79,105 This experimental therapy is being addition, due to the competitive binding by p15Ink4b for Cdk4 evaluated on terminal cancer patients with head and neck, and Cdk6, more p27Kip1 is released from its reservoir cyclin gastrointestinal, pancreatic and ovarian tumors.79,105 Another D-Cdk4/6 pool and binds to its real target, cyclin E-Cdk2. therapeutic approach using modified adenovirus or retrovirus The signal transduction pathway initiated by the growth- is to introduce wild-type p53 genes into p53− tumors, as the inhibitory cytokine TGF-␤ is not as clear as the ones induced infected cells should cease proliferation or die. Introgen by growth-stimulating cytokines, such as IL-3 or GM-CSF. The Therapeutic, Canji/Schering-Plough and other companies are first important TGF-␤ signaling mediator characterized was evualating this therapy with cancer patients that have lung, Mothers against decapentaplegic (Mad) in Drosophila cells.614 head and neck, ovarian and liver malignancies.79,105 This is not the same Mad which heterodimerizes with Max and inhibits Myc:Max heterodimers. So far, six Mad homologues have been identified in vertebrate cells, namely Smad- Apoptosis and cell death 1to6.615 Some kinases have also been associated with TGF- ␤-induced signaling which include MAPK, and the yet to be Apoptosis is an intrinsic mechanism of cell death in which a identified 57 kDa, 105 kDa, and 130 kDa kinases.616,617 The variety of stimuli can activate specific cellular factors to effec- TGF␤ receptor has an intrinsic serine/threonine kinase activity tively ‘dismantle’ the cell and facilitate its removal from a Review WL Blalock et al 1135

Figure 13 TGF-␤ induced signal transduction pathways. Potential interactions between IL-3 and TGF-␤ signal transduction pathways are indicated. TGF-␤ binds its receptor which contains an intrinsic serine/threonine kinase activity. This results in the activation of Smad2/3 which subsequently binds Smad4 and migrates to the nucleus to transactivate genes such as p21Cip1 and p15Ink4b. Ras can induce the expression of p21Cip1, p27Kip1, p16Ink4a and the cyclins. population without adversely affecting neighboring cells. and protein cleavage, increased intracellular Ca+2, loss of Apoptosis occurs as a normal process of cell elimination dur- mitochondrial transmembrane potential (␺) and release of ing development, homeostasis, and self-reacting immune cell cytochrome c (cyto c) from mitochondria, and externalization depletion.634–636 Apoptosis also plays a role in eliminating of phosphatidylserine from the inner plasma membrane cells exposed to environmental stress factors such as growth (Table 8). The most commonly accepted biochemical indi- factor withdrawal, irradiation, ischemia, and viral infection. cator of apoptosis is the cleavage of DNA into oligonucleoso- Dysregulation of apoptosis can lead to immortalization of cells mal-sized fragments.644–646 Single-strand nicks and cleavage and cancer, or inappropriate cell death as in AIDS-associated into 50–200 kbp-sized DNA may also occur before or in the T cell demise and some neurodegenerative diseases.637–639 An absence of oligonucleosome fragmentation.646 Apoptosis is overview of some of the factors, which can stimulate and/or generally characterized by one of more of these morphologi- repress apoptosis, is presented in Figure 14. Regardless of the cal and biochemical changes, but may vary with cell type, cell type or induction stimulus, apoptotic cells generally exhi- induction stimulus, and other factors. Some of the events bit similar morphological and biochemical changes, suggest- involved in the initiation of apoptosis are illustrated in ing a common execution program of effector and target mol- Figure 15. ecules.640–643 This systematic scheme of cell death involving Cell death which occurs by nonapoptotic mechanisms is a conserved repertoire of molecules supports the idea that characterized by increased membrane permeability and cell apoptosis may be controlled by therapeutic intervention, swelling, random degradation of the chromatin, and eventu- thereby reducing or preventing pathogenesis due to inappro- ally cell lysis. Apoptotic cells typically remain membrane- priate apoptosis. bound, even when they break apart, and therefore do not In general, morphological changes observed in cells elicit an inflammatory response. They are removed by local undergoing apoptosis include the loss of cytoplasmic volume macrophages or neighboring, nonprofessional phagocytic and shrinkage, whereas cells dying by necrosis tend to cells.647,648 The efficient recognition of apoptotic cells by swell.640 Chromatin condenses progressively, forming small, phagocytic cells appears to be mediated, in part by the exter- diffuse foci that may fuse and marginate against the nuclear nalization of phosphatidylserine during apoptosis.647,648 In membrane, and eventually collapse into a round, dense struc- contrast, cells that die by nonapoptotic mechanisms lyse and ture. The nucleus often breaks up into membrane-bound bod- elicit an immune response.648 ies, which may or may not contain chromatin. The cell itself may fissure into multiple membrane-bound apoptotic bodies Caspases and the regulation of apoptosis containing nuclear fragments, or shrink into a single, dense, round apoptotic body. Biochemical changes associated with The caspase family of proteases comprises the effector arm of apoptosis include specific patterns of DNA, ribosomal RNA the apoptotic pathway.649–651 Thirteen mammalian caspases, Review WL Blalock et al 1136

Figure 14 Overview of apoptosis in hematopoietic cells. Some of the molecular events and effectors involved in the apoptotic pathway are shown. Pro-apoptotic events and initiators are indicated at the top of the Figure. A variety of events and molecules can initiate or promote induction of apoptosis under normal and pathogenic conditions. Anti-apoptotic events and inhibitors are indicated at the bottom of the Figure and include both cellular and viral proteins. The ﬁnal steps of the apoptotic pathway are generally conserved and result in the activation of caspases and the cleavage of cell constituents required for viability.

have been identiﬁed thus far.652,653 These cysteine proteases can be triggered by the release of cyto c from mitochondria cleave substrates carboxy-terminal to an aspartate residue, the in response to apoptosis-inducing stimuli.664,670,671 Cyto c P1 site.654 However, caspases can be divided into three sub- binds to Apaf-1, which can interact with an upstream caspase, 665 groups based on differences in substrate preference dictated procaspase-9, and an apoptosis inhibitor, Bcl-xL. Binding of by the residues immediately amino-terminal to the P1 site.655 dATP to Apaf-1 is required for cleavage of procaspase-9, while

Caspases are synthesized as inactive proenzymes consisting binding of Bcl-xL can inhibit this cleavage. Once activated, of an amino-terminal prodomain and large and small subunits. caspase-9 then proteolytically activates downstream caspases, Caspase prodomains contain protein–protein interaction mod- like caspase-3, which directly cleaves life-sustaining cellular ules, which facilitate the association of multiple factors proteins. A diagram of the formation of the apoptosome is required for caspase activation in response to apoptosis- presented in Figure 17. inducing stimuli. Activation requires proteolytic removal of A caspase-3-like protease is also responsible for activating the prodomain and formation of a heterodimer between the a cytoplasmic endonuclease, CAD (caspase-activated large and small subunits. By forming tetramers these heterodi- deoxyribonuclease) which can generate the oligonucleosomal mers complete the active caspase.656,657 DNA fragments indicative of apoptosis.672 CAD is rendered Some caspase prodomains contain a death effector domain inactive by its association with two isoforms of a chaperone- (DED) through which they bind to another DED domain on like protein, ICAD/DFF-45 (inhibitor of CAD/DNA fragmen- an adapter protein (FADD/MORT1). This targets the caspase to tation factor), which inhibit CAD endonuclease activity by ligand-activated death receptors at the cell membrane where concealing the CAD nuclear localization sequence.670,672 caspase activation occurs in the death-inducing signaling Induction of apoptosis results in the cleavage and inactivation complex (DISC)658–663 (Figure 16). Other caspase prodomains of ICAD/DFF-45, the activation and nuclear translocation of contain a caspase recruitment domain (CARD) through which CAD, and fragmentation of DNA into oligonucleosomes. they bind to apoptotic protease activating factor-1 (Apaf-1) Another endonuclease, cyclophilin C may be involved in gen- during formation of a cytoplasmic caspase activating complex, erating the 50- to 200-kbp-sized DNA fragments.673 the apoptosome.664–667 In both cases, the multiple factors Downstream caspases cleave a variety of cellular substrates, required for caspase activation are assembled. Caspases are of which only a subset is linked functionally to the induction activated in a cascade in which ‘upstream’ caspases are acti- of apoptosis. Some of these caspase substrates become activated by cleavage in the apoptosome or DISC. These caspases vated by their cleavage and assist in the apoptotic process, in turn cleave and activate ‘downstream’ caspases whose sub- whereas others which contribute to cell morphology or sur- strates are cellular components critical to the life of the cell. vival functions are inactivated or destroyed. The binding of death ligands such as TNF␣, Fas, and TRAIL to their respective death receptors triggers DISC formation. Death receptors contain cytoplasmic death domains (DD) Protease targets in apoptotic cells which, when bound by their respective death ligand, recruit adapter proteins containing DD and DED domains.668,669 The Cleavage of certain kinases, eg PAK2/hPAK65, PKC isoforms DED domains of adaptor and pro-caspase proteins interact ␦ and ␪, and MEKK-1 by caspases renders them constitutively leading to autoproteolytic activation of the upstream caspases active and pro-apoptotic.674–679 Structural proteins, such as and priming of the caspase cascade. Apoptosome formation gelsolin, Gas2, and nuclear lamins, which are critical to main- Review WL Blalock et al 1137 Table 7 Abbreviations in cell cycle regulatory molecules and inhibitory drugs

Abbreviation Full name Application

B-myb Transcription factor involved in stimulating cell cycle progression, member of Myb gene family CDK Cyclin dependent kinase Serine/threonine kinase, forms complexes with cyclins Cyclin/CDK complex can phosphorylate Rb E2F Transcription factor responsible for gene activation required for progression

from G1 to S phase MDM2 Murine double minute 2 491-aa nuclear protein which inhibits p53-mediated cell cycle arrest and apoptosis, reduces the level of p53 by targeting p53 for degradation, its transcription is controlled by p53 p15Ink4b Inhibitor of cell cycle progression p16Ink4a Inhibitor of cell cycle progression p18Ink4c Inhibitor of cell cycle progression p19Ink4d Inhibitor of cell cycle progression p19ARF Controls the level of MDM2 protein p21Cip p21 Cdk-interacting protein Tumor suppressor; binds cyclin/cdk complexes and can inhibit their effects (in a concentration dependent manner); can be induced by p53 p27Kip1 Can inhibit the activities of cyclin/cdk complexes p27Kip2 Can inhibit the activities of cyclin/cdk complexes p53 Tumor suppressor gene, often deleted or mutated in human cancers; transcription factor controlling the expression of many genes, a 393 aa amino acid protein usually maintained in low levels in normal cells, phosphorylated in response to DNA damage, phosphorylation at S15 and S37 inhibits the ability of MDM2 to inhibit p53-dependent transactivation of target genes PCNA Proliferating cell nuclear antigen 261-aa protein involved in cell cycle progression, exists as a homotrimer, auxiliary factor for DNA polymerase ␦ and ⑀, involved in DNA replication and repair, may serve to load the DNA polymerase on to the DNA template, interacts with p21Cip1 Rb retinoblastoma protein Tumor suppressor; transcription factor involved in cell cycle progression; mutated in certain human tumors

Drug Cell cycle stage affected

NK1098P M phase Pironetin M phase

RK682 G1 to S phase Vinblastin Metaphase

taining cellular architecture, are key targets of caspases.680–685 plex nature of protease activation and substrate cleavage dur- Cleavage of these proteins leads to the morphological changes ing apoptosis is indicative of an elaborate system of checks associated with apoptosis, ie cytoplasmic shrinkage, disas- and balances regulating a cellular process with death as its sembly of the nucleus, and chromatin condensation. Caspases outcome. Although difficult to study, the complex specificity also cleave and inactivate apoptotic inhibitory proteins such of such a system is more conducive to the design of thera- as ICAD, Bcl-2 and Bcl-xL, and kinases involved in anti-apop- peutic interventions which can be targeted to specific cell totic signaling pathways, such as Raf-1 and Akt.672,686–690 types or proteins involved in pathogenic apoptosis without Caspases have been detected in almost all cell types, but their perturbing the ‘normal’ state of apoptotic regulation in other expression may be differentially regulated in response to cells. diverse stimuli. For some caspases, multiple splice variants and protein isoforms have been detected suggesting that various forms of caspases are involved in cleavage reactions. In Endogenous inhibitors of apoptosis addition, studies with caspase transgenic knock-out mice have indicated that caspases may have redundant functions in some Cells are protected from indiscriminate induction of death by cell types.691–698 endogenous inhibitors of apoptosis, which form an integral Two other proteases are also involved in apoptosis: gran- part of the apoptotic pathway. However, many of these inhibi- zyme B and the calpains.699–708 Granzyme B is a serine pro- tors are also implicated in the initiation and progression of tease found in cytotoxic granules of cytotoxic T cells and NK cancer. Apoptosis induced via death receptor pathways can cells that induces apoptosis of target cells by the cleavage and be inhibited by a DED-containing protein, FLIP, which com- activation of many of the caspases.699–706,708 Calpains are cal- petes for the DED of the FADD/MORT1 adaptor protein and cium-dependent cysteine proteases whose substrates include either displaces a procaspase from the DISC or prevents it PKC␥, ␣-fodrin (a cytoskeletal protein), focal adhesion kinase from binding (Figure 16).714–717 Interestingly, FLIP is overex- (FAK), Src and growth factor receptors, many of which are pressed in human metastatic melanomas.717 calmodulin-binding proteins.703 Calpains are involved in Death receptor-induced activation of caspases can also be some, but not all forms of cell death.704–708 Similarly, there is inhibited by the expression of decoy receptors for TRAIL, evidence of caspase-independent apoptosis.709–713 The com- which either lack a DD or contain a truncated, nonfunctional Review WL Blalock et al 1138 Table 8 Abbreviations used for pro- and anti-apoptotic factors

Abbreviation Function Size

Bcl-2 anti-apoptotic 239 aa

Bcl-XL anti-apoptotic 233 aa Bcl-w anti-apoptotic 193 aa Mcl-1 anti-apoptotic 350 aa A1 anti-apoptotic 175 aa Bag anti-apoptotic Bax pro-apoptotic 192 aa Bok pro-apoptotic 170 aa Bak pro-apoptotic 211 aa Bid pro-apoptotic 195 aa Bik pro-apoptotic 160 aa Blk pro-apoptotic 150 aa Hrk pro-apoptotic 91 aa

BimL pro-apoptotic 138 aa Bad pro-apoptotic 168 aa BNIP3 pro-apoptotic 219 and 194 aa

Abbreviation Full name Description

⌬⌿m Mitochondrial transmembrane potential AIF Apoptosis-inducing factor Protein released from mitochondria of apoptotic cells Apaf-1 Apoptotic protease activating factor-1 Apparent mammalian homologue of CED-4, activates caspase- 3 Apoptosome Cytoplasmic caspase-activating complex composed of cyto c, Apaf-1, dATP and pro-caspase-9 CAD Caspase-activated deoxyribonuclease Generates oligonucleosomal DNA fragments Calpain Calcium-dependent cysteine proteases whose targets may include many cytokine receptors CARD Caspase recruitment domain Domain in some caspases which mediates interactions with Apaf-1 Caspases Cysteine aspartases Family of proteases involved in apoptosis that cleave proteins on the carboxy side of aspartate residues, expressed as inactive precursors that are activated either by autocatalytic cleavage or by other caspases, can be inhibited by cellular, viral, synthetic caspase inhibitors, substrates include poly(ADP- ribose)polymerase (PARP), DNA dependent protein kinase, nuclear lamins, PKC␦, actin cIAP1,2 Cellular inhibitor of apoptosis-1 and -2 Members of a family of anti-apoptotic mammalian and viral proteins; inhibit caspases 3 and 7 CrmA Cytokine response modiﬁer Pox viral gene which can act as pseudosubstrate for caspase 1 and inhibit apoptosis Cyto c Cytochrome c 105-aa mitochondrial oxidative chain protein, involved in caspase cascade by release from mitochondrion and binding with Apaf-1 which allows Apaf-1 to bind and activate procaspase 9 which in turn activates caspase 3, release of cyto c from the mitochondrion is regulated by Bcl-2 family proteins DD Death domain Protein–protein interaction module present on death receptors and adaptor proteins DcR’s/Decoy receptors Death receptor-like proteins Bind TRAIL, but do not induce apoptosis because they lack or contain a nonfunctional DD, may be lost during malignant transformation DED Death effector domain Protein–protein interaction module present in some caspases which target them to ligand-activated death receptors and/or death adaptors DISC Death-inducing signaling complex Caspase-activating complex comprised of ligand-activated death receptor/adaptor proteins and caspases FADD/MORT1 Fas-associated death domain Adaptor protein which contains a DD through which it binds ‘activated’ death receptors and a DED through which it binds pro-caspases Fas Receptor for Fas-L; member of TNF receptor family, when bound by Fas-L can induce cell death Fas-L Fas ligand TNF family cytokine which can induce cell death by binding to Fas c-FLIP FADD-like inhibitory proteins, also Proteins which compete with the DED of FADD/MORT1, 2

known as Casper, I-FLICE, CASH, isoforms: FLIPL and FLIPs, FLIPs contain two DEDs, over- CLARP, MRIT expression generally protects cells from death receptor-induced apoptosis, in addition there are v-FLIPs Gas2 Growth arrest gene Microﬁlament protein cleaved during apoptosis Review WL Blalock et al 1139 Table 8 Continued

Gelsolin 782-aa-cytoskeletal protein cleaved during apoptosis, acts as an actin depolymerizing factor, plays a role in morphological changes, cleaved by caspases to release constitutively active fragments which cleave actin filaments Granzyme B Serine protease found in cytotoxic granules of cytotoxic T cells and NK cells ICAD/DFF45 Inhibitor of CAD/DNA fragmentation Inhibits CAD endonuclease activity and translocation to the nucleus factor NAIP Mammalian inhibitor of apoptosis NO Nitric oxide Endogenous inhibitor of caspases iNOS, eNOS, nNOS Inducible nitric oxide synthases Enzymes which catalyse the production of NO and its reactive species Nuclear lamins Structural elements of the nuclear envelope cleaved during apoptosis p35 Baculovirus 35-kDa protein which acts as a broad specturm pseudosubstrate of caspases and can inhibit apoptosis PAK2/hPAK65 P21-activated kinase Cleavage by caspases results in pro-apoptotic activity PARP Poly (ADP-ribose) polymerase A caspase substrate TNF, Lymphotoxin ␣ Tumor necrosis factor-␣ Growth factor which can induce apoptosis (or necrosis), binds to TNFR1 and promotes formation of receptor trimers TRAF2 TNF receptor-associated factor Adaptor protein activated by TNF receptor ligation, transduces its signal to NIK which stimulates NF-␬B TRAIL/Apo2L Family of mammalian death receptors, includes DR4/TRAIL- R1/DRS/TRAIL-R2/Trick2/KILLER XIAP/HILP1/MILHA X-linked IAP Mammalian inhibitor of apoptosis-inhibits caspases-3,7 z-DEVD-FMK z-DEVD-fluromethyl ketone A caspase inhibitor z-VAD-FMK z-VAD-fluormethyl ketone A caspase inhibitor

Figure 15 Interplay between signal transduction pathways, mitochondrial membrane proteins and caspase cascades. The effects of IL-3 induced signal transduction cascades (PKA, Raf and AKT) on the phosphorylation of Bad and the prevention of the caspase cascade. Solid bold lines within the mitochondrion indicate pro-apoptotic events, whereas dotted lines within and outside of the mitochondrion indicate anti-apoptotic events. Signal transduction originating from the ligand-bound receptor (active) can sometimes induce anti-apoptotic pathways. In contrast, the unoccupied cytokine receptor results in the induction of apoptosis and the activation of the caspase cascade. Bcl-2 may act to regulate the release of cyto c from the mitochondrion and/or may act directly by binding to the apoptosome or to cyto c. Some of the molecules present in the proposed apoptosome are indicated. Organelles are not drawn according to scale. Review WL Blalock et al 1140

Figure 16 Disc formation. Binding of death ligands to death receptors results in trimerization of death receptor (DR) extracellular and cytoplasmic domains. Association of the cytoplasmic DDs results in recruitment of adaptor proteins which bind the DDs of the DR and DEDs of procaspase-8, leading to activation of the caspase cascade and apoptosis. Death receptor-induced activation of caspases can be inhibited by: (1) the expression of decoy receptors, which either lack a DD or contain a truncated, nonfunctional DD; (2) viral or cellular FLIP molecules which have DED, but not DD domains; or (3) viral or cellular IAPs.

Figure 17 Formation of the apoptosome. The apoptosome is a cytoplasmic caspase-activating complex composed of cyto c, Apaf-1, dATP, and procaspase-9. In response to certain apoptotic stimuli, cyto c (¼¼¼¼¼) is released from mitochondria, an event associated with loss of mitochondrial transmembrane potential. The mechanism of cyto c release is unclear, but may occur via pores (permeability transition, PT pores) or leakage through membranes ruptured by swelling, or both. The apoptosome is formed by the association of Apaf-1 with pro-caspase 9 via their CARD domains. Binding of dATP to Apaf-1 (* ****)isrequired for cleavage of pro-caspase 9 to the active caspase 9, while binding of

Bcl-2 or Bcl-xL can inhibit this cleavage. Activated caspase 9 then proteolytically activates downstream caspases, like caspase 3, which cleave cellular substrates and lead to death of the cell. Review WL Blalock et al 1141 DD (Figure 16).718–724 Sequestration of TRAIL by decoy recep- four conserved motifs, Bcl-2 homology (BH) domains, BH1 to tors prevents DISC formation and TRAIL-induced apoptosis. BH4 as well as a transmembrane domain. The most closely Many tumor cell lines exhibit a specific sensitivity to TRAIL- related Bcl-2 proteins contain two to four of the BH domains induced apoptosis, suggestive of a loss of decoy receptors dur- (at least BH1 and BH2) and promote survival rather than 722–724 ing transformation. apoptosis. These include Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1. A family of mammalian inhibitors of apoptosis proteins The Bax subfamily is comprised of three proteins, Bax, Bak, (IAPs) has been identified and includes neuronal IAP (NIAP), and Bok, which are structurally similar to Bcl-2, but promote X-linked IAP (XIAP), c-IAP1, c-IAP2, and survivin.725–730 Cells apoptosis rather than survival. The BH3 subfamily includes in which these IAPs are overexpressed are protected from proteins that contain only the BH3 domain and promote 767,769–772 apoptosis induced by a variety of stimuli (Figures 14 and apoptosis: Bik, Blk, Hrk, BimL, Bad, Bid, and BNIP3. 16).726–730 Survivin is overexpressed in certain human malig- The Bcl-2 proteins function in a concentration-dependent nancies and has been postulated to contribute to their resist- manner by forming homo- and heterodimers with other family ance to apoptosis.729 IAPs exhibit caspase-specific inhibitory members via the BH domains.748 For example, when Bcl-2 is activities and different levels of expression, suggesting a in excess, Bcl-2/Bax heterodimers are formed, and apoptosis mechanism for differential sensitivity to apoptosis dependent is inhibited; when Bax predominates, Bax homodimers are upon the level of expression of certain IAPs.650 formed, and the cells are susceptible to apoptosis. The forma- Nitric oxide (NO) and NO reactive species can directly tion of dimers among family members appears to be some- inactivate multiple caspases by S-nitrosylating the catalytic what discriminate since some members form dimers with any cysteine residue.731–734 NO production can be produced by of the other members while some exhibit more limited associ- multiple cell types via the inducible synthase (iNOS) and cNos ations.772–781

enzymes. cNos is activated to produce NO following a cal- Many of the Bcl-2 related proteins, including Bcl-2, Bcl-xL, cium influx. The NO formed may then modulate the sensi- and Bax are localized in the outer mitochondrial membrane. tivity of cells to apoptosis by NO-mediated regulation of cas- Bcl-2 and related, pro-survival proteins can inhibit apoptosis pase activity. upstream of caspase activation, presumably by binding Apaf-1 during formation of the apoptosome, thus preventing caspase activation.665,782 However, these pro-survival proteins do not Exogenous inhibitors of apoptosis inhibit apoptosis induced by death receptor activation.783–785 Apoptosis is associated with distinct changes in the mito- ␺ Viral inhibitors of caspases have also been identified. These chondrion including a reduction of m and the release of cyto proteins are thought to inhibit or delay apoptosis induced by c and apoptosis inducing factor (AIF).786 Bcl-2-related proteins viral infection in order to maximize viral replication. The pox- are thought to be involved in regulating these changes by for- virus cytokine response modifier A (crmA) gene product acts ming pores (channels) in the membrane which allow the pass- as a pseudosubstrate for caspase-1 and interferes with the host age of cyto c and AIF.786–788 Both pro-apoptotic and pro-sur- inflammatory response to viral infection.735 It also inhibits the vival Bcl-2-related proteins form pores in vitro.759,789–794

activities of other caspases and blocks apoptosis induced by However, whereas Bcl-2 and Bcl-xL have been shown to a variety of stimuli.719,736–741 Baculovirus encodes an IAP, p35 inhibit cyto c release, Bax stimulates the release of cyto which, like CrmA, acts as a pseudosubstrate of casp- c.792,795 Furthermore, Bcl-2 can inhibit the capacity of Bax to ases.643,741,742 Unlike CrmA, p35 is a broad-spectrum inhibitor form pores.789–791 The functional signiﬁcance of the formation of apoptosis, and inhibits apoptosis induced by all stimuli of mitochondrial pores in the initiation or progression of 741,742 tested thus far. apoptosis is still controversial. Bcl-2 and Bcl-xL can bind cyto IL-3 deprivation of certain hematopoietic cell lines leads to c directly, suggesting that Bcl-2/Bcl-xL inhibition of apoptosis poly ADP-ribose polymerase (PARP) cleavage by caspases is accomplished by sequestering cyto c from apoptosomes and which can be blocked by caspase inhibitors but not by preventing caspase activation.796 The mechanism of Bcl-2- CrmA.743 In contrast, inhibition of caspases by CrmA can mediated regulation of apoptosis is still unclear, but like the increase the resistance of human hematopoietic cells to mul- regulation of caspases, it is likely to be complex with multiple chemotherapeutic agents.744 tiple checkpoints.

Bcl-2 family members and the regulation of apoptosis Cytokine regulation of apoptotic pathways

The bcl-2 gene was identiﬁed at the chromosomal breakpoint Cytokine stimulation suppresses apoptosis via different mech- 49,634,636,641,750 ␤ of t(14;18)-bearing follicular B cell lymphomas. Having been anisms. The carboxy-region of the IL-3R c translocated to a location near the enhancer elements of the chain has been associated with the IL-3-mediated prevention immunoglobulin heavy chain locus, the bcl-2 gene was tran- of apoptosis. Stimulation of appropriate target cells by IL-3 scriptionally enhanced, and the 26-kDa Bcl-2 protein was leads to phosphorylation of Bad and its sequesterization by overexpressed, contributing to malignant transform- 14–3-3 proteins.768,797,798 The unphosphorylated form of Bad ation.745,746 Most importantly for this review, Bcl-2 protects normally forms heterodimers with anti-apoptotic factors, such 768,798 hematopoietic cell lines from apoptosis following growth fac- as Bcl-2 or Bcl-xL, to induce cell death. Phosphorylation tor withdrawal. Bcl-2 is now known to be a member of a of Bad induced by IL-3 stimulation, releases Bad from Bcl-2

growing family of related proteins that play a pivotal role in and Bcl-xL. The kinase(s), which directly phosphorylates Bad, mediating whether or not apoptosis will proceed to com- is unknown; however, Raf, Akt and PKA have been speculated pletion in a stimulated cell.747–774 to be involved.284–286,768,797,798 Sequestering of Bad by 14–3-

The Bcl-2-related proteins share some structural similarities, 3 proteins allows Bcl-2 and Bcl-xL to bind Bax, resulting in the but are divided into subgroups based on their structural differ- prevention of apoptosis. Recent data has called into question ences and pro- vs anti-apoptotic activities.762 Bcl-2 contains whether this is the only means by which IL-3 and related cyto- Review WL Blalock et al 1142 kines may prevent apoptosis. Expression of ⌬p85, a dominant- 1.815–819 Bag-1 can increase Raf activity (as measured by in negative version of the PI3K subunit, in BAF/3 cells resulted vitro phosphorylation of GST-MEK) two- to 2.5-fold. Conceiv- in levels of Bad phosphorylation seen in unstimulated cells ably, a complex comprised of Bcl-2, Raf-1, and Bag-1 proteins even when these cells were treated with IL-3.799 Even so, these can activate a kinase on the mitochondrial membrane or tar- cells were still resistant to apoptosis suggesting at least one get other kinases to the mitochondrial membrane.815 Targets additional mechanism by which IL-3 prevents apoptosis. This for this anti-apoptotic complex have not been determined, but

secondary means may be a result of Bcl-2 or Bcl-XL phos- Raf was shown to phosphorylate Bad in vitro. However, the phorylation. Although some evidence has shown Bcl-2 phos- sites of phosphorylation were distinct from those leading to phorylation results in inactivation or cleavage by caspases 14–3-3 associations in response to growth factor signaling.798 generating a truncated Bcl-2 protein which now has pro-apop- Raf-1 may still be implicated in initialization of signal trans- totic properties, other evidence suggest phosphorylation of duction cascades that leads to Bad phosphorylation, but there Ser70 by the Ras/Raf/MAPK pathway is anti-apoptotic.870,871 is a necessity for an intermediate. Ras has also been shown This phosphorylation is correlated with enhanced Bcl-2 anti- to bind the Bcl-2 protein, however, the significance of this apoptotic activity and the subsequent association of protein association has not been determined.820,821 phosphatase 2A with Bcl-2. PP2A dephosphorylates Ser70 to Alternatively, the ability of Raf-1 to mediate the suppression return Bcl-2 anti-apoptotic activity to basal levels.870,871 of apoptosis may not be due to its interactions with Bcl-2 fam- Often the expression of the Bcl-2 family of proteins is regu- ily members but to an ability to initiate signal transduction lated by the transcription factor p53. p53 can induce Bax cascades required for continued cell survival. No interactions expression while repressing Bcl-2 expression, thereby between Bcl-2 and other members of the Raf family (A-Raf inducing apoptosis. Some anti-tumor drugs act by restoring and B-Raf) have been described; however, the importance of p53 expression to wild-type levels in order to enhance Bax these Raf family proteins in preventing apoptosis has been expression and induce apoptosis.800–802 demonstrated.378 Differences in apoptotic suppression may be cell type specific further confusing the issue. The ability to target components of Raf-mediated signal transduction path- Therapy based upon reduction of Bcl-2 expression ways by gene knockout will lead to a better understanding of apoptotic suppression. Understanding the influence of Raf on Many human tumors often show dysregulated Bcl-2 apoptosis could lead to information of clinical value. Sup- expression.752,753,803–810 Given the multiplicity of Bcl-2-related pression of apoptosis in cancerous cells is being considered as genes, it is reasonable to predict that many human tumors a means by which these cells avoid the activity of apoptosis- have mutated or aberrantly regulated apoptotic or anti-apop- inducing chemotherapeutic regimens.803–813 In accordance totic genes. The end result is a prevention of apoptosis and with this, Bcl-2 and Raf have been shown to influence drug uncontrolled growth, instead of death of the transformed cells. resistance. Thus, the study of signal transduction pathways A variety of approaches have been taken to harness Bcl-2 involving the Raf kinases is not only important for understand- overexpression (see Figure 4). The Genta Biotechnology Com- ing normal cell signaling, but also may lead to development pany has developed a strategy currently in clinical trials, of treatments that circumvent drug resistance. which uses antisense RNA to inhibit Bcl-2 expression. This strategy is being used to treat patients with a variety of solid tumors that over-express Bcl-2.79,105 The effects of treatment Cytokine-dependent cell lines as tools to understand signal of the MCF-7 breast cancer cell line with vectors expressing transduction and malignant transformation single chain antibodies to Bcl-2 has also shown promise in terms of increasing susceptibility to drug-induced cytotoxic- Cytokine-dependent hematopoietic cell lines isolated from ity.811 In addition, hammerhead ribozymes specific for Bcl-2 patients with hematopoietic malignancies4,822,823 and normal mRNA have been constructed and are being tested for their mouse tissues824–827 have been used to: (1) identify and clone efficacy in down-regulating Bcl-2 expression.812,813 Clearly cytokines and their cognate receptors; (2) elucidate the certain signal transduction molecules represent targets for respective cytokines modes of action; (3) understand cytokine- effective pro-apoptotic drugs. mediated signal transduction and the regulation of cell cycle progression; (4) identify many of the factors involved in apoptosis; (5) design and evaluate the ability of novel Suppression of apoptosis by Raf recombinant/chimeric cytokines to stimulate repopulation of hematopoietic cells; and (6) test the efficacy of novel anti- v-Raf has been shown to suppress apoptosis following IL-3 cancer drugs. These cell lines also serve as critical tools in withdrawal in the IL-3-dependent 32D.3 cell line.446 How- elucidating how viral and cellular oncogenes can alter the ever, v-Raf did not abrogate the cytokine dependency of these growth properties of hematopoietic cells and result in malig- cells. Bcl-2 protein expression was not altered by v-Raf over- nant transformation. expression, suggesting that suppression of apoptosis by v-Raf When cytokine-dependent cells are deprived of growth fac- 814 was not dependent on altered Bcl-2 expression. Instead Bcl- tors for 24 to 48 h, they enter the G0/G1 stage of the cell cycle. 2 was shown to co-immunoprecipitate with v-Raf in these When growth factors are added back to the media, the cells cells suggesting a functional link between the Raf kinase and re-enter the cell cycle and proliferate. However, if the cells the Bcl-2 protein in the suppression of apoptosis.446,814 This are deprived of growth factors for more than 24–48 h, they group subsequently showed that this interaction appears to undergo apoptosis.2–4,84–89 Murine cell lines undergo target Raf-1 to the mitochondrial membrane.797 This localiz- apoptosis after 24 h of cytokine deprivation, however, human ation presumably facilitates the kinase’s ability to recognize cells often take longer to die (48 to 72 h). Apoptosis in murine substrates involved in the control of cell death. Bag-1, which cells usually begins at 6 h post-starvation.84–89 If cytokines are encodes 36- and 50-kDa anti-apoptotic proteins generated by added back at or before this time, apoptosis can be avoided. alternative mRNA splicing, also forms complexes with Raf- However, after 12 to 24 h of cytokine deprivation, apoptosis Review WL Blalock et al 1143 is not completely reversible, and the percentage of viable cells attractive model systems to analyze the effects various onco- decreases to approximately zero by 24 to 48 h of cytokine genes have on signal transduction and leukemogenesis.2,3 deprivation, again depending on the origin of the cell line. A The growth factor-dependency of certain cytokine-depen- diagram of the regulation of growth by cytokines and onco- dent cell lines can be abrogated after infection by retroviruses genes in cytokine-dependent hematopoietic cell lines is encoding the following activated viral oncogenes: v-abl,86,829– presented in Figure 18. 834 v-erb-B,835–837 v-fms,838,839 v-src,840–842 v-fps,843 v- The TF-1 cell line was isolated from an adult with an raf,445,446 v-Ha-ras,844–848 or human oncogenes resulting from erythroleukemia and requires either IL-3 or GM-CSF for pro- chromosomal translocations: BCR-ABL,844,845,849–856 TEL- liferation.822 The TF-1 cell line will differentiate into more ABL,857–859 TEL-Jak.860 The efficiency of abrogation of cyto- mature erythroid, macrophage, megakarocytic and granulo- kine dependency varies depending upon the particular oncog- cytic cells with an appropriate differentiation-inducing ene and cell line. In studies with v-abl and v-src oncogenes, regime. In the presence of TPO, they differentiate into megak- the requirement of the oncogene for abrogation of cytokine aryoctes. When TF-1 cells are cultured in hemin and erythro- dependency was demonstrated by the use of temperature- poietin, they differentiate into more mature erythroid cells. In sensitive (ts) mutants. In these ts oncogene-infected cells, contrast, if they are treated with phorbol esters, they differen- abrogation of cytokine dependency occurred at the permissive tiate into more mature monocytic cells. The cytokine depen- temperature, whereas the cells were cytokine-dependent at dency of TF-1 cells can be relieved after infection with retro- the non-permissive temperature.834,840,842,852 A diagram of viruses encoding certain oncogenes.378 Thus, this cell line some of the oncogenes demonstrated to abrogate the cytokine serves as a model for examining the effects of oncogenes on dependency of hematopoietic cells and where they function hematopoietic cell growth and differentiation. The M07/E cell in signal transduction pathways, is presented in Figure 19. line was derived from a patient with a megakaryoblastic tumor With the exception of the GTP-binding protein encoded by and resembles megakaryocytic cells.823 These cells undergo v-Ha-Ras, all the previously mentioned oncogenes encode polyploidization, a characteristic of megakaryocytic cells, protein kinases, which interact with additional molecules upon treatment with the appropriate inducing agent.824 within signaling pathways to constitutively activate/deregulate The FDC-P1 cell line was recovered from the spleen of a the cell cycle and promote growth and survival. Interestingly, DBA/2 mouse and grows when either IL-3 or GM-CSF is pro- most of these oncogenes transduce some of their proliferative vided.825 The FL5.12 and BAF/3 cell lines were derived from effects through the Raf-MEK-ERK kinase pathway, suggesting murine fetal liver and possess characteristics of early that subtle disruptions of this pathway can abrogate the cyto- lymphoid/myeloid precursor cells.826,827 Spontaneous factor- kine dependency of established hematopoietic cell lines. independent cells are rarely (Ͻ10−7) recovered from the FDC- Abrogation of cytokine dependency by the v-abl,v-Ha-ras,v- P1, FL5.12 and BAF/3 cell lines. These cell lines are, in fms,v-fps,v-src, and v-raf oncogenes has been associated addition, non-tumorigenic upon injection into immuno- with malignant transformation of cells as indicated by the for- compromised mice.2,3 The 32D cell line is a murine multipo- mation of tumors in immunocompromised mice.829,830,838,843 tential (erythroid/neutrophil/basophil), IL-3 dependent cell line Over-expression of protein kinase C epsilon, but not protein derived from mice.828 These cells can be induced to differen- kinase C delta, will suppress apoptosis and induce Bcl-2 tiate into more mature granulocytes upon withdrawal of IL-3 expression in human TF-1 cells.861–863 and addition of G-CSF.828 Infection of these cells with onco- The raf and mek oncogenes are less effective in abrogating genes such as v-abl blocks G-CSF-induced differentiation.829 the cytokine dependency of hematopoietic cells than other These properties make cell lines such as those mentioned activated oncogenes (eg v-Ha-ras,v-src, and v-

Figure 18 Cytokine-dependent hematopoietic cell lines as tools to investigate apoptosis, cell cycling and cell transformation. Cytokine- dependent cells can be used to study various aspects of cell cycle progression, apoptosis, differentiation, signal transduction and malignant transformation. The ability of certain hematopoietic cells to undergo differentiation is indicated. The ability to monitor cell cycle progression by cytokine withdrawal and re-addition of cytokines is shown. Different genetic mechanisms, which can result in the abrogation of cytokine dependency of hematopoietic cells are shown in (b). Additional mutations may be required to induce malignant transformation. Review WL Blalock et al 1144

Figure 19 Transformation of hematopoietic cells by oncogenes. The sites at which various oncogenes act in signal transduction and apoptotic pathways are indicated in this ﬁgure. Transforming oncogenes are indicated in red.

abl).445,446,844,850 Some studies have shown that Raf and MEK cytokine synthesis remains controversial even after many over-expression will not relieve the growth factor dependency years of investigation. This is probably due to differences in of certain IL3-dependent cell lines.445,446,865 The reasons for the cell types analyzed and the sensitivities of the various this inability to efficiently abrogate cytokine dependency are detection methods. Many oncogene-transformed growth fac- not clear. However, it may be due to the positioning of Raf tor-independent cells synthesize autocrine cytokines as and MEK downstream of Src, Fms, Erb-B, v-Ha-Ras and v-Abl detected by sensitive RT-PCR analyses.360,840,842,849,853 How- in the signal transduction pathways. Thus Raf and MEK may ever, the significance of this cytokine synthesis remains not be able to induce as many ‘alternative/additional’ path- obscure because it is usually not at high enough levels to sup- ways as these upstream tyrosine kinases and v-Ha-Ras. Thus, port growth. The role of autocrine growth factor synthesis in cells with mutated raf or mek genes may not be as malignant autocrine transformation should be evaluated by more thor- as cells with a mutated tyrosine-kinase and may require ough techniques such as monitoring the effects on anti-sense additional genetic mutations to become phenotypically trans- cytokine RNAs on autocrine proliferation. However, many formed. hematopoietic cell lines synthesize multiple cytokines, each Growth factors promote survival of cells by mobilization of of which could contribute to autocrine growth. This could immediate-early genes that lead to the expression of genes make the analysis of the effects of autocrine production of necessary for cell growth (eg glucose transporter isoforms) and growth factors more difficult to interpret. the prevention of apoptosis (eg Bcl-2).49,218,864 Also growth More recent studies on the ability of oncogenes to abrogate factors stimulate the expression of genes which may normally the cytokine dependency of hematopoietic cells have been serve to inhibit cell growth (eg Cis, p53, p21, Rb). Autocrine performed with activated and viral cellular oncogenes inserted growth of hematopoietic cells results when a cell has acquired into retroviral vectors containing either the oncogene-estrogen the ability to produce growth factors required for its own sur- receptor (ER)327,374,375,377 or the oncogene androgen-receptor vival. Autocrine growth factor production has been observed (AR).869 Studies performed with v-erb-B:ER, v-src:ER, ⌬Raf:ER in TF-1 hematopoietic and NIH-3T3 fibroblast cells after trans- inducible constructs have demonstrated that the activated formation by an active raf oncogene.378,865 Thus, the presence viral oncogene is necessary for abrogation of cytokine depen- of a constitutively active Raf/MEK/MAPK pathway can result in dency since the cells reverted to cytokine dependency when autocrine transformation. The presence of an autocrine growth the steroid hormone was removed.378 These conditional con- factor loop has also been observed in acute myeloblastic leu- structs enable investigators to distinguish between biochemi- kemia (AML).866–868 The study of autocrine growth factor pro- cal effects which are due to the activated oncogene and effects duction in response to activated oncogenes will likely identify which result from the cytokine. We have determined that potential targets that will allow for the rational development human TF-1 cells infected with ⌬Raf:ER oncogenes secrete of compounds aimed at disrupting these autocrine loops in GM-CSF at sufficient levels to promote autocrine growth. With human malignancies. conditionally active oncogenes it is possible to directly moni- The ability of the activated oncogenes to induce autocrine tor the effects of certain oncogenes on signal transduction cas- Review WL Blalock et al 1145 cades as well as other pathways which may be induced by in vitro induction of 20␣-hydroxysteroid dehydrogenase in the oncogene. splenic lymphocytes from athymic mice by a unique lymphokine. J Immunol 1981; 126: 2184–2189. 6 Schrader JW, Lewis SJ, Clark-Lewis I, Culvenor JG. The persisting (P) cell: histamine content, regulation by a T cell-derived factor, Significance origin from a bone marrow precursor, and relationship to mast cells. Proc Natl Acad Sci USA 1981; 78: 323–327. Many of the therapeutic regimens now available for cancer 7 Nabel G, Galli SJ, Dvorak AM, Dvorak HF, Cantor H. Inducer T patients are highly cytotoxic making the development of new lymphocytes synthesize a factor that stimulates proliferation of and less toxic therapies essential to improve the care of these cloned mast cells. Nature 1981; 291: 332–334. 8 Bazill GW, Haynes M, Garland J, Dexter TM. Characterization patients. The use of hematopoietic cytokines in cancer ther- and partial purification of a hemopoietic cell growth factor in apy, which can be used to overcome myelosuppression WEHI-3 cell conditioned medium. Biochem J 1983; 210: 747– induced by anti-cancer drugs, has been well documented and 759. will clearly increase with the development of recombinant 9 Dy M, Lebel B, Kamoun P, Hamburger J. Histamine production chimeric cytokines which can simultaneously stimulate mul- during the anti-allograft response. Demonstration of a new lym- tiple growth factor receptors. However, under certain circum- phokine enhancing histamine synthesis. J Exp Med 1981; 153: stances, it may be critical to eliminate cells which express 293–309. 10 Schrader JW, Clark-Lewis I. A T cell-derived factor stimulating receptors to a particular cytokine. The design of chimeric multipotential hemopoietic stem cells: molecular weight and dis- cytokine-toxin molecules or blocking antibodies may allow tinction from T cell growth factor and T cell-derived granulocyte– the effective elimination of specific hematopoietic tumors. macrophage colony-stimulating factor. J Immunol 1982; 129: Alternatively, the combined use of anti-sense RNAs and 30–35. ribozymes may prove useful in the therapy of certain leukem- 11 Ihle JN, Keller J, Oroszlan S, Henderson LE, Copeland TD, Fitch ias. With the use of cytokine-dependent hematopoietic cell F, Prystowsky MB, Goldwasser E, Schrader JW, Palaszynski E, Dy M, Lebel B. Biological properties of homogeneous interleukin 3. lines, we have gained an understanding of how cytokines I. Demonstration of WEHI-3 growth factor activity, mast cell transduce their regulatory information. The elucidation of the growth factor activity, P cell-stimulating factor activity, colony- structure and function of cytokine receptors and their down- stimulating factor activity, and histamine-producing cell-stimulat- stream signal transduction cascades has provided insights into ing factor activity. J Immunol 1983; 131: 282–287. how we can target specific molecules to eliminate the growth 12 Metcalf D, Begley CG, Johnson GR, Nicola NA, Lopez AF, Willi- of leukemic cells. Understanding how malignant cells evade amson DJ. Effects of purified bacterially synthesized murine apoptosis and proliferate in the absence of cytokine(s), which multi-CSF (IL-3) on hematopoiesis in normal adult mice. Blood 1986; 68: 46–57. they previously required, will lead to the development of 13 Chang JM, Metcalf D, Lang RA, Gonda TJ, Johnson GR. Nonneo- novel approaches to treat cancer. Many signal transduction plastic hematopoietic myeloproliferative syndrome induced by targets have been identified, and pharmaceutical companies dysregulated multi-CSF (IL-3) expression. Blood 1989; 73: are designing inhibitors which will suppress these molecules. 1487–1497. Some of these ‘non-traditional’ drugs are indeed novel in nat- 14 Cockayne DA, Bodine DM, Cline A, Nienhuis AW, Dunbar CE. ure as they can be ribozymes that eliminate specific mRNAs Transgenic mice expressing antisense interleukin-3 RNA develop a B-cell lymphoproliferative syndrome or neurologic dysfunction. or recombinant single chain antibodies which target certain Blood 1994; 84: 2699–2710. proteins. The understanding that has been gained in signal 15 Mach N, Lantz CS, Galli SJ, Reznikoff G, Mihm M, Small C, transduction, cell cycle regulatory and anti-apoptotic pathway Granstein R, Beissert S, Sadelain M, Mulligan RC, Dranoff G. studies has led to and continues to direct the development of Involvement of interleukin-3 in delayed-type hypersensitivity. truely novel therapies to treat leukemia and other cancers. Blood 1998; 91: 778–783. 16 Chiang CS, Powell HC, Gold LH, Samimi A, Campbell IL. Macrophage/microglial-mediated primary demyelination and motor disease induced by the central nervous system production Acknowledgements of interleukin-3 in transgenic mice. J Clin Invest 1996; 97: 1512–1524. This work was supported in part by a grant from the National 17 Weng TC, Tanaka J, Peng H, Desaki J, Matsuda S, Maeda N, Institute of Health (R01 CA51025) to JAM and a Scientist Fujita H, Sato K, Sakanaka M. Interleukin 3 prevents delayed neu- Development Grant from the American Heart Association ronal death in the hippocampal CA1 field. J Exp Med 1988; 188: (993099N) to RAF. We sincerely thank Ms Catherine Spruill 635–649. 18 Campbell IL. Structural and functional impact of transgenic for the outstanding artwork. expression of cytokines in the CNS. Ann NY Acad Sci 1988; 840: 83–96. 19 Tabita T, Chui DH, Fan JP, Shirabe T, Konishi Y. Interleukin-3 References and interleukin-3 receptors in the brain. Ann NY Acad Sci 1998; 840: 107–116. 1 Arai K-I, Lee F, Miyajima A, Miyatake S, Arai N, Yokota T. Cyto- 20 Farese AM, Williams DE, Seiler FR, MacVittie TJ. Combination kines: coordinators of immune and inflammatory responses. protocols of cytokine therapy with interleukin-3 and granulo- Annu Rev Biochem 1990; 59: 783–836. cyte–macrophage colony-stimulating factor in a primate mode of 2 Wang XY, McCubrey JA. Regulation of interleukin-3 expression radiation-induced marrow aplasia. Blood 1993; 82: 3012–3018. in normal and autocrine transformed hematopoietic cells. Int 21 Maurer AB, Ganzer A, Buhl R, Seipelt G, Ottmann OG, Mentzel Oncol 1997; 10: 989–1001. U, Geissler RG, Hoelzer D. Restoration of impaired cytokine 3 Wang XY, Steelman LS, McCubrey JA. Abnormal activation of secretion from monocytes of patients with myelodysplastic syn- cytokine gene expression by intracisternal type A particle trans- dromes after in vivo treatment with GM-CSF or IL-3. Leukemia position: effects of mutations which result in autocrine growth 1993; 7: 1728–1733. stimulation and malignant transformation. Cytokin Cell Mol Ther 22 Williams DE, Park LS. Hematopoietic effects of granulocyte– 1997; 3: 3–19. macrophage colony-stimulating factor/interleukin-3 fusion pro- 4 Drexler HG. Expression of Flt3 receptor and response to Flt3 tein. Cancer 1991; 67: 2705–2707. ligand by hematopoietic cells. Leukemia 1996; 10: 588–599. 23 Bhalla K, Tang C, Ibrado AM, Grant S, Tourkina E, Holladay C, 5 Ihle JN, Pepersack L, Rebar L. Regulation of T cell differentiation: Hughes M, Mahoney ME, Huang Y. Granulocyte–macrophage Review WL Blalock et al 1146 colony-stimulating factor/interleukin-3 fusion protein (PIXY 321) 39 DiPersio JF, Vredenburg J, Copelan EA, Prosper F, Sola C, Lill enhances high-dose Ara-C-induced programmed cell death or M, Bolwell BJ, Santos VR, Collins DM, Baum CM. Prospective, apoptosis in human myeloid leukemia cells. Blood 1992; 80: randomized study of peripheral blood stem cell (PBSC) mobiliz- 2883–2890. ation with daniplestim plus G-CSF versus placebo plus G-CSF in 24 Petrucci MT, De Felice L, Ricciardi MR, Ariola C, Mascolo MG, breast cancer patients. Blood 1998; 92 (Suppl I): 269a. Fenu S, Tafuri A. Stem cell factor and PIXY-321 in acute lym- 40 DiPersio JF, Abboud CN, Winter JN, Bensinger W, Collins DM, phoblastic leukemia: in vitro study on proliferative effects and Peppard T, Santos VR, Baum CM. Phase I/II study of mobilization apoptosis. Cytokin Mol Ther 1996; 2: 225–230. of PBSC by SC-68420 in patients with breast cancer or lym- 25 O’Shaughnessy JA, Tolcher A, Riseberg D, Vanzon D, Zujewski phoma. Blood 1997; 90: (Suppl I): 97a. J, Noone M, Gossard M, Danforth D, Jacobson J, Chang V, Gold- 41 DiPersio JF, Gordon L, Frenette G, Denes A, Collins DM, Peppard spiel B, Keegan P, Giusti R, Cowan KH. Prospective, randomized T, Santos VR, Baum CM. Phase I/II study to determine the safety trial of 5-flurouracil, leucovorin, doxorubicin, and cyclophos- and tolerability of SC-70935 in patients with relapsed lymphoma phamide chemotherapy in combination with the interleukin- receiving ESHAP. Blood 1997; 90: (Suppl I): 173a. 3/granulocyte-macrophage colony-stimulating factor (GM-CSF) 42 Siegel N, Vilani-Price D, Abrams M, Galluppi G, Abegg A, Bur- fusion protein (PIXY321) versus GM-CSF in patients with nette B, Finn R, Frazier R, Heeren R, Sobacke S, Woodward H, advanced breast cancer. Blood 1996; 87: 2205–2211. Giri J, Kahn L, Pegg L, Staten N, Braford-Goldberg S, Summers, 26 Thomas JW, Baum CM, Hood WF, Klein B, Monahan JB, Paik Smith W, Farese A, MacVitte, McKearn J. Pharmokinetic differ- K, Staten N, Abrams M, McKearn JP. Potent interleukin 3 receptor ences observed for assorted promegapoietin molecules in rhesus agonist with selectively enhanced hematopoietic activity relative monkey studies. Blood 1998; 92 (Suppl II): 175b. to recombinant human interleukin 3. Proc Natl Acad Sci USA 43 Giri JG, Smith WG, Kahn LE, Streeter PR, Minster NI, Baum CM, 1995; 92: 3779–3783. Lind L, Farese AM, Lin J, Roecklein B, MacVittie TJ, McKearn JP. 27 MacVittie TJ, Farese AM, Heodin F, Grab LB, Baum CM, McKe- Promegapoietin, a chimeric growth factor for megakaryocyte and arn JP. Combination therapy for radiation-induced bone marrow platelet restoration. Blood 1997; 90 (Suppl I): 58a. aplasia in nonhuman primates using synthokine SC-55494 and 44 Doshi PD, Huynh MS, Favara JP, Thomas JW, Abegg AL, Kahn recombinant human granulocyte colony-stimulating factor. LE, Monahan JB, McKearn JP, Giri JG. Induction of early gene Blood 1996; 87: 4129–4135. expression by a chimeric cytokine with agonistic activities for 28 Farese AM, Herodin F, McKearn JP, Baum C, Burton E, MacVittie human IL-3 and c-mpl receptors. Blood 1996; 88 (Suppl I): 199a. TJ. Acceleration of hematopoietic reconstitution with a synthetic 45 Farese AN, MacVittie TJ, Lind LB, Smith WG, McKearn JP. The cytokine (SC-55494) after radiation-induced bone marrow apl- combined administration of daniplestim and mpl ligand aug- asis. Blood 1996; 87: 581–591. ments the hematopoietic reconstitution observed with single 29 Fleming WH, Lankford-Turner P, Turner CW, Wong J, Storbert cytokine administration in a nonhuman primate model of myelo- E, McKearn JP. Administration of daniplestim and granulocyte suppression. Stem Cells 1998; 16 (Suppl 2): 143–154. colony-stimulating factor for the mobilization of hematopoietic 46 Streeter PR, Ishioka G, Kahn LE, Lie WR, Evans RJ, Sette A, Fikes J, progenitor cells in nonhuman primates. Biol Blood Marrow Alexander J, Woulfe SL, McKearn JP. Progenipoietin, a chimeric Transplant 1999; 5: 8–14. growth factor that promotes the in vivo expansion of functional 30 Klein BK, Feng Y, McWherter CA, Hood WF, Paik K, McKearn dendritic cells. Blood 1998; 92: (Suppl I): 338a. JP. The receptor binding site of human interleukin-3 defined by 47 Woulfe SL, Streeter PR, Kahn LE, Minster NI, MacVittie TJ, Farese mutagenesis and molecular modeling. J Biol Chem 1997; 272: AM, McKearn JP. Expansion of functionally competent multi-lin- 22630–22641. eage hematopoietic cells using the novel chimeric hematopoietic 31 Merlin S, Rowold E, Abeegg A, Berlund C, Klover J, Staten N, growth factor, progenipoietin. Blood 1998; 92: (Suppl I): 377a. McKearn JP, Lee SC. Phage presentation and affinity selection of 48 Fleming WH, Mulcahy JM, McKearn JP, Streeter PR. Progenipoie- a deletion mutant of human interleukin-3. Appl Biochem Biotech tin mobilizes substantial numbers of transplantable hematopo- 1997; 67: 199–214. ietic stem cells. Blood 1998; 92: (Suppl. II): 286b. 32 Feng Y, Hood WF, Forgey RW, Abegg AL, Caparon MH, Thiele 49 Kinoshita T, Yokota T, Arai K-I, Miyajima A. Suppression of apop- BR, Leimgruber RM, McWherter CA. Multiple conformations of totic death in hematopoietic cells by signalling through the IL- a human interleukin-3 variant. Protein Sci 1997; 6: 1777–1782. 3/GM-CSF receptors. EMBO J 1995; 14: 266–275. 33 McWherter CA, Feng Y, Zurfluh LL, Klein BK, Baganoff MP, Pol- 50 Nimer S, Zhang J, Avraham H, Miyazaki Y. Transcriptional regu- azzi JO, Hood WF, Paik K, Abegg AL, Grabbe ES, Shieh JJ, Don- lation of interleukin-3 expression in megakaryocytes. Blood nelly AM, McKearn JP. Circular permutation of the granulocyte 1996; 88: 66–74. colony-stimulating factor receptor agonist domain of myelopoie- 51 Guba SC, Stella G, Turka LA, June CH, Thompson CB, Emerson tin. Biochemistry 1999; 38: 4564–4571. SG. Regulation of interleukin 3 gene induction in normal human 34 Feng Y, Minnerly JC, Zurfluh LL, Joy WD, Hood WF, Abegg AL, T cells. J Clin Invest 1989; 84: 1701–1706. Grabbe ES, Shieh JJ, Thurman TL, McKearn JP, McWherter CA. 52 Park J-H, Kaushansky K, Levitt L. Transcriptional regulation of Circular permutation of granulocyte colony-stimulating factor. interleukin 3 (IL3) in primary human T lymphocytes. Role of AP- Biochemistry 1999; 38: 4553–4563. 1- and octamer-binding proteins in control of IL3 gene 35 Monahan JB, Hood WF, Joy WD, Caparon MH, Bradford-Gold- expression. J Biol Chem 1993; 268: 6299–6308. berg S, Klein BK, Staten NR, Pegg LE, Bauer SC, Mina MS, Hirsch 53 Clipstone NA, Crabtree GR. Identification of calcineurin as a key JL, Giri JG, McKearn JP. Receptor binding and signaling charac- signalling enzyme in T-lymphocyte activation. Nature 1992; 357: teristics of MPO-1 (SC-68420) – multifunctional IL-3 and G-CSF 695–697. receptor agonist. Exp Hematol 1996; 24: 714a. 54 Link E, Kerr LD, Schreck R, Zabel U, Verma I, Baeuerle PA. Pur- 36 Smith WG, Giri JG, Abegg A, Donnelly A, Paik K, Hood WF, ified I kappa B is inactivated upon dephosphorylation. J Biol Monahan JB, Welply J, Polazzi J, Caparon M, Klein B, Farese AM, Chem 1992; 267: 239–246. Lind LB, Dacquel-Smith L, MacVittie TJ, McKearn JP. Preclinical 55 Nakano H, Shindo M, Sakon S, Nishinaka S, Mihara M, Yagita pharmacology of the myelopoietin leridistim, a multifunctional H, Okumura K. Differential regulation of I-␬B kinase ␣ and ␤ agonist of the IL-3 and G-CSF receptors. Blood 1998; 92: (Suppl by two upstream kinases NF-␬B inducing kinase and mitogen- I): 380a. activated protein kinase/ERK kinase kinase-1. Proc Natl Acad Sci 37 MacVittie TJ, Farese AM, Dacquel Smith J, Casey DB, Smith WG, USA 1998; 95: 3537–3542. Woulfe SL, Streeter PR, McKearn JP. Members of the progenipoie- 56 Nadler SG, Eversol AC, Tepper MA, Cleveland JS. Elucidating the tins, a family of Flt-3 ligand and G-CSF receptor agonists mobil- mechanism of action of the immunosuppressant 15-deoxyspergu- ize hematopoietic cells with different kinetics and efficiency in alin. Ther Drug Monitor 1995; 17: 700–703. nonhuman primates. Blood 1998; 92 (Suppl I): 682a. 57 Foxwell B, Browne K, Bondeson J, Clarke C, de Martin R, Bren- 38 MacVittie TJ, Farese AM, Davis TA, Lind LB, McKearn JP. Mobil- nan F, Feldmann M. Efficient adenoviral infection with I-␬B␣ ization of CD34+ and hematopoietic clonogenic cells in normal reveals that macrophage tumor necrosis factor alpha production nonhuman primates using myelopoietin, synthokine, G-CSF and in rheumatoid arthritis is NF-␬B dependent. Proc Natl Acad Sci synthokine plus G-CSF. Blood 1996; 88 (Suppl I): 642a. USA 1998; 95: 8211–8215. Review WL Blalock et al 1147 58 Sigal NH, Dumont FJ. Cyclosporin A, FK-506, and rapamycin: demethylation and mRNA expression are heritable in pharmacologic probes of lymphocyte signal transduction. Annu CD44(high)CD8+ T cells. J Exp Med 1998; 188: 103–107. Rev Immunol 1992; 10: 519–560. 77 Largaespada DA, Brannan CI, Jenkins NA, Copeland NG. NF1 59 Mathey-Prevot B, Andrews NC, Murphy HS, Kreissman SG, deficiency causes Ras-mediated granulocyte/macrophage col- Nathan DG. Positive and negative elements regulate human ony-stimulating factor hypersensitivity and chronic myeloid leu- interleukin 3 expression. Proc Natl Acad Sci USA 1990; 87: kemia. Nat Genet 1996; 12: 137–143. 5046–5050. 78 Dehbi M, Bedard PA. Regulation of gene expression in oncogen- 60 John S, Marais R, Child R, Light Y, Leonard WJ. Importance of ically transformed cells. Biochem Cell Biol 1992; 70: 980–997. low affinity Elf-1 sites in the regulation of lymphoid-specific 79 Barinaga M. From bench top to bedside. Science 1997; 278: inducible gene expression. J Exp Med 1996; 183: 743–750. 1036–1039. 61 Arai N, Naito Y, Watanabe M, Masuda ES, Yamaguchi-Iwai Y, 80 van Dam H, Huguier S, Kooistra K, Baguet J, Vial E, van der Eb Tsuboi A, Heike T, Matsuda I, Yokota K, Koyano-Nakagawa N. AJ, Herrlich P, Angel P, Castellazzi M. Autocrine growth and Activation of lymphokine genes in T cells: roles of cis-acting anchorage independence: two complementing Jun-controlled DNA elements that respond to T cell activation signals. Pharma- genetic program of cell transformation. Genes Dev 1998; 12: col Therapeut 1992; 55: 303–318. 1227–1239. 62 Davies K, TePas EC, Nathan DG, Mathey-Prevot B. Interleukin- 81 Ikushima S, Inukai T, Inaba T, Nimer SD, Cleveland JL, Look AT. 3 expression by activated T cells involves an inducible, T-cell- Pivotal role for the NFIL3/E4BP3 transcription factor interleukin specific factor and an octamer binding protein. Blood 1993; 81: 3-mediated survival of pro-B lymphocytes. Proc Natl Acad Sci 928–934. USA 1997; 94: 2609–2614. 63 Taylor DS, Laubach JP, Nathan DG, Mathey-Prevot B. 82 Ueffing M, Lovric J, Philipp A, Mischak H, Kolch W. Protein kin- Cooperation between core binding factor and adjacent promoter ase C-epsilon associates with the Raf-1 kinase and induces the elements contributes to the tissue-specific expression of interleu- production of growth factors that stimulate Raf-1 activity. Oncog- kin-3. J Biol Chem 1996; 271: 14020–14027. ene 1997; 24: 2921–2927. 64 Nishida J, Yoshida M, Arai K-I, Yokota T. Definition of a GC-rich 83 Oldham SM, Clark GJ, Gangarosa LM, Coffey RJ Jr, Der CJ. Acti- motif as regulatory sequence of the human IL-3 gene: coordinate vation of the Raf-1/MAP kinase cascade is not sufficient for Ras regulation of the IL-3 gene by CLE2/GC box of the GM-CSF gene transformation of RIE-1 epithelial cells. Proc Natl Acad Sci USA in T cell activation. Int Immunol 1991; 3: 245–254. 1996; 93: 6924–6928. 65 Koyano-Nakagawa N, Nishida J, Baldwin D, Arai K-I, Yokota T. 84 Grimaldi JC, Meeker TC. The t(5:14) chromosomal translocation Molecular cloning of a novel human cDNA encoding a zinc in a case of acute lymphocytic leukemia joins the interleukin-3 finger protein that binds to the interleukin-3 promoter. Mol Cell gene to the immunoglobulin heavy chain gene. Blood 1989; 73: Biol 1994; 14: 5099–5107. 2081–2085. 66 Himes SR, Katsikeros R, Shannon MF. Costimulation of cytokine 85 Meeker TC, Hardy D, Willman C, Hogan T, Abrams J. Activation gene expression in T cells by the human T-cell of the interleukin-3 gene by chromosome translocation in acute leukemia/lymphotropic virus type 1 trans activator Tax. J Virol lymphocytic leukemia with eosinophilia. Blood 1990; 76: 1996; 70: 4001–4008. 285–289. 67 Gerondakis S, Strasser A, Metcalf D, Grigoriadis G, Scheerlinck 86 McCubrey JA, Holland G, McKearn J, Risser R. Abrogation of JY, Grumont RJ. Rel-deficient T cells exhibit defects in production factor-dependence in two IL-3-dependent cell lines can occur by of interleukin 3 and granulocyte–macrophage colony-stimulating two distinct mechanisms. Oncogene Res 1989; 4: 97–109. factor. Proc Natl Acad Sci USA 1996; 93: 3405–3409. 87 Algate PA, McCubrey JA. Autocrine transformation of hemopo- 68 Ryan GR, Vadas MA, Shannon MF. T-cell functional regions of ietic cells resulting from cytokine message stabilization after the human IL-3 proximal promoter. Mol Reprod Dev 1994; 39: intracisternal A particle transposition. Oncogene 1993; 8: 1221–1232. 200–207. 88 Mayo MW, Wang X-Y, Algate PA, Arana GF, Hoyle PE, Steelman 69 Park JH, Levitt L. Overexpression of the mitogen-activated pro- LS, McCubrey JA. Synergy between AUUUA motif disruption and tein kinase (ERK1) enhances T cell cytokine gene expression: role enhancer insertion results in autocrine transformation of interleu- of AP1, NF-AT, and NF-␬B. Blood 1993; 82: 2470–2477. kin-3-dependent hematopoietic cells. Blood 1995; 86: 3139– 70 Gottschalk LR, Giannola DM, Emerson SG. Molecular regulation 3150. of the human IL-3 gene: inducible T cell-restricted expression 89 Wang X-Y, McCubrey JA. Malignant transformation induced by requires intact AP-1 and Elf-1 nuclear protein binding sites. J Exp cytokine genes: a comparison of the abilities of germline and Med 1993; 178: 1681–1692. mutated interleukin 3 genes to transform hematopoietic cells by 71 Leiden JM, Wang C-Y, Petryniak B, Markovitz DM, Nabel GJ, transcriptional and posttranscriptional mechanisms. Cell Growth Thompson CB. A novel Ets-related transcription factor, Elf-1, Diff 1996; 7: 487–500. binds to human immunodeficiency virus type 2 regulatory 90 Wang XY, McCubrey JA. Differential effects of retroviral long ter- elements that are required for inducible trans activation in T cells. minal repeats on interleukin-3 gene expression and autocrine J Virol 1992; 66: 5890–5897. transformation. Leukemia 1997; 11: 1711–1725. 72 Cameron S, Taylor DS, TePas EC, Speck NA, Mathey-Prevot B. 91 Wang X-Y, McCubrey JA. Characterization of proteins binding Identification of a critical regulatory site in the human interleu- specifically to the interleukin 3 (IL-3) mRNA 3′ untranslated kin-3 promoter by in vivo footprinting. Blood 1994; 83: 2851– region in IL-3-dependent and autocrine transformed cells. Leuke- 2859. mia 1998; 12: 520–531. 73 Zhang W, Zhang J, Kornuc M, Kwna K, Frank R, Nimer SD. Mol- 92 Stoecklin G, Hahn S, Moroni C. Functional hierarchy of AUUUA ecular cloning and characterization of NF-IL3A, a transcriptional motifs in mediating rapid interleukin-3 mRNA decay. J Biol Chem activator of the human interleukin-3 promoter. Mol Cell Biol 1994; 269: 28591–28597. 1995; 15: 6055–6063. 93 Gillis P, Malter JS. The adenosine-uridine binding factor recog- 74 Cockerill PN, Shannon MF, Bert AG, Ryan GR, Vadas MA. The nizes the AU-rich elements of cytokine, lymphokine, and oncog- granulocyte–macrophage colony-stimulating factor/interleukin 3 ene mRNAs. J Biol Chem 1991; 266: 3172–3177. locus is regulated by an inducible cyclosporin A-sensitive 94 Bohjanen PR, Petryniak B, June CH, Thompson CB, Lindsten T. enhancer. Proc Natl Acad Sci USA 1993; 90: 2466–2470. An inducible cytoplasmic factor (AU-B) binds selectively to 75 Beltman J, McCormick F, Cook SJ. The selective protein kinase C AUUUA multimers in the 3′ untranslated region of lymphokine inhibitor, Ro-31–8220, inhibits mitogen-activated protein kinase mRNA. Mol Cell Biol 1991; 11: 3288–3295. phosphatase-1 (MKP-1) expression, and activates Jun N-terminal 95 Brewer G. An A+U-rich element RNA-binding factor regulates c- kinase. J Biol Chem 1996; 271: 27018–27024. myc mRNA stability in vitro. Mol Cell Biol 1991; 11: 2460–2466. 76 Fitzpatrick DR, Shirley KM, McDonald LE, Bielefeldt-Ohmann H, 96 Zhang W, Wagner BJ, Ehrenman K, Schaefer AW, DeMaria CT, Kay GF, Kelso A. Distinct methylation of the interferon gamma Crater D, DeHaven K, Long L, Brewer G. Purification, charac- (IFN-gamma) and interleukin 3 (IL-3) genes in newly activated terization, and cDNA cloning of an AU-rich element RNA-bind- primary CD8+ T lymphocytes: regional IFN-gamma promoter ing protein, AUF1. Mol Cell Biol 1993; 13: 7652–7665. Review WL Blalock et al 1148 97 Kiledjian M, DeMaria CT, Brewer G, Novick K. Identification of 117 Pai LH, Pastan I. Clinical trials with Pseudomonas exotoxin AUF1 (heterogenous nuclear ribonucleoprotein D) as a compo- immunotoxins. Curr Top Micro Immun 1998; 234: 83–96. nent of the ␣-globin mRNA stability complex. Mol Cell Biol 118 Winkler U, Barth S, Schnell R, Diehl V, Engert A. The emerging 1997; 17: 4870–4876. role of immunotoxins in leukemia and lymphoma. Ann Oncol 98 DeMaria CT, Brewer G. AUF1 binding affinity to A+U-rich 1997; 8 (Suppl. 1): 136–146. elements correlates with rapid mRNA degradation. J Biol Chem 119 Sachs L. The control of hematopoiesis and leukemia: from basic 1996; 271: 12179–12184. biology to the clinic. Proc Natl Acad Sci USA. 1996; 93: 99 Hamilton BJ, Nagy E, Malter JS, Arrick BA, Rigby WFC. Associ- 4743–4749. ation of heterogeneous nuclear ribonucleoprotein A1 and C pro- 120 Larson RA, LeBeau MM, Vardiman JW, Rowley JD. Myeloid leu- teins with reiterated AUUUA sequences. J Biol Chem 1993; 268: kemia after hematotoxins. Environ Health Pros 1996; 104 (Suppl 8881–8887. 6): 1303–1307. 100 Henics T, Sanfridson A, Hamilton BJ, Nagy E, Rigby WFC. 121 Palaszynski EW, Ihle JN. Evidence for specific receptors for Enhanced stability of interleukin-2 mRNA in MLA 144 cells: interleukin 3 on lymphokine dependent cell lines established possible role of cytoplasmic AU-rich sequence-binding proteins. from long-term bone marrow cultures. J Immunol 1984; 132: J Biol Chem 1994; 269: 5377–5383. 1872–1878. 101 Hirch HH, Nair APK, Backenstoss V, Moroni C. Interleukin-3 122 Miyajima A, Mui AL-F, Ogorochi T, Sakamaki K. Receptors for mRNA stabilization by a trans-acting mechanism in autocrine granulocyte–macrophage colony-stimulating factor, interleukin- tumors lacking interleukin-3 gene rearrangements. J Biol Chem 3, and interleukin-5. Blood 1993; 82: 1960–1974. 1995; 270: 20629–20635. 123 Kitamura T, Sato N, Arai K-I, Miyajima A. Expression cloning of ␤ 102 Hirsch HH, Backenstoss V, Moroni C. Impaired interleukin-3 the human IL-3 receptor cDNA reveals a shared subunit of the mRNA decay in autocrine mast cell tumors after transient cal- human IL-3 and GM-CSF receptors. Cell 1991; 66: 1165–1174. cium ionophore stimulation. Growth Factors 1996; 13: 99–110. 124 Hara T, Miyajima A. Two distinct functional high affinity recep- 103 Nair APK, Hahn S, Banholzer R, Hirsch HH, Moroni C. Cyclos- tors for mouse interleukin-3 (IL-3). EMBO J 1992; 11: 1875– porin A inhibits growth of autocrine tumor cell lines by destabil- 1884. izing interleukin-3 mRNA. Nature 1994; 369: 239–242. 125 Gorman DM, Itoh N, Kitamura T, Schreurs J, Yonehara S, Yahara 104 Banhozer R, Nair APK, Hirsch HH, Ming XF, Moroni C. Rapamy- I, Arai K-I, Miyajima A. Cloning and expression of a gene enco- cin destabilizes interleukin-3 mRNA in autocrine tumor cells by ding an interleukin 3 receptor-like protein: identification of a mechanism requiring an intact 3′ untranslated region. Mol Cell another member of the cytokine receptor gene family. Proc Natl Biol 1997; 17: 3254–3260. Acad Sci USA 1990; 87: 5459–5463. 105 Roush W. On the biotech pharm, a race to harvest new cancer 126 Kremer E, Baker E, D’Andrea RJ, Slim R, Phillips H, Moretti PA, cures. Science 1997; 278: 1039–1040. Lopez AF, Petit C, Vadas MA, Sutherland GR, Goodall GJ. A 106 Rogers SY, Bradbury D, Kozlowski R, Russell NH. Evidence for cytokine receptor gene cluster in the X-Y pseudoautosomal internal autocrine regulation of growth in acute myeloblastic leu- region. Blood 1993; 82: 22–28. kemia cells. Exp Hematol 1994; 22: 593–598. 127 Kitamura TK, Hayashida K, Sakamaki K, Yokota T, Arai K-I, Miya- 107 Lopez AF, Shannon MF, Barry S, Phillips JA, Cambareri B, Dot- jima A. Reconstitution of functional receptors for human tore M, Simmons P, Vadas MA. A human interleukin 3 analog granulocyte/macrophage colony-stimulating factor (GM-CSF): with increased biological and binding activities. Proc Natl Acad evidence that the protein encoded by the AIC2B cDNA is a sub- Sci USA 1992; 89: 842–846. unit of the murine GM-CSF receptor. Proc Natl Acad Sci USA 108 Iversen PO, Rodwell RL, Pitcher L, Taylor KM, Lopez AF. Inhi- 1991; 88: 5082–5086. bition of proliferation and induction of apoptosis in juvenile mye- 128 Kitamura T, Takaku F, Miyajima A. IL-1 up-regulates the lomocytic leukemic cells by the granulocyte–macrophage col- expression of cytokine receptors on a factor-dependent human hemopoietic cell line, TF-1. Int Immunol 1991; 3: 571–577. ony-stimulating factor analogue E21R. Blood 1996; 88: 2634– 129 Miyajima A, Kitamura T, Harada N, Yokota T, Arai K-I. Cytokine 2639. receptors and signal transduction. Annu Rev Immunol 1992; 10: 109 Gibson I. Antisense approaches to the gene therapy of cancer – 295–331. ‘Recnac’. Cancer Metas Rev 1996; 15: 287–299. 130 Barry SC, Bagley CJ, Phillips J, Dottore M, Cambareri B, Moretti 110 Mukhopadhyay T, Roth JA. Antisense regulation of oncogenes in P, D’Andrea R, Goodall GJ, Shannon MF, Vadas MA, Lopez AF. human cancer. Crit Rev Oncol 1996; 7: 151–190. Two contiguous residues in human interleukin-3, Asp21 and 111 Scanlon KJ, Ohta Y, Ishida H, Kijima H, Ohkawa T, Kaminski Glu22, selectively interact with the alpha- and beta-chains of A, Tsai J, Horng G, Kashani-Sabet M. Oligonucleotide-mediated its receptor and participate in function. J Biol Chem 1994; 269: modulation of mammalian gene expression. FASEB J 1995; 9: 8488–8492. 1288–1296. 131 D’Andrea R, Rayner J, Moretti P, Lopez A, Goodall GJ, Gonda 112 Mercola D, Cohen JS. Antisense approaches to cancer gene ther- TJ, Vadas M. A mutation of the common receptor subunit for apy. Cancer Gene Therapy 1995; 2: 47–59. interleukin-3 (IL-3), granulocyte–macrophage colony-stimulating 113 Rozemuller H, Terpstra W, Rombouts EJC, Lawler M, Byrne C, factor, and IL-5 that leads to ligand independence and tumorig- FitzGerald JP, Kreitman RJ, Wielenga JJ, Lowenberg B, Touw, IP, enicity. Blood 1994; 10: 2802–2808. Hagenbeck A, Martens ACM. GM-CSF receptor targeted treat- 132 Hannemann J, Hara T, Kawai M, Miyajima A, Ostertag W, ment of primary AML in SCID mice using Diphtheria toxin fused Stocking C. Sequential mutations in the interleukin-3 to huGM-CSF. Leukemia 1998; 12: 1962–1970. (IL3)/granulocyte–macrophage colony-stimulating factor/IL5 114 Rozemuller H, Rombouts EJC, Touw IP, FitzGerald dJP, Kreitman receptor ␤-subunit genes are necessary for the complete conver- RJ, Hagenbeck A, Martens ACM. In vivo targeting of leukemic ␤ sion to growth autonomy mediated by a truncated c subunit. cells using diptheria toxin fused to murine GM-CSF. Leukemia Mol Cell Biol 1995; 15: 2402–2412. 1998; 12: 710–718. 133 Nishinakamura R, Nakayama N, Hirabayashi Y, Inoue T, Aud D, 115 Terpstra W, Rozemuller H, Breems DA, Rombouts EJ, Prins A, McNeil T, Azuma S, Yoshida S, Toyada Y, Arai K-I, Miyajima A, FitzGerald DJ, Kreitman RJ, Wielenga JJ, Ploemacher RE, Lowen- ␤ Murray R. Mice deficient for the IL-3/GM-CSF/IL-5 c receptor berg B, Hagenbeek A, Martens AC. Diphtheria toxin fused to exhibit lung pathology and impaired immune response, while ␤ granulocyte–macrophage colony-stimulating factor eliminates IL3 receptor-deficient mice are normal. Immunity 1995; 2: acute myeloid leukemia cells with the potential to initiate leuke- 211–222. mia in immunodefficient mice, but spares normal hematopoietic 134 Nicola NA, Robb L, Metcalf D, Cary D, Drinkwater CC, Begley stem cells. Blood 1997; 90: 3735–3742. CG. Functional inactivation in mice of the gene for the interleu- 116 Hall PD, Willingham MC, Kreitman RJ, Frankel AE. DT388-GM- kin-3 (IL-3)-specific receptor ␤-chain: implications for IL-3 and CSF, a novel fusion toxin consisting of a truncated diptheria toxin the mechanism of receptor transmodulation in hematopoietic fused to human granulocyte–macrophage colony-stimulating fac- cells. Blood 1996; 87: 2665–2674. tor, prolongs host survival in a SCID model of acute myeloid 135 Cooke KR, Nishinakamura R, Martin TR, Kobzik L, Brewer J, leukemia. Leukemia 1999; 13: 629–633. Whitsett JA, Bungard D, Murray R, Ferrara JL. Persistence of pul- Review WL Blalock et al 1149 monary pathology and abnormal lung function in IL-3/GM- stimulating factor receptor (G-CSF-R) gene in a case of acute CSF/IL-5 beta c receptor-deficient mice despite correction of myeloid leukemia results in the overexpression of a novel G-CSF- alveolar proteinosis after BMT. Bone Marrow Transplant 1997; R isoform. Blood 1995; 85: 902–911. 20: 657–662. 152 Horiike S, Yokata S, Nakao M, Iwai T, Sasai Y, Kaneko H, Tani- 136 Robb L, Drinkwater CC, Metcalf D, Li R, Kontgen F, Nicola NA, wai M, Kashima K, Fujii H, Abe T, Misawa S. Tandem dupli- Begley CG. Hematopoietic and lung abnormalities in mice with cation of the FLT3 receptor gene are associated with leukemic a null mutation of the common ␤ subunit of the receptors for transformation of myelodysplasia. Leukemia 1997; 11: 1442– granulocyte–macrophage colony-stimulating factor and interleu- 1446. kins 3 and 5. Proc Natl Acad Sci USA 1995; 92: 9565–9569. 153 Tsujimura T, Kanakura Y, Kitamura Y. Mechanisms of constitut- 137 Dranoff G, Crawford AD, Sadelain M, Ream B, Rashid A, Bron- ive activation of c-kit receptor tyrosine kinase. Leukemia 1997; son RT, Dickersin GR, Bachurski CJ, Mark EL, Whitsett JA, Mulli- 11 (Suppl 3): 396–398. gan RC. Involvement of granulocyte–macrophage colony-stimul- 154 Tobal K, Pagliuca A, Bhatt B, Bailey N, Layton DM, Mufti GJ. ating factor in pulmonary homeostasis. Science 1994; 264: Mutations of the human FMS gene (M-CSF receptor) in myelod- 713–716. ysplastic syndromes and acute myeloid leukemia. Leukemia 138 Stanley E, Lieschke GJ, Grail D, Metcalf D, Hodgson G, Gall 1997; 4: 486–489. JAM, Maher DW, Cebon J, Sinickas V, Dunn AR. 155 Ridge SA, Worwood M, Oscier D, Jacobs A, Padua RA. FMS Granulocyte/macrophage colony-stimulating factor-deficient mutations in myelodysplastic, leukemic, and normal subjects. mice show no major perturbation of hematopoiesis but develop Proc Natl Acad Sci USA 1990; 87: 1377–1380. a characteristic pulmonary pathology. Proc Natl Acad Sci USA 156 Piao X, Paulson R, van der Geer P, Pawson T, Bernstein A. 1994; 91: 5592–5596. Oncogenic mutation in the Kit receptor tyrosine kinase alters 139 Nishinakamura R, Miyajima A, Mee PJ, Tybulewicz VLJ, Murry substrate specificity and induces degradation of the protein tyro- R. Hematopoiesis in mice lacking the entire granulocyte–macro- sine phosphatase SHP-1. Proc Natl Acad Sci USA 1996; 93: phage colony-stimulating factor/interleukin-3/interleukin-5 func- 14665–14669. tions. Blood 1996; 88: 2458–2464. 157 Li Q, Kondoh G, Inafuku S, Nishimune Y, Hakura A. Abrogation 140 Metcalf D, Mfsud S, Di Rago L, Robb L, Nicola NA, Alexander of c-kit/Steel factor-dependent tumorigenesis by kinase defective WA. The biological consequences of excess GM-CSF levels in mutants of the c-kit receptor: c-kit kinase defective mutants as transgenic mice also lacking high-affinity receptors for GM-CSF. candidate tools for cancer gene therapy. Cancer Res 1996; 56: Leukemia 1998; 12: 353–363. 4343–4346. 141 Brown MA, Harrison-Smith M, DeLuca E, Begley CG, Gough 158 Kondoh G, Hayasaka N, Li Q, Nishimune Y, Hacker A. An in NM. No evidence for GM-CSF receptor alpha chain gene vivo model for tyrosine kinase autocrine/paracrine activation: mutation in AML-M2 leukemias which have lost a sex chromo- auto-stimulated KIT receptor acts as a tumor promoting factor some. Leukemia 1994; 8: 1774–1779. in papillomavirus-induced tumorigenesis. Oncogene 1995; 10: 142 Decker J, Fiedler W, Samalecos A, Hossfeld DK. Absence of 341–347. point mutations in the extracellular domain of the alpha subunit 159 Rohrschneider LR, Bourette RP, Lioubin MN, Algate PA, Myles of the GM-CSF receptor in a series of patients with acute myeloid GM, Carlberg K. Growth and differentiation signals regulated by leukemia (AML). Leukemia 1995; 9: 185–188. the M-CSF receptor. Mol Reprod Dev 1997; 46: 96–103. 143 Freeburrn RW, Gale RE, Wagner HM, Linch DC. Analysis of the 160 Glover HR, Baker DA, Celetti A, Dibb NJ. Selection of activating coding sequence for the GM-CSF receptor alpha and beta chains mutations of c-fms in FDC-P1 cells. Oncogene 1995; 11: in patients with juvenile chronic myeloid leukemia (JCML). Exp 1347–1356. Hematol 1997; 25: 306–311. 161 Arland M, Fiedler W, Samalecos A, Hossfeld DK. Absence of 144 Maroc N, Rottapel R, Rosnet O, Marchetto S, Lavezzi C, Man- point mutations in a functionally important part of the extracellu- noni P, Birnbaum D, Dubreuil P. Biochemical characterization lar domain of the c-kit proto-oncogene in a series of patients with and analysis of the transforming potential of the FLT3/FLK2 acute myeloid leukemia. Leukemia 1994; 8: 498–501. receptor tyrosine kinase. Oncogene 1993; 8: 909–918. 162 Carlberg K, Rohrschneider L. The effects of activating mutations 145 Tidow N, Pilz C, Kasper B, Welte K. Frequency of point on dimerization, tyrosine phosphorylation and internalization of mutations in the gene for the G-CSF receptor in patients with the macrophage colony stimulating factor receptor. Mol Biol Cell chronic neutropenia undergoing G-CSF therapy. Stem Cells 1994; 5: 81–95. 1997; 15 (Suppl. 1): 113–119. 163 Stephenson J, Mufti GJ, Yoshida Y. Myelodysplastic syndromes: 146 Carapeti M, Soede-Bobok A, Hochhaus A, Sill M, Touw IP, Gold- from morphology to molecular biology. Part II. The molecular man JM. Rarity of dominant-negative mutations of the G-CSF genetics of myelodysplasia. Int J Hematol 1993; 57: 99–112. receptor in patients with blast crisis of chronic myeloid leukemia 164 Roussel MF, Downing JR, Rettenmier CW, Sherr CJ. A point or de novo acute leukemia. Leukemia 1997; 11: 1005–1008. mutation in the extracellular domain of the human CSF-1 recep- 147 Tidow N, Pilz C, Teichmann B, Muller-Brechlin A, Germe- shausen M, Kasper B, Rauprich P, Sykora KW, Welte K. Clinical tor (c-fms proto-oncogene product) activates its transforming relevance of point mutations in the cytoplasmic domain of the potential. Cell 1988; 55: 979–988. granulocyte colony-stimulating factor receptor gene in patients 165 Gale RE, Freeburn RW, Khwaja A, Chopra R, Linch DC. A trunc- with severe congenital neutropenia. Blood 1997; 89: 2369– ated isoform of the human beta chain common to the receptors 2375. for granulocyte–macrophage colony-stimulating factor, interleu- 148 McLemore ML, Poursine-Laurent J, Link DC. Increased granulo- kin-3 (IL-3), and IL-5 with increased mRNA expression in some cyte colony-stimulating factor responsiveness but normal resting patients with acute leukemia. Blood 1998; 91: 54–63. granulopoiesis in mice carrying a targeted granulocyte colony- 166 Rajotte D, Cadieux C, Haman A, Wilkes BC, Clark SC, Hercus stimulating factor receptor mutation derived from a patient with T, Woodcock JA, Lopez A, Hoang T. Crucial role of the residue ′ ′ severe congenital neutropenia. J Clin Invest 1998; 102: 483–492. R280 at the F –G loop of the human granulocyte/macrophage 149 Dong F, Brynes RK, Tidow N, Welte K, Lowenberg B, Touw IP. colony-stimulating factor receptor alpha chain for ligand recog- Mutations in the gene for the granulocyte colony-stimulating-fac- nition. J Exp Med 1997; 185: 1939–1950. tor receptor in patients with acute myeloid leukemia preceded 167 Inhorn RC, Carlesso N, Durstin M, Frank DA, Griffin JD. Identifi- by severe congenital neutropenia. New Engl J Med 1995; 333: cation of a viability domain in the granulocyte/macrophage col- 487–493. ony-stimulating factor receptor beta-chain involving tryosine- 150 Dong F, Hoefsloot LH, Schelen AM, Broeders CA, Meijer Y, 750. Proc Natl Acad Sci USA 1995; 92: 8665–8669. Veerman AJ, Touw IP, Lowenberg B. Identification of a nonsense 168 Matsuguchi T, Zhao Y, Lilly MD, Kraft AS. The cytoplasmic mutation in the granulocyte colony-stimulating factor receptor in domain of granulocyte–macrophage colony-stimulating factor severe congenital neutropenia. Proc Natl Acad Sci USA 1994; (GM-CSF) receptor alpha subunit is essential for both GM-CSF- 91: 4480–4484. mediated growth and differentiation. J Biol Chem 1997; 272: 151 Dong F, van Paassen M, van Buitenen C, Hoefsloot LH, Lowen- 17450–17459. berg B, Touw IP. A point mutation in the granulocyte colony- 169 Okuda K, Smith L, Griffin JD, Foster R. Signaling functions of the Review WL Blalock et al

1150 ␤ tyrosine residues in the c chain of the granulocyte–macrophage and activation of Btk and Tec tyrosine kinases by gp130, a signal colony-stimulating factor receptor. Blood 1997; 90: 4759–4766. transducer of the interleukin-6 family of cytokines. Blood 1995; 170 Itoh T, Muto A, Watanabe S, Miyajima A, Yokota T, Arai K. Gra- 85: 627–633. nulocyte–macrophage colony-stimulating factor provokes RAS 188 Tsubokawa M, Tohyama Y, Tohyama K, Asahi M, Inazu T, Naka- activation and transcription of c-fos through different modes of mura H, Saito H, Yamamura H. Interleukin-3 activates Syk in a signaling. J Biol Chem 1996; 271: 7587–7592. human myeloblastic leukemia cell line, AML 193. Euro J 171 Lia F, Rajote D, Clark SC, Hoang T. A dominant negative gra- Biochem 1997; 249: 792–796. nulocyte–macrophage colony-stimulating factor receptor alpha 189 Stomski FC, Woodchock JM, Zacharakis B, Bagley CJ, Sun Q, chain reveals the multimeric structure of the receptor complex. Lopez AF. Identification of a Cys motif in the common beta chain J Biol Chem 1996; 271: 28287–28293. of the interleukin 3, granulocyte–macrophage colony-stimulating 172 Bone H, Dechert U, Jirik F, Schrader JW, Welham MJ. SHP1 and factor, and interleukin 5 receptor essential for disulfide-linked ␤ SHP2 protein-tyrosine phosphatases associate with c, after receptor hetreodimerization and activation of all three receptors. interleukin-3-induced receptor tyrosine phosphorylation. Identi- J Biol Chem 1998; 273: 1192–1199. fication of potential binding sites and substrates. J Biol Chem 190 Stomski FC, Sun Q, Bagley CJ, Woodcock J, Goodall G, Andrews 1997; 272: 14470–14476. RK, Berndt MC, Lopez AF. Human interleukin-3 (IL-3) induces 173 Yi T, Mui AL, Krystal G, Ihle JN. Hematopoietic cell phosphatase disulfide-linked IL-3 receptor alpha- and beta-chain heterodimer- associates with the interleukin-3 (IL-3) receptor beta chain and ization, which is required for receptor activation but not high- down-regulates IL-3-induced tyrosine phosphorylation and affinity binding. Mol Cell Biol 1996; 16: 3035–3046. mitogenesis. Mol Cell Biol 1993; 13: 7577–7586. 191 Anderson SM, Jorgensen B. Activation of src-related tyrosine kin- 174 Quelle FW, Sato N, Witthuhn BA, Inhorn RC, Eder M, Miyajima ases by IL-3. J Immunol 1995; 155: 1660–1670. ␤ A, Griffin JD, Ihle JN. Jak2 associates with the c chain of the 192 Watanabe S, Ishida S, Koike K, Arai K. Characterization of cis- receptor for granulocyte–macrophage colony-stimulating factor, regulatory elements of the c-myc promoter responding to human and its activation requires the membrane-proximal region. Mol GM-CSF or mouse interleukin 3 in mouse pro B cell BA/F3 cells Cell Biol 1994; 14: 4335–4341. expressing the human GM-CSF receptor. Mol Biol Cell 1995; 6: 175 Jaster R, Zhu Y, Pless M, Bhattachary S, Mathey-Prevot B, D’And- 627–636. rea AD. JAK2 is required for induction of the murine DUB-1 193 Barry SC, Korpelainen E, Sun Q, Stomski FC, Moretti PA, Wakao gene. Mol Cell Biol 1997; 17: 3364–3372. H, D’Andrea RJ, Vadas MA, Lopez AF, Goodall GJ. Roles of the 176 Yoshimura A. The CIS/JAB family; novel negative regulators of N and C terminal domains of the interleukin-3 receptor alpha JAK signaling pathways. Leukemia 1998; 12: 1851–1857. chain in receptor function. Blood 1997; 89: 842–852. 177 Tian SS, Tapley P, Sincich C, Stein RB, Rosen J, Lamb P. Multiple 194 Nicola NA, Smith A, Robb L, Metcalf D, Begley CG. The struc- signaling pathways induced by granulocyte colony-stimulating tural basis of biological actions of the GM-CSF receptor. The factor involving activation of JAKs, STAT5 and/or STAT3 are molecular basis of cellular defense mechanisms. Ciba Foun- required for regulation of three distinct classes of immediate- dation Symp 1997; 204: 19–27. early genes. Blood 1996; 88: 4435–4444. 195 Doyle SE, Gasson JC. Characterization of the role of the human 178 Rao P, Mufson RA. A membrane proximal domain of the human granulocyte–macrophage colony-stimulating factor receptor interleukin-3 receptor beta c subunit that signals DNA synthesis alpha subunit in the activation of JAK2 and STAT5. Blood 1998; in NIH 3T3 cells specifically binds a complex of Src and Janus 92: 867–876. family kinases and phosphatidylinositol 3-kinase. J Biol Chem 196 Hynes NE, Groner G. A target for tumor-directed therapy. Nature 1995; 270: 6886–6893. Med 1995; 1: 631–632. 179 Jucker M, Feldman RA. Identification of a new adapter protein 197 Wells W, Beerli R, Hellmann P, Schmidt M, Marte BM, Kornilova that may link the common ␤ subunit of the receptor for ES, Hekele A, Mendelsohn J, Groner B, Hynes NE. EGF receptor granulocyte/macrophage colony-stimulating factor, interleukin and p185erbB-2-specific single-chain antibody toxins differ in (IL)-3, and IL-5 phosphatidylinositol 3-kinase. J Biol Chem 1995; their cell-killing activity on tumor cells expressing both receptor 270: 27817–27822. proteins. Int J Cancer 1995; 60: 137–144. 180 Azam M, Erdjument-Bromage H, Kreider BL, Xia M, Quelle F, 198 Sedlacek HH. Vaccination for treatment of tumors; a critical Basu R, Saris C, Tempst P, Ihle JN, Schindler C. Interleukin-3 comment. Crit Rev Oncogen 1994; 5: 555–587. signals through multiple isoforms of Stat5. EMBO J 1995; 14: 199 Janson CH. Biology of monoclonal antibodies in tumor therapy. 1402–1411. Med Oncol Tum Pharmacother 1993; 10: 21–23. 181 Pratt JC, Weiss M, Sieff CA, Shoelson SE, Burakoff SJ, Ravichand- 200 Jucker M, Feldman RA. Identification of a new adapter protein ran KS. Evidence for a physical association between the Shc-PTB that may link the common beta subunit of the receptor for ␤ domain and the c chain of the granulocyte–macrophage colony- granulocyte/macrophage colony-stimulating factor, interleukin stimulating factor receptor. J Biol Chem 1996; 271: 12137– (IL)-3, and IL-5 to phosphatidylinositol 3-kinase. J Biol Chem 12140. 1995; 270: 27817–27822. 182 Matsuguchi T, Inhorn RC, Carlesso N, Xu G, Druker B, Griffin 201 McKearn TJ. Radioimmunodetection of solid tumors. Future hor- JD. Tyrosine phosphorylation of p95Vav in myeloid cells is regu- izons and applications for radioimmunotherapy. Cancer 1993; lated by GM-CSF, IL-3 and steel factor and is constitutively 71: 4302–4313. increased by p210BCR/ABL. EMBO J 1995; 14: 257–265. 202 Russell PJ, Plomley J, Shon IH, O’Grady H, Pearce N. Mono- 183 Sato N, Sakamaki K, Terada N, Arai K-I, Miyajima A. Signal trans- clonal antibodies for intravesicle radioimmunotherapy of human duction by the high-affinity GM-CSF receptor: two distinct cyto- bladder cancer. Cell Biophys 1993; 2: 27–47. plasmic regions of the common ␤ subunit responsible for differ- 203 Ragnhammar P, Fagerberg J, Frodin JE, Hjelm AL, Lindemalm ent signaling. EMBO J 1993; 12: 4181–4189. C, Magnusson I, Masucci G, Mellstedt H. Effect of monoclonal 184 Liu L, Cutler RL, Krystal G. Identification and characterization of antibody 17–1A and GM-CSF in patients with advanced colorec- an interleukin-3 receptor-associated serine/threonine kinase. J tal carcinoma – long-lasting, complete remissions can be Biol Chem 1995; 270: 22422–22427. induced. Int J Cancer 1993; 53: 751–758. 185 Yoshimura A, Ohkubo T, Kiguchi T, Jenkins NA, Gilbert DJ, 204 Sun Q, Woodcock JM, Rapoport A, Stomski FC, Korpelainen EI, Copeland NG, Hara T, Miyajima A. A novel cytokine-inducible Bagley CJ, Goodall GJ, Smith WB, Gamble JR, Vadas MA, Lopez gene CIS encodes an SH2-containing protein that binds to tyro- AF. Monoclonal antibody 7G3 recognizes the N-terminal sine-phosphorylated interleukin 3 and erythropoietin receptors. domain of the human interleukin-3 (IL-3) receptor alpha-chain EMBO J 1995; 14: 2816–2826. and functions as a specific IL-3 receptor antagonist. Blood 1996; 186 Takahashi-Tezuka M, Hibi M, Fujitani Y, Fukada T, Yamaguchi 87: 83–92. T, Hirano T. Tec tyrosine kinase links the cytokine receptors to 205 Morla AO, Schreurs J, Miyajima A, Wang JYJ. Hematopoietic PI-3 kinase probably through JAK. Oncogene 1997; 14: 2273– growth factors activate the tyrosine phosphorylation of distinct 2278. sets of proteins in interleukin-3-dependent murine cell lines. Mol 187 Matsuda T, Takahashi-Tezuka M, Fukada T, Okuyama Y, Fujitani Cell Biol 1988; 8: 2214–2218. Y, Tsukada S, Mano H, Hirai H, Witte ON, Hirano T. Association 206 Kanakura Y, Druker B, Cannistra SA, Furukawa Y, Torimoto Y, Review WL Blalock et al 1151 Griffin JD. Signal transduction of the human granulocyte–macro- 225 Ihle JN. Cytokine receptor signaling. Nature 1995; 377: 591– phage colony-stimulating factor and interleukin-3 receptors 594. involves tyrosine phosphorylation of a common set of cytoplas- 226 Wilks AF. Two putative protein-tyrosine kinases identified by mic proteins. Blood 1990; 76: 706–715. application of the polymerase chain reaction. Proc Natl Acad Sci 207 Silvennoinen O, Witthuhn BA, Quelle FW, Cleveland JL, Yi T, USA 1989; 86: 1603–1607. Ihle JN. Structure of the murine Jak2 protein-tyrosine kinase and 227 Firmbach-Kraft I, Byers M, Shows T, Dalla-Favera R, Krolewski its role in interleukin 3 signal transduction. Proc Natl Acad Sci JJ. tyk2, prototype of a novel class of non-receptor tyrosine kinase USA 1993; 90: 8429–8433. genes. Oncogene 1990; 5: 1329–1336. 208 Carroll MP, Clark-Lewis I, Rapp UR, May WS. Interleukin-3 and 228 Heim MH, Kerr IM, Stark GK, Darnell JE Jr. Contribution of Stat granulocyte–macrophage colony-stimulating factor mediate SH2 groups to specific interferon signalling by the Jak-Stat path- rapid phosphorylation and activation of cytosolic c-Raf. J Biol way. Science 1995; 267: 1347–1349. Chem 1990; 265: 19812–19817. 229 Nakamura N, Chin H, Miyasaka N, Miura O. An epidermal 209 Terada K, Kaziro Y, Satoh T. Ras-dependent activation of c-Jun growth factor receptor/Jak2 tyrosine kinase domain chimera N-terminal kinase/stress-activated protein kinase in response to induces tyrosine phosphorylation of Stat 5 and transduces a interleukin 3 stimulation in hematopoietic BaF3 cells. J Biol growth signal in hematopoietic cells. J Biol Chem 1996; 271: Chem 1997; 272: 4544–4548. 19483–19488. 210 Kinoshita T, Shirouzu M, Kamiya A, Hashimoto K, Yokoyama S, 230 Barinaga M. Two major signal pathways meet at MAP-kinase. Miyajima A. Raf/MAPK and rapamycin-sensitive pathways Science 1995; 269: 1673. mediate the anti-apoptotic function of p21Ras in IL-3-dependent 231 Winston LA, Hunter T. Jak2, Ras, and Raf are required for acti- hematopoietic cells. Oncogene 1997; 15: 619–627. vation of extracellular signal-regulated kinase/mitogen-activated 211 Duronio V, Welham MJ, Abraham S, Dryden P, Schrader JW. protein kinase by growth hormone. J Biol Chem 1995; 270: Ras p21 activation via hemopoietin receptors and c-kit requires 30837–30840. Ras tyrosine kinase activity but not tyrosine phosphorylation of p21 232 David M, Petricoin E, Benjamin C, Pine R, Weber MJ, Larner, GTPase-activating protein. Proc Natl Acad Sci USA 1992; 89: AC. Requirement for MAP kinase (ERK2) activity in interferon 1587–1591. alpha- and interferon beta-stimulated gene expression through 212 Rapp UR, Troppmair J, Carroll M, May S. Role of Raf-1 protein Stat proteins. Science 1995; 269: 1721–1723. kinase in IL-3 and GM-CSF-mediated signal transduction. Curr 233 Migone TS, Lin JX, Cereseto A, Mulloy JC, O’Shea JJ, Franchini Topics Micro Immuno 1990; 166: 129–139. G, Leonard WJ. Constitutively activated Jak-Stat pathway in T 213 Okuda K, Sanghera JS, Pekech SL, Kanakura Y, Hallek M, Griffin cells transformed with HTLV-1. Science 1995; 269: 79–81. JD, Druker BJ. Granulocyte–macrophage colony-stimulating fac- 234 Danial NN, Pernis A, Rothman PB. Jak-Stat signaling induced by tor, interleukin-3, and steel factor induce rapid tyrosine phos- the v-abl oncogene. Science 1995; 269: 1875–1877. phorylation of p42 and p44 MAP kinase. Blood 1992; 79: 235 Ilaria RL, Van Etten RA. p210 and p190 (BCR/ABL) induce the 2880–2887. tyrosine phosphorylation and DNA binding activity of multiple 214 McCubrey JA, Steelman LS, Risser RG, McKearn JP. Structure and specific Stat family members. J Biol Chem 1996; 271: 31704– expression of the T cell receptor gamma locus in pre-B and early 31710. hemopoietic cells. Eur J Immunol 1989; 19: 2303–2308. 236 Murakami Y, Nakano S, Niho Y, Hamasaki, Izuhara K. Constitut- 215 McCubrey JA, Steelman LS, Sandlin G, Riddle RS, Ways DK. ive activation of Jak-2 and Tyk-2 in a v-Src-transformed human Effects of phorbol esters on an interleukin-3-dependent cell line. gallbladder adenocarcinoma cell line. J Cell Physiol 1998; 175: Blood 1990; 76: 63–72. 220–228. 216 McCubrey JA, Steelman LS, McKearn JP. Interleukin-3 and phor- 237 Campbell GS, Yu CL, Jove R, Carter-Su C. Constitutive activation bol esters induce different patterns of immediate–early gene of JAK1 in Src-transformed cells. J Biol Chem 1997; 272: expression in an interleukin-3-dependent cell line. Oncogene 2591–2594. Res 1991; 6: 1–12. 238 O’Shea JJ, Notarangelo LD, Johnston JA, Candotti F. Advances 217 Ways DK, Qin W, Riddle RS, Garris TD, Bennett TE, Steelman in the understanding of cytokine signal transduction: the roles of LS, McCubrey JA. Differential effect of phorbol esters and IL-3 Jaks and STATs in immunoregulation and the pathogenesis of on protein kinase C isoform content and kinase activity in the immunodeficiency. J Clin Immunol 1997; 17: 431–447. FDC-P1 cell line. Blood 1991; 78: 2633–2641. 239 Paul WE. Interleukin 4: signalling mechanism and control of T 218 White MK, DeVente JE, Robbins PJ, Canupp DM, Mayo MW, cell differentiation. Ciba Foundation Symp 1997; 204: 208–216. Steelman LS, McCubrey JA. Differential regulation of glucose 240 Chen M, Cheng A, Chen YQ, Hymel A, Hanson EP, Kimmel L, transporter expression in hematopoietic cells by oncogenic trans- Minami Y, Taniguchi T, Changelian PS, O’Shea JJ. The amino formation and cytokine stimulation. Oncol Rep 1994; 1: 17–26. terminus of JAK3 is necessary and sufficient for binding to the 219 McCubrey JA, Algate PA, Mayo MW, Arana G, Wang X-Y, Pre- common gamma chain and confers the ability to transmit vost KD, White M, Steelman LS. Differential effects of tumor promoters and cytokines on proto-oncogene expression in a hema- interleukin 2-mediated signals. Proc Natl Acad Sci USA 1997; topoietic cytokine-dependent cell line. Oncol Rep 1994; 1: 94: 6910–6915. 285–300. 241 Berridge MJ. Lymphocyte activation in health and disease. Crit 220 Mayo MW, Steelman LS, McCubrey JA. Phorbol esters support Rev Immunol 1997; 17: 155–178. the proliferation of a hematopoietic cell line by upregulating c- 242 Riedy MC, Dutra AS, Blake TB, Modi W, Lal BK, Davis J, Bosse Jun expression. Oncogene 1994; 9: 1999–2008. A, O’Shea JJ, Johnston JA. Genomic sequence, organization, and 221 White MK, McCubrey JA. Changes in glucose transport associa- chromosomal localization of human JAK3. Genomics 1996; 37: ted with malignant transformation. Int J Oncol 1995; 7: 701–712. 57–61. 222 McCubrey JA, Steelman LS, Mayo MW, Algate PA, Dellow RA, 243 Izuhara K, Heike T, Otsuka T, Yamaoka K, Mayumi M, Imamura Kaleko M. Growth promoting effects of insulin-like growth fac- T, Niho Y, Harada N. Signal transduction pathway of interleukin- tor-1 on hematopoietic cells: overexpression of introduced IGF- 4 and interleukin-13 in human B cells derived from X-linked sev- 1 receptor abrogates IL-3-dependency of murine factor depen- ere combined immunodeficient patients. J Biol Chem 1996; 271: dent cells by a ligand-dependent mechanism. Blood 1991; 78: 619–622. 921–929. 244 Leonard WJ. Dysfunctional cytokine receptor signaling in severe 223 Brown CB, Pihl CE, Murray EW. Oligomerization of the soluble combined immunodeficiency. J Invest Med 1996; 44: 304–311. granulocyte–macrophage colony-stimulating factor receptor: 245 Rodig SJ, Meraz M, White JM, Lampe PA, Riley JK, Arthur CD, identification of the functional ligand-binding species. Cytokine King KL, Sheehan KCF, Yin L, Pennica D, Johnson EM, Schreiber 1997; 9: 219–225. RD. Disruption of the Jak1 gene demonstrates obligatory and 224 Lia F, Rajotte D, Clark SC, Hoang T. A dominant negative gra- nonredundant roles of the Jaks in cytokine-induced biologic nulocyte–macrophage colony-stimulating factor receptor alpha responses. Cell 1998; 93: 373–383. chain reveals the multimeric structure of the receptor complex. 246 Parganas E, Wang D, Stravopodis D, Topham DJ, Marine J-C, J Biol Chem 1996; 271: 28287–28293. Teglund S, Vanin EF, Bodner S, Colamonici OR, van Deurson Review WL Blalock et al 1152 JM, Grosveld G, Ihle JN. Jak2 is essential for signalling through mann CM. Inhibition of acute lymphoblastic leukemia by a Jak- a variety of cytokine receptors. Cell 1998; 93: 385–395. 2 inhibitor. Nature 1996; 379: 645–648. 247 Thomis DC, Lee W, Berg LJ. T cells from Jak3-deficient mice 265 Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni have intact TCR, but increased apoptosis. J Immunol 1997; 159: G, Nicoletti I, Grignani F, Pawson T, Pelicci PG. A novel trans- 4708–4719. forming protein (Shc) with an SH2 domain is implicated in mitog- 248 Sohn SJ, Forbush KA, Nguyen N, Witthuhn B, Nosaka T, Ihle JN, enic signal transduction. Cell 1992; 70: 93–104. Perlmutter RM. Requirement for Jak3 in mature T cells: its role 266 Blaikie P, Immanuel D, Wu J, Li NX, Yajnik V, Margolis B. A in regulation of T cell homeostasis. J Immunol 1998; 160: region in Shc distinct from the SH2 domain can bind tyrosine- 2130–2138. phosphorylated growth factor receptors. J Biol Chem 1994; 269: 249 Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted disrup- 32031–32034. tion of mouse Stat1 gene results in compromised innate immun- 267 Kavanaugh WM, Williams LT. An alternative to SH2 domains ity to viral disease. Cell 1996; 84: 443–450. for binding tyrosine-phosphorylated proteins. Science 1994; 266: 250 Simpson SJ, Shah S, Comiskey M, de Jong YP, Wang B, Mizogu- 1862–1865. chi E, Bhan AK, Terhorst C. T cell-mediated pathology in two 268 Lioubin MN, Algate PA, Tsai S, Carlberg K, Aebersold R, models of experimental colitis depends predominantly on the Rohrschneider LR. p150Ship, a signal transduction molecule with interleukin 12/signal transducer and activator of transcription inositol polyphosphate-5-phosphatase activity. Genes Dev 1996; (Stat)-4 pathway, but is not conditional on interferon gamma 10: 1084–1095. expression by T cells. J Exp Med 1998; 187: 1225–1234. 269 Corey S, Eguinoa A, Puyana-Theall K, Bolen JB, Cantley L, Molli- 251 Feldman GM, Rosenthal LA, Liu X, Hayes MP, Wynshaw-Boris nedo F, Jackson TR, Hawkins PT, Stephens LR. Granulocyte– A, Leonard WJ, Hennighausen L, Finbloom DS. STAT5-deficient macrophage colony-stimulating factor stimulates both associ- mice demonstrate in granulocyte–macrophage colony-stimulat- ation and activation of phosphoinositide 3OH-kinase and src- ing factor-induce proliferation and gene expression. Blood 1997; related tyrosine kinase(s) in human myeloid derived cells. EMBO 90: 1768–1776. J 1993; 12: 2681–2690. 252 Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis 270 Ohta T, Kinoshita T, Naito M, Nozaki T, Masutani M, Tsurouo D, Wang D, Brown M, Bodner S, Grosveld G, Ihle J. Stat 5a and T, Miyajima A. Requirement of the caspase-3/CPP32 protease Stat5b proteins have essential and nonessential, or redundant, cascade for apoptotic death following cytokine deprivation in roles in cytokine responses. Cell 1998; 93: 841–850. hematopoietic cells. J Biol Chem 1997; 272: 23111–23116. 253 Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida, 271 Suzuki J, Kaziro Y, Koide H. An activated mutant of R-Ras N, Kihimoto T, Akira S. Targeted disruption of the mouse Stat3 inhibits cell death caused by cytokine deprivation in BaF3 cells gene leads to embryonic lethality. Proc Natl Acad Sci USA 1997; in the presence of IGF-1. Oncogene 1997; 15: 1689–1697. 94: 3801–3804. 272 Ahmed N, Kansara M, Berridge MV. Acute regulation of glucose 254 Takeda K, Kamanaka M, Tanaka T, Kishimoto T, Akira S. transport in a monocyte–macrophage cell line: Glut-3 affinity for Impaired IL-13-mediated functions of macrophages in STAT6- glucose is enhanced during the respiratory burst. Biochem J deficient mice. J Immunol 1996; 157: 3220–3222. 1997; 327: 369–375. 255 Kaplan MH, Schindler U, Smiley ST, Grusby MJ. Stat6 is required 273 Craddock BL, Welham MJ. Interleukin-3 induces association of for mediating responses to IL-4 and for development of Th2 cells. the protein-tyrosine phosphatase SHP2 and phosphatidylinositol Immunity 1996; 4: 313–319. 3-kinase with a 100-kDa tyrosine-phosphorylated protein in 256 Takeda K, Tanaka T, Shi W, Matsumoto M, Minami M, Kashiwa- hematopoietic cells. J Biol Chem 1997; 272: 29281–29289. mura S, Nakanishi K, Yoshida N, Kishimoto T, Akira S. Essential 274 Anderson SM, Burton EA, Koch BL. Phosphorylation of Cbl fol- role of Stat6 in IL-4 signalling. Nature 1996; 380: 627–630. lowing stimulation with interleukin-3 and its association with 257 Matsumoto A, Masuhara M, Mitsui K, Yokouchi M, Ohtsubo M, Grb2, Fyn and phosphatidylinositol 3-kinase. J Biol Chem 1997; Misawa H, Miyajima A, Yoshimura A. CIS, a cytokine inducible 272: 739–745. SH2 protein, is a target of the JAK-STAT5 pathway and modulates 275 Minshall C, Arkins S, Freund GG, Kelly KW. Requirement for STAT5 activation. Blood 1997; 89: 3148–3154. phosphatidylinositol-3-kinase to protect hemopoietic progenitors 258 Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mit- against apoptosis depends upon the extracellular survival factor. sui K, Matsumoto A, Tanimura S, Ohtsubo M, Misawa H, Miya- J Immunol 1996; 156: 939–947. zaki T, Leonor N, Taniguchi T, Fujita T, Kanakura Y, Komiya S, 276 Kubota Y, Angelotti T, Niederfellner G, Herbst R, Ullrich A. Acti- Yoshimura A. A new protein containing an SH2 domain that vation of phosphatidlyinositol-3-kinase is necessary for differen- inhibits JAK kinases. Nature 1997; 387: 921–924. tiation of FDC-P1 cells following stimulation of type III receptor 259 Starr R, Wilson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, tyrosine kinases. Cell Growth Diff 1998; 9: 247–256. Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ. A 277 Ahmed NN, Grimes HL, Bellacosa A, Chan TO, Tsichlis PN. family of cytokine-inducible inhibitors of signalling. Nature Transduction of interleukin-2 anti-apoptotic and proliferative sig- 1997; 387: 917–921. nals via Akt protein kinase. Proc Natl Acad Sci USA 1997; 94: 260 Masuhara M, Sakamoto H, Suzuki R, Yasukawa H, Mitsui K, 3627–3632. Wakioka T, Tanimura S, Sasaki A, Misawa H, Yokouchi M, 278 Srivastava RK, Srivastava AR, Korsmeyer SJ, Nesterova M, Cho- Ohsubo M, Yoshimura A. Cloning and characterization of novel Chung YS, Longo DL. Involvement of microtubules in the regu- CIS family genes. Biochem Biophys Res Comm 1997; 239: lation of Bcl2 phosphorylation and apoptosis through cyclic 439–446. AMP-dependent protein kinase. Mol Cell Biol 1998; 18: 3509– 261 Sakamoto H, Yasukawa K, Masuhara M, Tanimura S, Sasaki A, 3517. Yuge K, Ohtusbo M, Ohtsuka A, Fujita T, Ohta T, Furukawa Y, 279 Scheid MP, Duronio V. Dissociation of cytokine-induced phos- Iwase S, Yamada H, Yosimura A. A Janus kinase inhibitor, JAB, phorylation of Bad and activation of PKB/akt: involvement of is an interferon-␥-inducible gene and confers resistance to inter- MEK upstream of Bad phosphorylation. Proc Natl Acad Sci USA ferons. Blood 1998; 92: 1668–1676. 1998; 95: 7439–7444. 262 Wang D, Stravopodis D, Teglund S, Kitazawa J, Ihle JN. Naturally 280 del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. occurring dominant negative variants of Stat5. Mol Cell Biol Interleukin-3-induced phosphorylation of BAD through the pro- 1996; 16: 6141–6148. tein kinase Akt. Science 1997; 278: 687–689. 263 Nielsen M, Kaltoft K, Nordahl M, Ropke C, Geisler C, Mustelin 281 Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterfield MD. T, Dobson P, Svejgaard A, Odum N. Constitutive activation of a Phosphoinositide-3-kinases: a conserved family of signal trans- slowly migrating isoform of Stat3 in mycosis fungoides: tyrphos- ducers. Trends Biochem Sci 1997; 22: 267–272. tin AG490 inhibits Stat 3 activation and growth of mycosis fungo- 282 Duronio V, Scheid MP, Ettinger S. Downstream signalling events ides tumor cell lines. Proc Natl Acad Sci USA 1997; 94: regulated by phosphatidylinositol 3-kinase activity. Cell Signal 6764–6749. 1998; 10: 233–239. 264 Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot 283 Pullen N, Thomas G. The modular phosphorylation and acti- Z, Leeder JS, Freedman M, Cohen A, Gazit A, Levitzki A, Roif- vation of P70S6K. FEBS Lett 1997; 410: 78–82. Review WL Blalock et al 1153 284 Downward J. Mechanisms and consequences of activation of viable motheaten mice have mutations in the haematopoietic protein kinase B/Akt. Curr Opin Cell Biol 1998; 10: 262–267. cell phosphatase gene. Nat Genet 1993; 4: 124–129. 285 Songyang Z, Baltimore D, Cantley LC, Kaplan DR, Franke TF. 305 Lydon NB, Mett H, Mueller M, Becker M, Cozens RM, Stover D, Interleukin-3-dependent survival by the Akt protein kinase. Proc Daniels D, Traxler P, Buchdunger E. A potent protein-tyrosine Natl Acad Sci USA 1997; 94: 11345–11350. kinase inhibitor which selectively blocks proliferation of epider- 286 Downward J. Ras signalling and apoptosis. Curr Opin Gen Dev mal growth factor receptor-expressing tumor cells in vitro and in 1988; 8: 49–54. vivo. Int J Cancer 1998; 76: 54–163. 287 Yanagihara Y, Basaki Y, Ikizawa K, Kajiwara K. Possible role of 306 Wang YEY, Zhang X, Lebwohl M, DeLeo V, Wei H. Inhibition nuclear factor-kappa B activity in germline C epsilon transcrip- of ultraviolet B (UVB)-induced c-fos and c-jun expression in vivo tion in a human Burkitt lymphoma B cell line. Cell Immunol by a tyrosine kinase inhibitor genistein. Carcinogenesis 1998; 19: 1997; 176: 66–74. 649–654. 288 Damen JE, Liu L, Rosten P, Humphries RK, Jefferson AB, Majerus 307 Couet J, Sargiacomo M, Lisanti MP. Interaction of a receptor tyro- PW, Krystal G. The 145-kDa protein induced to associate with sine kinase, EGF-R, with caveolins. Caveolin binding negatively Shc by multiple cytokines is an inositol tetraphosphate and phos- regulates tyrosine and serine/threonine kinase activities. J Biol phatidylinositol 3,4,5-trisphosphate 5-phosphatase. Proc Natl Chem 1997; 272: 30429–30438. Acad Sci USA 1996; 93: 1689–1693. 308 Fredenhagen A, Petersen F, Tintelnot-Blomley M, Rosel J, Mett 289 Gulbins E, Coggeshall KM, Baier G, Katzav S, Burn P, Altman H, Hug P. Semichliodinol A and B: inhibitors of HIV-1 protease A. Tyrosine kinase-stimulated guanine nucleotide exchange and EGF-R protein tyrosine kinase related to asterriquinones pro- activity of Vav in T cell activation. Science 1993; 260: 822–825. duced by the fungus Chrysosporium merdarium. J Antibiot 1997; 290 Gulbins E, Coggeshall KM, Langlet C, Baier G, Bonnefoy-Berard 50: 395–401. N, Burn P, Wittinghofer A, Katzav S, Altman A. Activation of Ras 309 Jones HE, Dutkowski CM, Barrow D, Harper ME, Waleling AE, in vitro and in intact fibroblasts by the Vav guanine nucleotide Nicholson RI. New EGF-R selective tyrosine kinase inhibitor exchange protein. Mol Cell Biol 1994; 14: 906–913. reveals variable growth responses in prostate carcinoma cell 291 Adams JM, Houston H, Allen J, Lints T, Harvey R. The hematopo- lines PC-3 and DU-145. Int J Cancer 1997; 71: 1010–1018. ietically expressed vav proto-oncogene shares homology with the 310 Cuadrado A, Bruder JT, Heidaran MA, App H, Rapp UR, Aaron- dbl GDP-GTP exchange factor, the bcr gene and a yeast gene son SA. H-Ras and Raf-1 cooperate in transformation of NIH3T3 (CDC24) involved in cytoskeletal organization. Oncogene 1992; fibroblasts. Oncogene 1993; 8: 2443–2448. 7: 611–618. 311 Saez R, Chan AM-L, Miki T, Aaronson SA. Oncogenic activation 292 Caponigro F, French RC, Kaye SB. Protein kinase C, a worthwhile of human R-Ras by point mutations analogous to those of proto- target for anticancer drugs. Anti-Cancer Drugs 1997; 8: 26–33. type H-Ras oncogenes. Oncogene 1994; 9: 2977–2982. 293 Das M, Stenmark KR, Ruff LJ, Dempsey EC. Selected isozymes 312 Wittinghofer A, Nassar N. How Ras-related proteins talk to their of PKC contribute to augmented growth of fetal and neonatal effectors. Trends Biochem Sci 1996; 21: 488–491. bovine PA adventitial fibroblasts. Am J Physiol 1997; 273: 313 Vojtek AB, Hollenberg SM, Cooper JA. Mammalian Ras interacts L1276–L1284. directly with the serine/threonine kinase Raf. Cell 1993; 74: 294 Harris TE, Persaud SJ, Jones PM. Atypical isoforms of PKC and 205–214. insulin secretion from pancreatic beta-cells: evidence using Go 314 Marshall M. Interactions between Ras and Raf: key regulatory 6976 and Ro 31–8220 as PKC inhibitors. Biochem Biophys Res proteins in cellular transformation. Mol Repro Dev 1995; 42: Comm 1996; 227: 672–676. 493–499. 295 Liu L, Damen JE, Ware MD, Krystal G. Interleukin-3 induces the 315 Brtva TR, Drugan JK, Ghosh S, Terrell RS, Cambell-Burk S, Bell association of the inositol 5-phosphatase SHIP with SHP2. J Biol RM, Der CJ. Two distinct Raf domains mediate interaction with Chem 1997; 272: 10998–11001. Ras. J Biol Chem 1995: 270: 9809–9812. 296 Damen JE, Liu L, Wakao H, Miyajima A, Rosten P, Jefferson AB, 316 Drugan JK, Khosravi-Far R, White MA, Der CJ, Sung YJ, Hwang Majerus PW, Krosl J, Humphries RK, Krystal G. The role of Y-H, Cambell SL. Ras interaction with two distinct binding erythropoietin receptor tyrosine phosphorylation in erythropoie- domains in Raf-1 may be required for Ras transformation. J Biol tin-induced proliferation. Leukemia 1995; 11 (Suppl 3): 423– Chem 1996; 271: 233–237. 425. 317 Cox AD, Der CJ. Farnesyltransferase inhibitors and cancer treat- 297 Giuriato S, Payrastre B, Drayer AL, Plantavid M, Woscholski R, ment: targeting simply Ras? Biochem Biophys Acta 1997; 1333: Parker P, Erneux C, Cap H. Tyrosine phosphorylation and relo- F51–F71. cation of SHIP are integrin-mediated in thrombin-stimulated 318 Hernandez-Alcoceba R, Saniger L, Campos J, Nunez MC, Kha- human blood platelets. J Biol Chem 1997; 272: 26857–26863. less F, Gallo MA, Espinosa A, Lacal JC. Choline kinase inhibitors 298 Lamkin TD, Walk SF, Liu L, Damen JE, Krystal G, Ravichandran as a novel approach for antiproliferative drug design. Oncogene KS. Shc interaction with Src homology 2 domain containing 1997; 15: 2289–2301. inositol phosphatase (SHIP) in vivo requires the Shc-phospho- 319 Byk G, Lelievere Y, Duchesne M, Clerc FF, Scherman D, Guitton tyrosine binding domain and two specific phosphotyrosines on JD. Synthesis and conformational analysis of peptide inhibitors SHIP. J Biol Chem 1997; 272: 10396–10401. of farnesyltransferase. Bioorg Med Chem 1997; 5: 115–124. 299 Pei D, Wang J, Walsh CT. Differential functions of the two Src 320 Sepp-Lorenzino L, Ma Z, Rands E, Kohl NE, Gibbs JB, Oliff A, homology 2 domains in protein tyrosine phosphatase SH-PTP1. Rosen N. A peptidomimetic inhibitor of farnesyl:protein transfer- Proc Natl Acad Sci USA 1996; 93: 1141–1145. ase blocks the anchorage-dependent and -independent growth 300 D’Ambrosio D, Fong DC, Cambier JC. The SHIP phosphatase of human tumor cell lines. Cancer Res 1995; 55: 5302–5309. becomes associated with Fc gammaRIIB1 and is tyrosine phos- 321 Heimbrook DC, Oliff A. Therapeutic intervention and signaling. phorylated during ‘negative’ signaling. Immunol Lett 1996; 54: Curr Opin Cell Biol 1998; 10: 284–288. 77–82. 322 Gibbs JB, Oliff A. The potential of farnesyltransferase inhibitors 301 Pei D, Lorenz U, Klingmuller U, Neel BG, Walsh CT. Intramol- as cancer chemotherapeutics. Ann Rev Pharmacol Toxicol 1997; ecular regulation of protein phosphatase SH-PTP1: a new func- 37: 143–166. tion for Src homology 2 domains. Biochemistry 1994; 33: 323 Khosravi-Far R, White MA, Westwick JK, Solski PA, Chrzanow- 15483–15493. ska-Wodnicka M, Van Aelst L, Wigler MH, Der CJ. Oncogenic 302 Helgason CD, Damen JE, Rosten P, Grewal R, Sorensen P, Chap- Ras activation of Raf/mitogen-activated protein kinase-inde- pel SM, Borowski A, Jirik F, Krystal G, Humphries RK. Targeted pendent pathways is sufficient to cause tumorigenic transform- disruption of SHIP leads to hemopoietic perturbations, lung path- ation. Mol Cell Biol 1996; 16: 3923–3933. ology, and a shortened life span. Genes Dev 1998; 12: 1610– 324 Morrison DK. Mechanisms regulating Raf-1 activity in signal 1620. transduction pathways. Mol Rep Dev 1995; 42: 507–514. 303 Yi T, Cleveland JL, Ihle JN. Identification of novel protein tyrosine 325 Heidecker G, Ko¨lch W, Morrison DK, Rapp UR. The role of Raf- phosphatases of hematopoietic cells by polymerase chain reac- 1 phosphorylation in signal transduction. Adv Cancer Res 1992; tion amplification. Blood 1991; 78: 2222–2228. 58: 53–73. 304 Tsui HW, Siminovitch KA, de Souza L, Tsui FI. Motheaten and 326 Mark GE, Mac IR, Digan ME, Ambrosio L, Perimon N. Drosophi- Review WL Blalock et al 1154 lia melanogaster homologs of the Raf oncogene. Mol Cell Biol signal-regulated kinases 1 and 2 (ERK1/ERK2) and STAT1 alpha. 1987; 7: 2134–2140. J Biol Chem 1996; 271: 1111–1117. 327 Pritchard CA, Bolin L, Slattery R, Murray RL, McMahon M. Post- 349 Toigoe T, O’Connor R, Santoli D, Reed JC. Interleukin-3 regu- natal lethality and neurological and gastrointestinal defects in lates the activity of the LYN protein-tyrosine kinase in myeloid- mice with targeted disruption of the A-Raf protein kinase. Curr committed leukemic cell lines. Blood 1992; 80: 617–624. Biol 1996; 6: 614–617. 350 Hanazono Y, Chiba S, Sasaki K, Mano H, Miyajima A, Arai K-I, 328 Storm SM, Brennscheidt U, Sithanandam G, Rapp UR. Raf onco- Yazaki Y, Hirai H. c-fps/fes protein-tyrosine kinase is implicated genes in carcinogenesis. Crit Rev Oncog 1990; 2: 1–8. in a signaling pathway triggered by granulocyte-macrophage col- 329 Wojnowski L, Zimmer AM, Beck TW, Hahn H, Bernal R, Rapp ony-stimulating factor and interleukin-3. EMBO J 1993; 12: UR, Zimmer A. Endothelial apoptosis in Braf-deficient mice. Nat 1641–1646. Genet 1997; 16: 293–297. 351 Yousefi S, Hoessli DC, Blaser K, Mills GB, Simon HU. Require- 330 Muszynski KW, Ruscetti FW, Heidecker G, Rapp U, Troppmair ment of Lyn and Syk tyrosine kinases for the prevention of J, Gooya JM, Keller JR. Raf-1 protein is required for growth factor- apoptosis by cytokines in human eosinophils. J Exp Med 1996; induced proliferation of hematopoietic cells. J Exp Med 1995; 183: 1407–1414. 181: 2189–2199. 352 Pazdrak K, Schreiber D, Forsythe P, Justement L, Alam R. The 331 Clark GJ, Drugan JK, Rossman KL, Carpenter JW, Rogers-Graham intracellular signal transduction mechanism of interleukin 5 in K, Fu H, Der C, Campbell SL. 14–3-3␨ negatively regulates Raf- eosinophils: the involvement of the lyn tyrosine kinase and the 1 activity by interactions with the Raf-1 cysteine-rich domain. J Ras-Raf-1-MEK-microtubule-associated protein kinase pathway. Biol Chem 1997; 272: 20990–20993. J Exp Med 1995; 181: 1827–1834. 332 Mott HR, Carpenter JW, Zhong S, Ghosh S, Bell RM, Campbell 353 Chao TS, Abe M, Hershenson MB, Gomes I, Rooner MR. Src SL. The solution structure of the Raf-1 cysteine-rich domain: a tyrosine kinase mediates stimulation of Raf-1 and mitogen-acti- novel Ras and phospholipid binding site. Proc Natl Acad Sci USA vated protein kinase by the tumor promoter thapsigargin. Mol 1996; 93: 8312–8317. Cell Biol 1997; 57: 3168–3173. 333 Warne PH, Viciana PR, Downward J. Direct interaction of Ras 354 Diaz B, Barnard D, Filson A, MacDonald S, King A, Marshall M. and the amino-terminal region of Raf-1 in vitro. Nature 1993; Phosphorylation of Raf-1 serine 338-serine 339 is an essential 364: 352–355. regulatory event for Ras dependent activation and biological sig- 334 Zhang X, Settleman J, Kyriakis JM, Takeuchi-Suzuki E, Elledge SJ, naling. Mol Cell Biol 1997; 17: 4509–4516. Marshall MS, Bruder JT, Rapp UR, Avruch J. Normal and oncog- 355 Pena LA, Fuks Z, Kolesnick R. Stress-induced apoptosis and the enic p21ras proteins bind to the amino-terminal regulatory sphingomyelin pathway. Biochem Pharmacol 1997; 53: 615– domain of c-Raf-1. Nature 1993; 364: 308–365. 621. 335 Hu CD, Kariya K, Tamada M, Akasaka K, Shirouza M, Yokoyama 356 Spiegel S, Foster D, Kolesnick R. Signal transduction through S, Kataoka T. Cysteine-rich region of Raf-1 interacts with acti- lipid second messenger. Curr Opin Cell Biol 1996; 8: 159–167. vator domain of post-translationally modified Ha-Ras. J Biol 357 Canagarajah BJ, Khokhlatchev A, Cobb MH, Goldsmith EJ. Acti- Chem 1995; 270: 30274–30277. vation mechanism of the MAP kinase ERK2 by dual phosphoryl- 336 Fabian JR, Daar IO, Morrison DK. Critical tyrosine residues regu- ation. Cell 1997; 90: 859–869. late the enzymatic and biological activity of Raf-1 kinase. Mol 358 Marshall CJ. Specificity of receptor tyrosine kinase signaling: Cell Biol 1993; 13: 7170–7179. transient vs sustained extracellular signal-regulated kinase acti- 337 Marais R, Light Y, Paterson H, Marshall CJ. Ras recruits Raf-1 to vation. Cell 1995; 80: 179–185. the plasma membrane for activation by tyrosine phosphorylation. 359 Bruder JT, Heidecker G, Rapp UR. Serum-, TPA-, and Ras- EMBO J 1995; 14: 101–110. induced expression from AP-1/Ets-driven promoters requires Raf- 338 Cai H, Smola U, Wixler V, Eisenmann-Tappe I, Diaz-Meco MT, 1 kinase. Genes Dev 1992; 6: 545–556. Moscat J, Rapp U, Cooper GM. Role of diacylglycerol-regulated 360 Griffin JD, Ranbaldi A, Vellenga E, Young DC, Ostapovicz D, protein kinase C isotypes in growth factor activation of the Raf- Cannistra SA. Secretion of interleukin-1 by acute myeloblastic 1 protein kinase. Mol Cell Biol 1997; 17: 732–741. leukemia cells in vitro induces endothelial cells to secrete col- 339 Xia M, Mukhopadhyay NK, Inhorn RC, Barber DL, Rose PE, Lee ony-stimulating factors. Blood 1987; 70: 1218–1221. RS, Narsimhan RP, D’Andrea AD, Griffin JD, Roberts TM. The 361 Janknecht R, Ernst WH, Pingould V, Nordheim A. Activation of cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a TCF Elk-1 by MAP kinases. EMBO J 1993; 12: 5097–5104. p21ras-dependent manner. Proc Natl Acad Sci USA 1996; 93: 362 Marais R, Wynne J, Treisman R. The SRF accessory protein Elk- 11681–11686. 1 contains a growth factor-regulated transcriptional activation 340 Marshall CJ. Raf gets it together. Nature 1996; 383: 127–128. domain. Cell 1993; 73: 381–393. 341 Farrar MA, Alberola-Ila J, Perlmutter RM. Activation of the Raf- 363 Miltenberger RJ, Farnham PJ, Smith DE, Stommel JM, Cornwell 1 kinase cascade by coumermycin induced dimerization. Nature MM. v-Raf activates transcription of growth-responsive pro- 1996; 383: 178–181. moters via GC-rich sequences that bind the transcription factor 342 Luo Z, Tzivion G, Belshaw PJ, Vavvas D, Marshall M, Avruch J. Sp1. Cell Growth Diff 1995; 549: 549–556. Oligomerization activates c-Raf-1 through a Ras-dependent 364 Xing J, Ginty DD, Greenberg ME. Coupling of the Ras-MAPK mechanism. Nature 1996; 383: 181–185. pathway to gene activation by Rsk2, a growth factor regulated 343 Daum G, Eisenmann-Tappe I, Fries H-W, Troppmair J, Rapp UR. CREB kinase. Science 1996; 273: 959–963. The ins and outs of Raf kinases. TIBS 1994; 19: 474–479. 365 Kanno T, Siebenlist U. Activation of nuclear factor-␬B via T cell 344 Yao B, Zhang Y, Delikat S, Mathias S, Basu S, Kolesnick R. Phos- receptor requires a Raf kinase and Ca2+ influx. J Immunol 1996; phorylation of Raf by ceramide-activated protein kinase. Nature 157: 5277–5283. 1995; 378: 307–310. 366 Fisher TL, Blenis J. Evidence for two catalytically active kinase 345 Zhang Y, Yao B, Delikat S, Bayoumy S, Lin XH, Basu S, McGin- domains in pp90rsk. Mol Cell Biol 1996; 16: 1212–1219. ley M, Chan-Hui PY, Lichenstein H, Kolesnick R. Kinase sup- 367 Nilsson M, Ford J, Bohm S, Toftgard R. Characterization of a pressor of Ras is ceramide-activated protein kinase. Cell 1997; nuclear factor that binds juxtaposed with ATF3/Jun on a com- 89: 63–72. posite response element specifically mediating induced tran- 346 Kolch W, Heidecker G, Kochs G, Hummel R, Vahidi H, Mischak scription in response to an epidermal growth factor/Ras/Raf sig- H, Finkenzeller G, Marme D, Rapp UR. Protein kinase C acti- naling pathway. Cell Growth Diff 1997; 8: 913–920. vates Raf-1 by direct phosphorylation. Nature 1993; 364: 249– 368 Xu S, Robbins D, Frost J, Dang A, Lange-Carter C, Cobb MH. 252. MEKK1 phosphorylates MEK1 and MEK2 but does not cause acti- 347 Marais R, Light Y, Mason C, Paterson H, Olson MF, Marshall CJ. vation of mitogen-activated protein kinase. Proc Natl Acad Sci Requirement of Ras-GTP-Raf complexes for activation of Raf-1 USA 1995; 92: 6808–6812. by protein kinase C. Science 1998; 280: 109–112. 369 Davis RJ, Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulev- 348 Singh K, Balligand JL, Fischer TA, Smith TW, Kelly RA. Regu- itch RJ. Independent human MAP kinase signal transduction lation of cytokine-inducible nitric oxide synthase in cardiac myo- pathways defined by MEK and MKK isoforms. Science 1995; cytes and microvascular endothelial cells. Role of extracellular 267: 682–685. Review WL Blalock et al 1155 370 Davis RJ. Transcriptional regulation by MAP kinases. Mol Rep the level of Raf activity with arrest mediated by p21Cip. Mol Cell Dev 1995; 42: 459–467. Biol 1997; 17: 5598–5611. 371 Johnson NL, Gardner AM, Diener KM, Lange-Carter CA, Gleavy 392 Dubois T, Rommel C, Howell S, Steinhussen U, Soneji Y, Mor- J, Jarpe MB, Minden A, Karin M, Zon LI, Johnson GL. Signal rice N, Moelling K, Aitken A. 14–3-3 is phosphorylated by casein transduction pathways regulated by mitogen-activated/ kinase I on residue 233. Phosphorylation at this site in vivo regu- extracellular response kinase kinase kinase induce cell death. J lates Raf/14–3-3 interaction. J Biol Chem 1997; 272: 28882– Biol Chem 1996; 271: 3229–3237. 28888. 372 Dudley DT, Pung L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic 393 Hsu SY, Kaipia A, Zhu L, Hsueh AJ. Interference of BAD (Bcl- inhibitor of the mitogen-activated protein kinase cascade. Proc xL/Bcl-2 associated death promoter)-induced apoptosis in mam- Natl Acad Sci USA 1995; 92: 7686–7689. malian cells by 14–3-3 isoforms and P11. Mol Endo 1997; 11: 373 Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. 1858–1867. PD098059 is a specific inhibitor of the activation of mitogen- 394 Zhang L, Wang H, Liu D, Liddington R, Fu H. Raf-1 kinase and activated protein kinase kinase in vitro and in vivo. J Biol Chem exoenzyme S interact with 14–3-3zeta through a common site 1995; 270: 27489–27494. involving lysine 49. J Biol Chem 1997; 272: 13717–13724. 374 Samuels ML, Weber MJ, Bishop JM, McMahon M. Conditional 395 Xing H, Kornfeld K, Muslin AJ. The protein kinase KSR interacts transformation of cells and rapid activation of the mitogen-acti- with 14–3-3 protein and Raf. Curr Biol 1997; 7: 294–300. vated protein kinase cascade by an estradiol-dependent human 396 Dubois T, Howell S, Amess B, Kerai P, Learmonth M, Madrazo Raf-1 protein kinase. Mol Cell Biol 1993; 13: 6241–6252. J, Chaudhri M, Rittinger K, Scarbel M, Soneji Y, Aitken A. Struc- 375 Pritchard CA, Samuels ML, Bosch E, McMahon M. Conditionally ture and sites of phosphorylation of 14-3-3 protein: role in coord- oncogenic forms of the A-Raf and B-Raf protein kinases display inating signal transduction pathways. J Prot Chem 1997; 16: different biological and biochemical properties in NIH-3T3 cells. 513–522. Mol Cell Biol 1995; 15: 9430–9442. 397 Yaffe MB, Rittinger K, Volina S, Caron PR, Aitken A, Leffers H, 376 Pritchard C, McMahon M. Raf revealed in life-or-death decisions. Gamblin SJ, Smerdon SJ, Cantley LC. The structural basis for 14– Nat Genet 1997; 16: 214–215. 3-3: phosphopeptide binding specificity. Cell 1997; 91: 961– 377 Bosch E, Cherwinski H, Peterson D, McMahon M. Mutations of 971. critical amino acids affect the biological and biochemical 398 Muslin AJ, Tanner JW, Allen PM, Shaw AS. Interaction of 14–3- properties of oncogenic A-Raf and Raf-1. Oncogene 1997; 11: 3 with signaling proteins is mediated by the recognition of phos- 1021–1034. phoserine. Cell 1996; 84: 889–897. 378 McCubrey JA, Steelman LS, Hoyle PA, Blalock WL, Weinstein- 399 Rommel C, Radziwill G, Lovric J, Noeldeke J, Heinicke T, Jones Oppenheimer CR, Franklin RA, Cherwinski H, Bosch E, McMa- D, Aitken A, Moelling K. Activated Ras displaces 14–3-3 protein hon M. Differential abilities of activated Raf oncoproteins to from the amino terminus of c-Raf-1. Oncogene 1996; 12: abrogate cytokine dependency, prevent apoptosis and induce 609–619. autocrine growth factor synthesis in human hematopoietic cells. 400 Freed E, Symons M, Macdonald SG, McCormick F, Ruggieri R. Leukemia 1998; 12: 1903–1929. Binding of 14–3-3 proteins to the protein kinase Raf and effects 379 Lee JE, Beck TW, Wojnowski L, Rapp UR. Regulation of A-Raf on its activation. Science 1994; 265: 1713–1716. expression. Oncogene 1996; 12: 1669–1677. 401 Fu H, Xia K, Pallas DC, Cui C, Conroy K, Narsimhan RP, Mamon 380 Stephens RM, Sithanandam G, Copelan TD, Kaplan DR, Rapp H, Collier RJ, Roberts TM. Interaction of the protein kinase Raf- UR, Morrison DK. 95-kilodalton B-Raf serine/threonine kinase: 1 with 14–3-3 proteins. Science 1994; 266: 126–129. identification of the protein and its major autophosphorylation 402 Thorson JA, Yu LWK, Hsu AL, Shih N-Y, Graves PR, Tanner JW, site. Mol Cell Biol 1992; 12: 3733–3742. Allen PM, Piwnica-Worms H, Shaw AS. 14–3-3 proteins are 381 Heidecker G, Huleihel M, Cleveland JL, Kolch W, Beck TW, required for maintenance of Raf-1 phosphorylation and kinase Lloyd P, Pawson T, Rapp UR. Mutational activation of c-Raf-1 activity. Mol Cell Biol 1998; 18: 5229–5238. and definition of the minimal transforming sequence. Mol Cell 403 Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukas- Biol 1990; 10: 2503–2512. awa K, Vande Woude GF, Ahn NG. Transformation of mam- 382 Ikawa S, Fukui M, Ueyama Y, Tamaoki N, Yamamoto T, malian cells by constitutively active MAP kinase kinase. Science Toyoshia K. B-Raf, a new member of the Raf family, is activated 1994; 265: 966–970. by DNA rearrangement. Mol Cell Biol 1988; 8: 2651–2654. 404 Weber MJ, Catling AD, Schaeffer HJ, Reuter CW, Reddy GR. A 383 Reuter CW, Catling AD, Jelinek T, Weber MJ. Biochemical analy- proline-rich sequence unique to MEK1 and MEK2 is required for sis of MEK activation in NIH-3T3 fibroblasts. Identification of B- Raf binding and regulates MEK function. Mol Cell Biol 1995; 15: Raf and other activators. J Biol Chem 1995; 270: 7644–7655. 5214–5225. 384 Fukui M, Yamamoto T, Kawai S, Mitsunobu F, Totoshima K. Mol- 405 Huang W, Kessler DS, Erikson RL. Biochemical and biological ecular cloning and characterization of an activated human c-Raf- analysis of MEK1 phosphorylation site mutants. Mol Biol Cell 1 gene. Mol Cell Biol 1987; 7: 1776–1781. 1995; 15: 237–245. 385 Williams JG, Roberts TM. Signal transduction pathways involving 406 Brunet A, Pages G, Pouyssegur J. Constitutively active mutants the Raf proto-oncogene. Cancer Metast Rev 1994; 13: 105–116. of MAP kinase kinase (MEK1) induce growth factor relaxation 386 Catling AD, Reuter CWM, Cox ME, Parsons SJ, Weber MJ. Partial and oncogenicity when expressed in fibroblasts. Oncogene purification of a mitogen-activated protein kinase kinase acti- 1994; 9: 3379–3387. vator from bovine brain. J Biol Chem 1994; 269: 30014–30021. 407 Krebs EG, Sege R, Seger D, Reszka AA, Munar ES, Eldar-Finkel- 387 Jaiswal RK, Moodie SA, Wolfman A, Landreth GE. The mitogen- man H, Dobrowolska G, Jensen AM, Campbell JS, Fischer EH. activated protein kinase cascade is activated by B-Raf in response Overexpression of mitogen-activated protein kinase kinase to nerve growth factor through interaction with p21Ras. Mol Cell (MAPKK) and its mutants in NIH 3T3 cells. Evidence that MAPKK Biol 1994; 14: 6944–6953. involvement in cellular proliferation is regulated by phosphoryl- 388 Lee JE, Beck TW, Brennscheidt U, DeGennaro LJ, Rapp UR. The ation of serine residues in its kinase subdomains VII and VIII. J complete sequence and promoter activity of the human A-Raf-1 Biol Chem 1994; 269: 25699–25709. gene (ARaf1). Genomics 1994; 20: 43–45. 408 Alessandrini A, Greulich H, Huang W, Erikson RL. MEK1 phos- 389 Ravi RK, Weber E, McMahon M, Williams JR, Baylin S, Mal A, phorylation site mutants activate Raf-1 in NIH 3T3 cells. J Biol Harter ML, Dillehay LE, Clauddio PP, Giordano A, Nelkin BD, Chem 1996; 271: 31612–31618. Mabry M. Activated Raf-1 causes cell cycle arrest in small cell 409 Bottorff D, Stang S, Agellon S, Stone JC. Ras signaling is abnormal lung cancer cells. J Clin Invest 1998; 101: 153–159. in c-Raf1 MEK1 double mutant. Mol Cell Biol 1995; 15: 390 Lloyd AC, Obermuller F, Staddon S, Barth CF, McMahon, Land 5113–5122. H. Cooperating oncogenes converge to regulate cyclin/Cdk com- 410 Okazaki K, Sagata N. MAP kinase activation is essential for plexes. Genes Dev 1997; 11: 663–677. oncogenic transformation of NIH3T3 cells by Mos. Oncogene 391 Woods D, Parry D, Cherwinski H, Bosch E, Lees E, McMahon 1995; 10: 1149–1157. M. Raf-induced proliferation or cell cycle arrest is determined by 411 Pham CD, Arlinghaus RB, Zheng CF, Guan KL, Singh B. Charac- Review WL Blalock et al 1156 terization of MEK1 phosphorylation by the v-Mos protein. Onco- of Raf oncogenes during lung carcinogenesis. Lung Cancer 1988; gene 1995; 10: 1683–1688. 4: 162–167. 412 Rapp UR, Troppmair J, Bruder JT, Munoz H, Lloyd PA. Kyriakis 432 Reynolds SH, Anna CK, Brown KC, Wiest JS, Beattie EJ, Pero RW, J, Banerjee P, Avruch J. Mitogen-activated protein Iglehart JD, Anderson MW. Activated protooncogenes in human kinase/extracellular signal-regulated protein kinase activation by lung tumors from smokers. Proc Natl Acad Sci USA 1991; 88: oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate 1085–1089. requires Raf and is necessary for transformation. J Biol Chem 433 Hubuchi T, Kinoshita H, Yamada H, Kakehi Y, Ogawa O, Wu 1994; 269: 7030–7035. W-J, Takahashi R, Sugiyama T, Yoshida O. Oncogene amplifi- 413 Kranitz LM, Burns LA, Sutor SL, Blenis J, Abraham RT. Interleu- cation in urothelial cancers with p53 gene mutation or MDM2 kin-2 triggers a novel phosphotidylinositol 3-kinase-dependent amplification. JNCI 1994; 86: 1331–1335. MEK activation. Mol Cell Biol 1995; 15: 3049–3057. 434 Nickell-Brady C, Hahn FF, Finch GI, Belinsky SA. Analysis of K- 414 Berra E, Diaz-Meco MT, Lozano J, Frutos S, Municio MM, San- Raf, p53 and c-Raf-1 mutations in beryllium-induced rat lung chez P, Sanz L. Evidence for a role of MEK and MAPK during tumors. Carcinogenesis 1994; 15: 257–262. signal transduction by protein kinase C zeta. EMBO J 1995; 14: 435 Cadrian U, You M, Goodrow T, Maronpot RR, Reynolds SH, 6157–6163. Anderson MW. Activation of protooncogenes in spontaneously 415 Marquardt B, Frith D, Stabel S. Signalling from TPA to MAP kin- occurring non-liver tumors from C57BL/6 X C3H F1 mice. Cancer ase requires protein kinase C, Raf and MEK: reconstitution of the Res 1991; 51: 1148–1153. signalling pathway in vitro. Oncogene 1994; 9: 3213–3218. 436 Jung M, Notarrio V, Dritschilo A. Mutations in the p53 gene in 416 Schaeffer HJ, Catling AD, Eblen ST, Collier LS, Krauss A, Weber radiation-sensitive and resistant human squamous carcinoma MJ. MP1: a MEK binding partner that enhances enzymatic acti- cells. Cancer Res 1992; 52: 6390–6393. vation of the MAP kinase cascade. Science 1998; 281: 1668– 437 Harris P, Morton CC, Guglielmi P, Li F, Kelly K, Latt SA. Mapping 1671. by chromosome sorting of several gene probes, including c-myc, 417 Whitmarsh AJ, Cavanagh J, Tournier C, Yasuda J, Davis RJ. A to the derivative chromosomes of a 3;8 translocation associated mammalian scaffolding complex that selectively mediates MAP with familial renal cancer. Cytometry 1986; 7: 589–594. kinase activation. Science 1998; 281: 1671–1674. 438 Pfeifer AMA, Jones RT, Bowden PE, Mann D, Spillare E, Klein- 418 Neubauer A, Greenberg P, Negrin R, Ginzton N, Liu E. Mutations Szanto AJP, Trump BF, Harris CC. Human bronchial epithelial in the Ras protooncogenes in patients with myelodysplastic syn- cells transformed by the c-Raf-1 and c-Myc protooncogenes dromes. Leukemia 1994; 8: 638–641. induce multidifferentiated carcinomas in nude mice: a model for 419 Buschle M, Janssen JW, Drexler H, Lyons B, Bartram CR. Evi- lung carcinogenesis. Cancer Res 1991; 51: 3793–3801. dence for a pluripotent stem cell origin of idiopathic myelo- 439 Eggstein S, Manthey G, Hirsch T, Bass F, Spech BUV, Farthmann fibrosis: clonal analysis of a case characterized by a N-ras gene EH. Raf-1 kinase, epidermal growth factor receptors, and mutant mutation. Leukemia 1988; 2: 658–660. Ras proteins in colonic carcinomas. Dig Dis Sci 1996; 41: 420 Flotho C, Valcamonica S, Mach-Pascual S, Schmahl G, Corral L, 1069–1075. Ritterbach J, Hasle H, Arico` M, Biondi A, Niemeyer CM. Ras 440 Callans LS, Naama H, Khandelwal M, Plotkin R, Jardines L. Raf-1 mutations and clonality analysis in children with juvenile myelo- protein expression in human breast cancer cells. Ann Surg Oncol monocytic leukemia (JMML). Leukemia 1999; 13: 32–37. 1995; 2: 38–42. 421 Kalra R, Paderanga DC, Olson K, Shannon KM. Genetic analysis 441 Okuda K, Matulonis U, Salgia R, Kavakura U, Druker B, Griffin consistent with the hypothesis that NF1 limits myeloid cell JD. Factor-independence of human myeloid leukemia cell lines growth through p21 ras. Blood 1994; 84: 3435–3439. is associated with increased phosphorylation of the protooncog- 422 Cline MJ, Jat PS, Foti A. Molecular mechanisms in the evolution ene Raf-1. Exp Hematol 1994; 22: 1111–1117. of chronic myelocytic leukemia. Leuk Lymphoma 1992; 7: 442 Schmidt CA, Oettle H, Ludwig WD, Serke S, Pawlaczyk-Peter B, Wilborn F, Binder LT, Huhn D, Siegert W. Overexpression of the 283–287. Raf-1 proto-oncogene in human myeloid leukemia. Leukemia 423 Cutler RE Jr, Morrison DK. Mammalian Raf-1 is activated by Res 1994; 18: 409–413. mutations that restore Raf signaling in Drosophila. EMBO J 1997; 443 Kolch W, Heidecker G, Lloyd P, Rapp U. Raf-1 protein kinase 16: 1953–1960. is required for growth of induced NIH-3T3 cells. Nature 1991; 424 Storm SM, Rapp UR. Oncogene activation: c-Raf-1 gene 349: 426–428. mutations in experimental and naturally occurring tumors. Tox- 444 Quresh SA, Joseph CK, Hendrickson M, Song J, Gupta R, Bruder icol Lett 1993; 67: 201–210. J, Rapp U, Foster DA. A dominant negative Raf-1 mutant prevents 425 Miyamoto K, Matsuoka M, Tomita N, Suzuki C, Sato Y, Ishi A, v-Src induced transformation. Biochem Biophys Res Comm Kitajima K-I, Nonaka H, Kondo, T, Hiraki Y, Kobayashi N. A B- 1993; 192: 969–975. cell line having chromosome 14 aberration at break band q11 445 Rapp UR, Troppmair J, Carrol M, May WS. Role of Raf-1 protein derived from an adult T-cell leukemia patient. Jpn J Cancer Res kinase in IL-3 and GM-CSF-mediated signal transduction. Curr 1988; 79: 12–16. Top Microbiol Immunol 1990; 166: 129–139. 426 Ishikawa F, Takaku F, Nagao M, Sugimura T. Rat c-Raf oncogene 446 Cleveland JL, Troppmair J, Puckham G, Askew DS, Lloyd P, Ihle activation by a rearrangement that produces a fused protein. Mol JN, Rapp UR. v-Raf suppresses apoptosis and promotes growth Cell Biol 1987; 7: 1226–1232. of interleukin-3-dependent myeloid cells. Oncogene 1994; 9: 427 Miyamoto K, Tomita N, Ishii A, Miyamoto N, Nonaka M, Kondo 2227–2234. T, Sugihara T, Yawata Y, Tada S, Tsubota T, Kitajima K-I, Kimura 447 Thiagalingam A, De Bustros A, Borges M, Jasti R, Compton D, I. Specific abnormalities of chromosome 14 in patients with Diamond L, Mabry M, Ball DW, Baylin SB, Nelkin BD. RREB- acute type of adult T cell leukemia/lymphoma. Int J Cancer 1987; 1, a novel zinc finger protein, is involved in the differentiation 40: 461–468. response to Ras in human medullary thyroid carcinomas. Mol 428 Ingvarsson S, Asker C, Szpirer J, Levan G, Klein G. Rat c-Raf Cell Biol 1996; 16: 5335–5345. oncogene is located on chromosome 4 and may be activated by 448 Benn J, Su F, Doria M, Schneider RJ. Hepatitis B virus HBx pro- sequences from chromosome 13. Somatic Cell Mol Gen 1987; tein induces transcription factor AP-1 by activation of extracellu- 14: 401–405. lar signal-regulated and c-Jun N-terminal mitogen-activated pro- 429 Chung GTY, Huang DP, Wai Lo K, Chan MKM, Wong FWS. tein kinases. J Virol 1996; 70: 4978–4985. Genetic lesion in the carcinogenesis of cervical cancer. 449 Doria M, Klein N, Lucito R, Schneider RJ. The hepatitis B virus Anticancer Res 1992; 12: 1485–1490. HBx protein is a dual specificity cytoplasmic activator of Ras and 430 Sithanandam G, Dean M, Brennscheidt U, Beck T, Gazdar A, nuclear activator of transcription factors. EMBO J 1995; 14: Minna JD, Brauch H, Abar B, Rapp UR. Loss of heterozygosity 4747–4757. at the c-Raf locus in small cell lung carcinoma. Oncogene 1989; 450 Ito T, Sasaki Y, Wands JR. Overexpression of human insulin 4: 451–455. receptor substrate 1 induces cellular transformation with acti- 431 Rapp UR, Huleihel M, Pawson T, Linnoila I, Minna JD, Hei- vation of mitogen-activated protein kinases. Mol Cell Biol 1996; decker G, Cleveland JL, Beck T, Forchhammer J, Storm SM. Role 16: 943–951. Review WL Blalock et al 1157 451 Oka H, Chatan Y, Hoshino R, Ogawa O, Kakehi Y, Terachi T, 470 Han J, Lee JD, Bibbs L, Ulevitch RJ. A MAP kinase targeted by Okada Y, Kawaichi M, Kohno M, Yoshida O. Constitutive acti- endotoxin and hyperosmolarity in mammalian cells. Science vation of mitogen-activated protein (MAP) kinases in human 1994; 265: 808–811. renal cell carcinoma. Cancer Res 1995; 55: 4182–4187. 471 Meier R, Rous J, Cuenda A, Nebreda AR, Cohen P. Cellular 452 Silvaraman VS, Wang H, Nuovo GJ, Malbon CC. Hyperexpres- stresses and cytokines activate multiple mitogen-activated-pro- sion of mitogen-activated protein kinase in human breast cancer. tein kinase kinase homologues in PC12 and KB cells. Eur J J Clin Invest 1997; 99: 1478–1483. Biochem 1996; 236: 796–805. 453 Patel BK, Ray S, Whiteside TL, Kasid U. Correlation of constitut- 472 Hibi M, Lin A, Smeal T, Minden A, Karin M. Identification of an ive activation of Raf-1 with morphological transformation and oncoprotein- and UV-responsive protein kinase that binds and abrogation of tyrosine phosphorylation of distinct sets of proteins potentiates the c-Jun activation domain. Genes Dev 1993; 7: in human squamous carcinoma cells. Mol Carcinogen 1991; 8: 2135–2148. 1–6. 473 Lange-Carter CA, Johnson GL. Ras-dependent growth factor 454 Riva C, Lavieille JP, Reyt E, Brambilla E, Lunardi J, Brambilla C. regulation of MEK kinase in PC12 cells. Science 1994; 265: Differential c-myc,c-jun,c-Raf, and p53 expression in squamous 1458–1461. cell carcinoma of the head and neck: implication in drug and 474 Marais R, Light Y, Paterson HF, Marshall CJ. Ras recruits Raf-1 to radioresistance. Eur J Cancer 1995; 31B: 384–391. the plasma membrane for activation by tyrosine phosphorylation. 455 Faris M, Ensoli B, Stahl N, Yancopoulos G, Nguyen A, Wang S, EMBO J 1995; 14: 3136–3145. Nel AE. Differential activation of the extracellular signal-regu- 475 Wang XZ, Ron D. Stress-induced phosphorylation and activation lated kinase, Jun kinase and Janus kinase-Stat pathways by of CHOP (GADD153) by p38 MAP kinase. Science 1996; 272: oncostatin M and basic fibroblast growth factor in AIDS-derived 1347–1349. Kaposi’s sarcoma cells. AIDS 1996; 10: 369–378. 476 Schwenger P, Alpert D, Skolnik EY, Vilcek J. Activation of p38 456 Silberman S, Janulis M, Schultz RM. Characterization of down- mitogen-activated protein kinase by sodium salicylate leads to stream Ras signals that induce alternative protease-dependent inhibition of tumor necrosis factor-induced I␬-B alpha phos- invasive phenotypes. J Biol Chem 1997; 272: 5927–5935. phorylation and degradation. Mol Cell Biol 1998; 18: 78–84. 457 Canman CE, Gilmer TM, Coutts SB, Kastan MB. Growth factor 477 Morley SJ, McKendrick L. Involvement of stress-activated protein modulation of p53-mediated growth arrest vs apoptosis. Genes kinase and p38/RK mitogen-activate protein kinase signaling Dev 1995; 9: 600–611. pathways in the enhanced phosphorylation of initiation factor 4E 458 Pirollo KF, Hao Z, Rait A, Ho CW, Chang EH. Evidence support- in NIH 3T3 cells. J Biol Chem 1997; 272: 17887–17893. ing a signal transduction pathway leading to the radiation-resist- 478 Gould GW, Cuenda A, Thompson FJ, Cohen P. The activation ant phenotype in human tumor cells. Biochem Biophys Res of distinct mitogen-activated protein kinase cascades is required Comm 1997; 230: 196–201. for the stimulation of 2-deoxyglucose uptake by interleukin-1 459 Simon C, Juarez J, Nicolson GL, Boyd D. Effect of PD 098059, and insulin-like growth factor-1 in KB cells. Biochem J 1995; a specific inhibitor of mitogen-activated protein kinase kinase, 311: 735–738. on urokinase expression and in vitro invasion. Cancer Res 1996; 479 Guan Z, Buckmn SY, Pentland AP, Templeton DJ, Morrison AR. 56: 5369–5374. Induction of cyclooxygenase-2 by the activated 460 Loda M, Capodieci P, Mishra R, Yao H, Corless C, Grigioni W, MEKK1→SEK1/MKK4→p38 mitogen-activated protein kinase Wang Y, Magi-Galluzzi C, Stork PJ. Expression of mitogen-acti- pathway. J Biol Chem 1998; 273: 12901–12908. vated protein kinase phosphatase-1 in the early phases of human 480 McIlroy J, Chen D, Wjasow C, Michaeli T, Backer JM. Specific epithelial carcinogenesis. Am J Pathol 1996; 149: 1553–1564. activation of p85-p110 phosphatidylinositol 3′-kinase stimulates 461 Magi-Galluzzi C, Mishra R, Fiorentino M, Montironi R, Yao H, DNA synthesis by Ras- and p70 S6 kinase-dependent pathways. Capodieci P, Wishnow K, Kaplan I, Stork PJ, Loda M. Mitogen- Mol Cell Biol 1997; 17: 248–255. activated protein kinase phosphatase 1 is overexpressed in pros- 481 Rodriguez-Viciana P, Marte BM, Warne PH, Downward J. Phos- tate cancers and is inversely related to apoptosis. Lab Invest phatidylinositol 3′ kinase: one of the effectors of Ras. Phil Trans 1997; 76: 37–51. Roy Soc London Series B: Biol Sci 1996; 351: 225–231. 462 Scimeca JC, Servant MJ, Dyer JO, Meloche S. Essential role of 482 Edelmann HM, Kuhne C, Petritsch C, Ballou LM. Cell cycle regu- calcium in the regulation of MAP kinase phosphatase-1 lation of p70 S6 kinase and p42/p44 mitogen-activated protein expression. Oncogene 1997; 15: 717–725. kinases in Swiss mouse 3T3 fibroblasts. J Biol Chem 1996; 271: 463 Bennett AM, Tonks NK. Regulation of distinct stages of skeletal 963–971. muscle differentiation by mitogen-activated protein kinases. 483 Terada N, Franklin RA, Lucas JJ, Blenis J, Gelfand EW. Failure Science 1997; 278: 1288–1291. of rapamycin to block proliferation once resting cells have 464 Krautwald S, Buscher D, Dent P, Ruthenberg K, Baccarini M. entered the cell cycle despite inactivation of p70 S6 kinase. J Suppression of growth factor-mediated MAP kinase activation by Biol Chem 1993; 268: 12062–12068. v-Raf in macrophages: a putative role for the MKP-1 phospha- 484 Shi Y, Frankel A, Radvanyi LG, Penn LZ, Miller RG, Mills GB. tase. Oncogene 1995; 10: 1187–1192. Rapamycin enhances apoptosis and increases sensitivity to cis- 465 Yan M, Dai T, Deak JC, Kyriakis JM, Zon LI, Woodgett JR, Tem- platin in vitro. Cancer Res 1995; 55: 1982–1988. pleton DJ. Activation of stress-activated protein kinase by MEKK1 485 Blenis J, Grammer T. Evidence for MEK-independent pathways phosphorylation of its activator SEK1. Nature 1994; 372: 798– regulating the prolonged activation of the ERK-MAP kinases. 800. Oncogene 1997; 14: 1635–1642. 466 Lin A, Minden A, Martinetto H, Claret FX, Lange-Carter C, Mer- 486 Gelfand EW, Ishizuka T, Oshiba A, Sakata N, Terada N, Johnson curio F, Johnson GL, Karin M. Identification of a dual specificity GL. Aggregation of the Fc⑀RI on mast cells stimulates c-Jun kinase that activates the Jun kinases and p38-Mpk2. Science amino-terminal kinase activity. J Biol Chem 1996; 271: 1995; 268: 286–290. 12762–12766. 467 Karin M, Minden A, Lin A, McMahon M, Lange-Carter C, Deri- 487 Liao DF, Duff JL, Daum G, Pelech SL, Berk BC. Angiotensin II jard B, Davis RJ, Johnson GL. Differential activation of ERK and stimulates MAP kinase kinase kinase activity in vascular smooth JNK mitogen-activated protein kinases by Raf-1 and MEKK. muscle cells, role of Raf. Circ Res 1993; 79: 1007–1014. Science 1994; 266: 1719–1723. 488 Calvo V, Wood M, Gjertson C, Vik T, Bierer BE. Activation of 468 Raitano AB, Halpern JR, Hambuch TM, Sawyers CL. The Bcr- 70-kDa S6 kinase, induced by the cytokines interleukin-3 and Abl leukemia oncogene activates Jun kinase and requires Jun for erythropoietin and inhibited by rapamycin, is not an absolute transformation. Proc Natl Acad Sci USA 1995; 92: 11746– requirement for cell proliferation. Eur J Immunol 1994; 24: 11750. 2664–2671. 469 Zanke BW, Rubie EA, Winnett E, Chan J, Randall S, Parsons M, 489 Hara K, Yonezawa K, Sakaue H, Kotani K, Kotani K, Kojima A, Boudreau K, McInnis M, Yan M, Templeton DJ, Woodgett JR. Waterfield MD, Kasuga M. Normal activation of p70 S6 kinase Mammalian mitogen-activated protein kinase pathways are regu- by insulin in cells overexpressing dominant negative 85kDa sub- lated through formation of specific kinase-activator complexes. unit of phosphoinositide 3-kinase. Biochem Biophys Res Comm J Biol Chem 1996; 271: 29876–29881. 1995; 208: 735–741. Review WL Blalock et al 1158 490 Price DJ, Grove JR, Calvo V, Avruch J, Bierer BE. Rapamycin- scription with cell cycle progression. Curr Opin Cell Biol 1994; induced inhibition of the 70-kilodalton S6 protein kinase. 6: 859–866. Science 1992; 257: 973–977. 515 Serrano M, Gomez-Lahoz E, Depinho RA, Beach D, Bar-Sagi D. 491 Sugiyama H, Papst P, Gelfand EW, Terada N. p70 S6 kinase sen- Inhibition of ras-induced proliferation and cellular transformation sitivity to rapamycin is eliminated by amino acid substitution of by p16INK4. Science 1995; 267: 249–252. Thr229. J Immunol 1996; 157: 656–660. 516 Medema RH, Herrera RE, Lam F, Weinberg RA. Growth sup- 492 Pullen N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, pression by p16INK4 requires functional retinoblastoma protein. Hemmings BA, Thomas G. Phosphorylation and activation of Proc Natl Acad Sci USA 1995; 92: 6289–6293. p70S6K by PDK1. Science 1998; 279: 707–710. 517 Weinberg RA. The retinoblastoma protein and cell cycle control. 493 Stephens L, Anderson K, Stokoe D, Erdjument-Bromage H, Cell 1995; 81: 323–330. Painter GF, Holmes AB, Gaffney PRJ, Reese CB, McCormick F, 518 Hofmann F, Livingston DM. CDK3 in conjunction with an as- Tempst P, Coadwell J, Hawkins PT. Protein kinase B kinases that yet-uncharacterized cyclin is also required for E2F-1-DP-1 heter- mediate phosphatidylinositol 3,4,5-Trisphosphate-dependent odimers to be rendered maximally active as transactivators. activation of protein kinase B. Science 1998; 279: 710–714. Genes Dev 1996; 10: 851–861. 494 Lenormand P, McMahon M, Pouyssegur J. Oncogenic Raf-1 acti- 519 Stillman B. Cell cycle control of DNA replication. Science 1996; vates the p70 S6 kinase via a mitogen-activated protein kinase- 274: 1659–1664. independent pathway. J Biol Chem 1996; 271: 15762–15768. 520 Zwicker J, Muller R. Cell-cycle regulation of gene expression by 495 Thomas G, Hall MN. Tor signalling and control of cell growth. transcriptional repression. Trends Genet 1996; 13: 3–6. Curr Opin Cell Biol 1997; 9: 782–787. 521 DeGregori J, Kowalik T, Nevins JR. Cellular targets for activation 496 Pardee AB. A restriction point for control of normal animal cell by the E2F1 transcription factor include DNA synthesis- and proliferation. Proc Natl Acad Sci USA 1974; 71: 1286–1290. G1/S-regulatory genes. Mol Cell Biol 1995; 15: 4215–4224. 497 Meyerson M, Harlow E. Identification of G1 kinase activity for 522 Zwicker J, Muller R. Cell cycle-regulated transcription in mam- Cdk6, a novel cyclin D partner. Mol Cell Biol 1994; 14: malian cells. Prog Cell Cycle Res 1995; 1: 91–99. 2077–2086. 523 Roberts JM, Koff A, Polyak K, Firpo E, Collins S, Ohtsubo M, 498 Pines J, Hunter T. Human cyclin A is adenovirus E1A-associated Massague J. Cyclins, Cdks, and cyclin kinase inhibitors. CSH Sym protein p60 and behaves differently from cyclin B. Nature 1990; Quant Bio 1994; 59: 31–38.

346: 760–763. 524 Sherr CJ. Mammalian G1 cyclins. Cell 1993; 73: 1059–1065. 499 Pines J, Hunter T. Human cyclins A and B1 are differentially 525 Sherr CJ, Kato J, Quelle DE, Matsuoka M, Roussel MF. D-type

located in the cell and undergo cell cycle-dependent nuclear cyclins and their cyclin-dependent kinases: G1 phase integrators transport. J Cell Biol 1991; 115: 1–17. of the mitogenic response. CSH Sym Quant Biol 1994; 59: 500 Ohtsubo M, Roberts J. Cyclin-dependent regulation of G1 in 11–19. mammalian fibroblasts. Science 1993; 259: 1908–1912. 526 Keyomarsi K, Toyofuku W, Fox MP. Deregulation of cyclin E in 501 Quelle DE, Ashmun RA, Shurtleff SA, Kato J-Y, Bar-Sagi D, Rous- breast cancer. Oncogene 1995; 11: 941–950. sel MF, Sherr CJ. Overexpression of mouse D-type cyclins accel- 527 Sgambato A, Han EK-H, Zhang Y-J, Moon RC, Santella RM, erates G1 phase in rodent fibroblasts. Genes Dev 1993; 7: Weinstein IB. Deregulated expression of cyclin D1 and other cell 1559–1571. cycle-related genes in carcinogen-induced rat mammary tumors. 502 Resnitzky D, Gossen M, Bujard H, Reed S. Acceleration of the Carcinogenesis 1995; 16: 2193–2198. G1/S phase transition by expression of cyclins D1 and E with an 528 Zwijsen RML, Klompmaker R, Wientjens E, Kristel PMP, Burg inducible system. Mol Cell Biol 1994; 14: 1669–1679. BVD, Michalides RJAM. Cyclin D1 triggers autonomous growth 503 Baldin V, Lukas J, Marcote, M, Pagano M, Draetta G. Cyclin D1 of breast cancer cells by governing cell cycle exit. Mol Cell Biol is a nuclear protein required for cell cycle progression in G1. 1996; 16: 2554–2560. Genes Dev 1993; 7: 812–821. 529 Hartwell LH, Kastan MB. Cell cycle control and cancer. Science 504 Ohtsubo M, Theodoras A, Schumacher J, Roberts J, Pagano M. 1994; 266: 1821–1828. Human cyclin E, a nuclear protein essential for the G1-to-S phase 530 Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations transition. Mol Cell Biol 1995; 15: 2612–2624. in the p53 tumor suppressor gene: clues to cancer etiology and 505 Girard F, Stausfeld U, Fernandez A, Lamb N. Cyclin A is required molecular pathogenesis. Cancer Res 1994; 54: 4855–4878. for the onset of DNA replication in mammalian fibroblasts. Cell 531 Nurse P. Universal control mechanism regulating onset of M- 1991; 67: 1169–1179. phase. Nature 1990; 344: 503–508. 506 Pagano M, Pepperkok P, Verde F, Ansorge W, Draetta G. Cyclin 532 El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent A is required at two points in the human cell cycle. EMBO J JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. Waf1, a poten- 1992; 11: 961–971. tial mediator of p53 tumor suppression. Cell 1993; 75: 817–825. 507 Walker D, Maller J. Role for cyclin A in the dependence of 533 Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 mitosis on completion of DNA replication. Nature 1991; 354: Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin- 314–317. dependent kinases. Cell 1993; 75: 805–816. 508 Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg 534 Hatakeyama M, Herrera RA, Makela T, Dowdy SF, Jacks T, RA. Regulation of retinoblastoma protein functions by ectopic Weinberg RA. The cancer cell and the cell cycle clock. CSH Sym expression of human cyclins. Cell 1992; 70: 993–1006. Quant Biol 1994; 59: 1–10. 509 Krude T, Jackman M, Pines J, Laskey RA. Cyclin/Cdk-dependent 535 Noda A, Ning, Y, Venable SF, Pereira-Smith OM, Smith JR. Clon- initiation of DNA replication in a human cell-free system. Cell ing of senescent cell-derived inhibitors of DNA synthesis using 1997; 88: 109–119. an expression screen. Exp Cell Res 1994; 211: 90–98. 510 Nevins JR. E2F: a link between the Rb tumor suppressor protein 536 Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. and viral oncoproteins. Science 1992; 258: 424–429. p21 is a universal inhibitor of cyclin kinases. Nature 1993; 366: 511 Neumann E, Flemington EK, Sellers WR, Kaelin WG Jr. Transcrip- 701–704. tion of the E2F-1 gene is rendered cell cycle dependent by E2F 537 Zhang H, Hannon GJ, Casso D, Beach D. p21 is a component DNA-binding sites within its promoter. Mol Cell Biol 1994; 14: of active cell cycle kinases. CSH Symp Quant Biol 1994; 59: 6607–6615. 21–29. 512 Geng Y, Eaton EN, Picon M, Roberts JM, Lundberg AS, Gifford 538 Guan K, Jenkins CW, Li Y, Nichols MA, Wu X, O’Keefe CL, Mat- A, Sardet C, Weinberg RA. Regulation of cyclin E transcription era AG, Xiong Y. Growth suppression by p18, a p16INK4/MTS1- by E2F and retinoblastoma protein. Oncogene 1996; 12: and p14INK4/MTS2-related CDK6 inhibitor, correlates with wild- 1173–1180. type pRb function. Genes Dev 1994; 8: 2939–2952. 513 Botz J, Zerfass-Thome K, Spitkovsky D, Delius H, Vogt B, Eilers 539 Hirai H, Roussel MF, Kato J, Aashmun RA, Sherr CJ. Novel INK4 M, Hatzigeorgiou A, Jansen-Durr P. Cell cycle regulation of the proteins, p19 and p18, are specific inhibitors of cyclin D-depen- murine cyclin E gene depends on an E2F binding site in the pro- dent kinases CDK4 and CDK6. Mol Cell Biol 1995; 15: 2672– moter. Mol Cell Biol 1996; 16: 3401–3409. 2681. 514 Lam EW-F, La Thangue NB. DP, E2F proteins: coordinating tran- 540 Okuda T, Hira H, Valentine VA, Shurtleff SA, Kidd VJ, Lahti JM, Review WL Blalock et al 1159 Sherr CJ, Downing JR. Molecular cloning, expression pattern, Gasparotto D, Carbone A, Boiocchi M. Human non-Hodgkin’s and chromosomal localization of human CDKN2D/INK4d, an lymphomas overexpress a wild-type form of p53 which is a func- inhibitor of cyclin D-dependent kinases. Genomics 1995; 29: tional transcriptional activator of the cyclin-dependent kinase 623–630. inhibitor p21. Blood 1997; 89: 2523–2528. 541 Waldman T, Lengauer C, Kinzler KW, Vogelstein B. Uncoupling 564 Deng C, Zhang P, Harper J, Elledge S, Leder P. Mice lacking of S phase and mitosis induced by anticancer agents in cells lack- p21CIP1/WAF1 undergo normal development, but are defective ing p21. Nature 1996; 381: 713–716. in G1 checkpoint control. Cell 1995; 82: 675–684. 542 Chan FKM, Zhang J, Chen L, Shapiro DN, Winoto A. Identifi- 565 Zen YX, el-Deiry WS. Regulation of p21WAF1/CIP1 expression cation of human/mouse p19, a novel Cdk4/Cdk6 inhibitor with by p53-independent pathways. Oncogene 1996; 12: 1557–1564. homology to p16INK4. Mol Cell Biol 1995; 15: 2682–2688. 566 Macleod KF, Sherry N, Hannon G, Beach D, Tokino T, Kinzler 543 Xiong Y, Zhang H, Beach D. Subunit rearrangement of the K, Vogelstein B, Jacks T. P53-dependent and independent cyclin-dependent kinases is associated with cellular transform- expression of p21 during cell growth, differentiation, and DNA ation. Genes Dev 1993; 7: 1572–1583. damage. Genes Dev 1995; 9: 935–944. 544 Lucas J, Parry D, Aagaard L, Mann DJ, Barkova J, Strauss M, 567 Moustakas A, Kardassis D. Regulation of the human Peters G, Bartek J. Retinoblastoma-protein-dependent cell-cycle p21/WAF1/CIP1 promoter in hepatic cells by functional interac- inhibition by the tumor suppressor p16. Nature 1995; 375: tions between Sp1 and Smad family members. Proc Natl Acad 503–506. Sci USA 1998; 95: 6733–6738. 545 Morse L, Chen D, Franklin D, Xiong Y, Chen-Kiang S. Induction 568 Lee SJ, Ha MJ, Lee J, Nguyen P, Choi YH, Pirnia F, Kang WK, of cell cycle arrest and B cell terminal differentiation by CDK Wang XF, Kim, SJ, Trepel JB. Inhibition of the 3-hydroxy-3- inhibitor p18 (INK4c) and IL-6. Immunity 1997; 6: 47–56. methylglutaryl-coenzyme A reductase pathway induces p53- 546 Hannon GJ, Beach D. P15INK4B is a potential effector of TGF- independent transcriptional regulation of p21 (WAF1/CIP1) in ␤ -induced cell cycle arrest. Nature 1994; 371: 257–261. human prostate carcinoma cells. J Biol Chem 1998; 273: 547 Haber DA. Splicing into senescence: the curious case of p16 and 10618–10623. ARF p19 . Cell 1997; 91: 555–558. 569 Xiao H, Hasegawa T, Miyaishi O, Ohkusu K, Isobe K. Sodium 548 Siebert R, Willers CP, Opalka B. Role of the cyclin-dependent butyrate induces NIH3T3 cells to senescence-like state and kinases 4 and inhibitor gene family p15, p16, p18 and p19 in enhances promoter activity of p21WAF/CIP1 in p53-independent leukemia and lymphoma. Leuk Lymphoma 1996; 23: 505–520. manner. Biochem Biophys Res Commun 1997; 237: 457–460. 549 Drexler HG. Review of alterations of the cyclin-dependent kinase 570 Halevy O, Novitch BG, Spicer DB, Skapek SX, Rhee J, Hannon inhibitor INK4 family genes p15, p16, p18, and p19 in human GJ, Beach D, Lasse AB. Correlation of terminal cell cycle arrest leukemia-lymphoma cells. Leukemia 1998; 12: 845–859. of skeletal muscle with induction of p21 by MyoD. Science 550 Hirama T, Koeffler HP. Role of the cyclin-dependent kinase 1995; 267: 1018–1020. inhibitors in the development of cancer. Blood 1995; 86: 841– 571 Polyak K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, 854. Koff A. P27kip, a cyclin-Cdk inhibitor, links TGF-␤ and contact 551 Williams ME, Whitefield M, Swerdlow SH. Analysis of the cyclin- inhibition to cell cycle arrest. Genes Dev 1994; 8: 9–22. dependent kinase inhibitors p18 and p19 in mantle-cell lym- 572 Toyoshima H, Hunter T. P27, a novel inhibitor of G1 cyclin/Cdk phoma and chronic lymphocytic leukemia. Ann Oncol 1997; 2 protein kinase activity, is related to p21. Cell 1994; 78: 67–74. (Suppl. 8): 71–73. 573 Lee MH, Reynisdo´ttir I, Massague´ J. Cloning of p57KIP2, a 552 Zhang H, Xiong Y, Beach D. Proliferating cell nuclear antigen cyclin-dependent kinase inhibitor with unique domain structure and p21 are components of multiple cell cycle kinase com- and tissue distribution. Genes Dev 1995; 9: 639–649. plexes. Mol Biol Cell 1993; 4: 897–906. 574 Matsuoka S, Edwards M, Bai C, Parker S, Zhang P, Baldini A, 553 Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, Harper JW, Elledge SJ. P57KIP2, a structurally distinct member Elledge SJ, Reed SI. P53-dependent inhibition of cyclin-depen- of the p21CIP1 Cdk inhibitor family, is a candidate tumor sup- dent kinase activities in human fibroblasts during radiation- pressor gene. Genes Dev 1995; 9: 650–662. induced G1 arrest. Cell 1994; 76: 1013–1023. 575 Watanabe H, Pan ZQ, Schreiber-Agus N, DePinho RA, Hurwitz 554 Gu Y, Turek CW, Morgan DO. Inhibition of CDK2 activity in J, Xiong Y. Suppression of cell transformation by the cyclin- vivo by an associated 20K regulatory subunit. Nature 1993; 366: dependent kinase inhibitor p57KIP2 requires binding to prolifer- 707–710. ating cell nuclear antigen. Proc Natl Acad Sci USA 1998; 95: 555 Zhang H, Hannon GJ, Beach D. P21-containing cyclin kinases 1392–1397. exist in both active and inactive states. Genes Dev 1994; 8: 576 Blain SW, Montalvo E, Massague´ J. Differential interaction of the 1750–1758. cyclin-dependent kinase (Cdk) inhibitor p27Kip1 with cyclin A- 556 Harper JW, Elledge SJ, Keyomarsi K, Dynlacht B, Tsai LH, Zhang, Cdk2 and cyclin D2-Cdk4. J Biol Chem 1997; 272: 25863– P, Dobrowolski S, Bai C, Connell-Crowley L, Swindell E, Fox 25872. MP, Wei N. Inhibition of cyclin-dependent kinases by p21. Mol Bio Cell 1996; 6: 387–400. 577 Soos TJ, Kiyokawa H, Yan JS, Rubin MS, Giordano A, DeBlasio 557 Flores-Rozas H, Kelman Z, Dean FB, Pan Z, Harper WJ, Elledge A, Bottega S, Wong B, Mendelsohn J, Koff A. Formation of p27– SJ, O’Donnell M, Hurwitz J. Cdk-interacting protein 1 directly CDK complexes during the human mitotic cell cycle. Cell binds with proliferating cell nuclear antigen and inhibits DNA Growth Diff 1996; 7: 135–146. replication catalyzed by the DNA polymerase and holoenzyme. 578 Poon RYC, Toyoshima H, Hunter T. Redistribution of the CDK Proc Natl Acad Sci USA 1994; 91: 8655–8659. inhibitor p27 between different cyclin–CDK complexes in the 558 Waga S, Hannon GJ, Beach D, Stillman B. The p21 inhibitor of mouse fibroblast cell cycle and in cells arrested with pvastatin cyclin-dependent kinases controls DNA replication by interac- or ultraviolet irradiation. Mol Biol Cell 1996; 6: 1197–1213. tion with PCNA. Nature 1994; 369: 574–578. 579 Reynisdo´ttir I, Massague´ J. The subcellular locations of p15Ink4b 559 Li R, Waga S, Hannon GJ, Beach D, Stillman B. Differential and p27Kip1 coordinate their inhibitory interactions with Cdk4 effects by the p21 CDK inhibitor on PCNA dependent DNA repli- and Cdk2. Genes Dev 1997; 11: 492–503. cation and repair. Nature 1994; 371: 534–537. 580 Sheaff RJ, Groudine M, Gordon M, Roberts JM, Clurman BE. 560 Shivji MKK, Grey SJ, Strausfeld UP, Wood RD, Blow JJ. Cip1 Cyclin E-Cdk2 is a regulator of p27Kip1. Genes Dev 1997; 11: inhibits DNA replication but not PCNA-dependent nucleotide 1464–1478. excision repair. Curr Biol 1994; 4: 1062–1068. 581 Zetterberg A, Larsonn O, Wiman Kg. What is the restriction 561 Isaacs JS, Chiao C, Merrick BA, Selkirk JK, Barrett JC, Weissman point? Curr Opin Cell Biol 1995; 7: 835–842. BE. P53-dependent p21 induction following gamma-irradiation 582 Lavoie JN, L’Allemain G, Brunet A, Mu¨ller R, Pouysse´gur J. without concomitant p53 induction in a human peripheral neur- Cyclin D1 expression is regulated by the p42/p44MAPK and oepithelioma cell line. Cancer Res 1997; 57: 2986–2992. negatively by the p38/HOGMAPK pathway. J Biol Chem 1996; 562 Kim TK. In vitro transcriptional activation of p21 promoter by 271: 20608–20616. p53. Biochem Biophys Res Commun 1997; 234: 300–302. 583 Liu J, Chao J, Jiang M, Ng S, Yen JJ, Yang-Yen H. Ras transform- 563 Maestro R, Gloghini A, Doglioni C, Piccinin S, Vukosavljevic T, ation results in an elevated level of cyclin D1 and acceleration Review WL Blalock et al 1160 of G1 progression in NIH 3T3 cells. Mol Cell Biol 1995; 15: using an estradiol-dependent fusion protein delta Raf-1:ER. Meth 3654–3663. Mol Biol 1997; 85: 137–151. 584 Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, 606 Leof EB, Proper JA, Goustin AS, Shipley GD, DiCorleto PE, Moses Pestell RG. Transforming p21ras mutants and c-Ets-2 activate the HL. Induction of c-sis mRNA and activity similar to platelet- cyclin D1 promoter through distinguishable regions. J Biol Chem derived growth factor by transforming growth factor beta: a pro- 1995; 270: 23589–23597. posed model for indirect mitogenesis involving autocrine 585 Akatas H, Cai H, Cooper GM. Ras links growth factor signaling activity. Proc Natl Acad Sci USA 1986; 83: 2453–2457. to the cell cycle machinery via regulation of cyclin D1 and the 607 Centrella M, McCarthy TL, Canalis E. Transforming growth fac- Cdk inhibitor p27kip1. Mol Cell Biol 1997; 17: 3850–3857. tor-␤ is a bifunctional regulator of replication and collagen syn- 586 Filmus J, Robles AI, Shi W, Wong MJ, Colombo LL, Conti CJ. thesis in osteoblast-enriched cell cultures from fetal rat bone. J Induction of cyclin D1 overexpression by activated ras. Oncog- Biol Chem 1987; 288: 2869–2874. ene 1994; 9: 3627–3633. 608 Battegay EJ, Raines EW, Seifert RA, Bowen-Pope, DF, Ross R. 587 Winston J, Dong F, Pledger WJ. Differential modulation of G1 TGF-␤ induces bimodal proliferation of connective tissue cells cyclins and the Cdk inhibitor p27Kip1 by platelet-derived growth via complex control of an autocrine PDGF loop. Cell 1990; 63: factor and plasma factors in density-arrested fibroblasts. J Biol 515–524. Chem 1986; 271: 11253–11260. 609 Laiho M, DeCaprio JA, Ludlow JW, Livingston DM, Massague´ J. 588 Winston JT, Coats SR, Wang Y, Pledger WJ. Regulation of the Growth inhibition by TGF-␤ linked to suppression of retinoblas- cell cycle machinery by oncogenic ras. Oncogene 1996; 12: toma protein phosphorylation. Cell 1990; 62: L175–L185. 127–134. 610 Geng Y, Weinberg RA. Transforming growth factor ␤ effects on 589 Barbacid M. Ras gene. Annu Rev Biochem 1987; 56: 779–827. expression of G1 cyclins and cyclin-dependent protein kinases. 590 Cahill MA, Janknecht R, Nordheim A. Signalling pathways: jack Proc Natl Acad Sci USA 1993; 90: 10315–10319. ␤ of all cascades. Curr Biol 1996; 6: 16–19. 611 Ewen ME, Sluss HK, Whitehouse LL, Livingston DM. TGF- inhi- 591 Franza BR Jr, Maruyama K, Garrels JI, Ruley HE. In vitro estab- bition of Cdk4 synthesis is linked to cell cycle arrest. Cell 1993; lishment is not a sufficient prerequisite for transformation by acti- 74: 1009–1020. vated Ras oncogenes. Cell 1986; 44: 409–418. 612 Datto MB, Li Y, Panus JF, Howe DJ, Xiong Y, Wang XF. Trans- ␤ 592 Hirakawa T, Ruley HE. Rescue of cells from ras oncogene- forming growth factor induces the cyclin-dependent kinase induced growth arrest by a second complementing oncogene. inhibitor p21 through a p53-independent mechanism. Proc Natl Proc Natl Acad Sci USA 1988; 85: 1519–1523. Acad Sci USA 1995; 92: 5545–5549. 593 Ridley AJ, Davis JB, Stroobant P, Land H. Transforming growth 613 Peters G. Cell cycle: stifled by inhibitions. Nature 1994; 371: factors-␤1 and ␤2 are mitogens for rat Schwann cells. J Cell Biol 204–205. 1989; 109: 3419–3424. 614 Raftery LA, Twombly V, Wharton K, Gelbart WM. Genetic 594 Bar-Sagi D, Feramisco JR, Microinjection of the ras oncogene screens to identify elements of the decapentaplegic signaling pathway in Drosophila. Genetics 1995; 139: 241–254. protein into PC12 cells induces morphological differentiation. ␤ Cell 1985; 42: 841–848. 615 Derynck R, Feng X-H. TGF- receptor signaling. Biochim Biophy 595 Benito M, Porras A, Nebreda AR, Santos E. Differentiation of Acta 1997; 1333: F105-F150. 616 Yan Z, Winawer S, Friedman E. Two different signal transduction 3T3-L1 fibroblasts to adipocytes induced by transfection of ras ␤ oncogenes. Science 1991; 253: 565–568. pathways can be activated by transforming growth factor 1in epithelial cells. J Biol Chem 1994; 229: 13231–13237. 596 Serrano M, Lin AW, McCurrach ME, Beach D, Lowe S. Oncog- ␤ enic ras provokes premature cell senescence associated with 617 Hartsough ME, Mulder KM. Transforming growth factor- acti- accumulation of p53 and p16ink4a. Cell 1997; 88: 593–602. vation of p44mapk in proliferating cultures of epithelial cells. J 597 Blagosklonny MV, Schulte TW, Nguyen P, Mimnaugh EG, Trepel Biol Chem 1995; 270: 7117–7124. 618 Macias-Silva M, Abdollah S, Hoodless P, Pirone R, Attisano L, J, Neckers L. Taxol induction of p21Waf1 and p53 requires c-raf- Wrana JL. MADR-2 is a substrate of the TGF-␤ receptor and its 1. Cancer Res 1995; 55: 4623–4626. phosphorylation is required for nuclear accumulation and sig- 598 Pumiglia KM, Decker SJ. Cell cycle arrest mediated by the naling. Cell 1996; 87: 1215–1224. MEK/mitogen-activated protein kinase pathway. Proc Natl Acad 619 Zhang Y, Feng XH, Wu RY, Derynck R. Receptor-associated Mad Sci USA 1997; 94: 448–452. homologs synergize to induce TGF-␤ response. Nature 1996; 599 Wood KW, Qi H, D’Arcangelo G, Armstrong RC, Roberts TM, 382: 168–172. Halegoua S. The cytoplasmic raf oncogene induces a neuronal 620 Kramer IM, Koornneeff I, de Laat SW, van den Eijnden-van Raaj phenotype in PC12 cells: a potential role for cellular raf kinases AJ. TGF-␤ induces phophorylation of the cyclic AMP responsive in neuronal growth factor signal transduction. Proc Natl Acad element binding protein in ML-CCL64 cells. EMBO J 1991; 10: Sci USA 1993; 90: 5016–5020. 1083–1089. 600 Yen A, Williams M, Platko JD, Der C, Hisska M. Expression of 621 Alevizopoulos A, Dussere Y, Tsai-Pflugfelder M, von der Weid activated Raf accelerates cell differentiation and PB protein T, Wahli W, Mermod N. A proline-rich TGF-␤-responsive tran- down-regulation but not hypophosphorylation. Eur J Cell Biol scriptional activator interacts with histone H3. Genes Dev 1995; 1994; 65: 103–113. 9: 3051–3066. 601 O’Shea CC, Crompton T, Rosewell IR, Hayday AC, Owen MJ. 622 Juan G, Gong J, Traganos F, Darzynkiewicz Z. Unscheduled Raf regulates positive selection. Eur J Immunol 1996; 26: expression of cyclins D1 and D3 in human tumor cell lines. Cell 2350–2355. Prolif 1996; 29: 259–266. 602 McCarthy SA, Chen D, Yang B-S, Garcia Ramirez JJ, Cherwinski 623 Skorski T, Nieborowska-Skorska M, Szczylik C, Kanakaraj P, Per- H, Chen X-R, Klagsbrun M, Hauser CA, Ostrowski MC, McMa- rotti D, Zon G, Gewirtz A, Perussia B, Calabretta B. c-Raf-1 hon M. Rapid phosphorylation of Ets-2 accompanies mitogen- serine/threonine kinase is required in BCR/ABL-dependent and activated protein kinase activation and the induction of heparin- normal hematopoiesis. Cancer Res 1995; 55: 2275–2278. binding epidermal growth factor gene expression by oncogenic 624 Osada H. Development of new antitumor reagents which block Raf-1. Mol Cell Biol 1997; 17: 2410–2412. the cell cycle progression. Gan Kagaku Ryoho 1997; 24: 603 McCarthy SA, Samuels ML, Pritchard CA, Abraham JA, McMa- 1541–1546. hon M. Rapid induction of heparin binding epidermal growth 625 Kondoh M, Usui T, Kobayashi S, Tsuchiya K, Nishikawa K, Nishi- factor/diphtheria toxin receptor by Ras and Raf oncogenes. kiori T, Mayumi T, Osada H. Cell cycle arrest and antitumor Genes Dev 1995; 9: 1953–1964. activity of pironetin and its derivatives. Cancer Lett 1998; 126: 604 Samuels ML, McMahon M. Inhibition of platelet-derived growth 29–32. factor- and epidermal growth-mediated mitogenesis and sig- 626 Hamaguchi T, Sudo T, Osada H. RK-682, a potent inhibitor of naling in 3T3 cells expressing ⌬Raf-ER, an estradiol-regulated tyrosine phosphatase, arrested the cell cycle progression at G1 from of Raf-1. Mol Cell Biol 1994; 14: 7855–7866. phase. FEBS Lett 1995; 372: 54–58. 605 McCarthy SA, Aziz N, McMahon M. Identification of immediate– 627 Haraguchi T, Kaneda T, Hiraoka Y. Dynamics of chromosomes early gene targets of the Raf-1 serine/threonine protein kinase and microtubules visualized by multiple-wavelength fluor- Review WL Blalock et al 1161 escence imaging in living mammalian cells: effects of mitotic 652 Alnemri ES, Livingston DJ, Nicholson DW, Salvesen G, inhibitors on cell cycle progression. Genes to Cells 1997; 2: Thornberry NA, Wong WW, Yuan J. Human ICE/CED-3 protease 369–380. nomenclature. Cell 1996; 87: 171. 628 Anderson HJ, Coleman JE, Andersen RJ, Roberge M. Cytotoxic 653 Wang S, Miuyra M, Jung Y-K, Zhu H, Li E, Yuan J. Murine cas- peptides hemiasterlin, hemiasterlin A and hemiasterlin B induce pase-11, and ICE-interacting protease, is essential for the acti- mitotic arrest and abnormal spindle formation. Cancer Chemo vation of ICE. Cell 1998; 92: 501–509. Pharma 1997; 39: 223–226. 654 Black RA, Kronheim SR, Merriam JE, March CJ, Hopp TP. A pre- 629 Gong J, Traganos F, Darzynkiewicz Z. Discrimination of G2 and aspartate-specific protease from human leukocytes that cleaves mitotic cells by flow cytometry based on different expression of pro-interleukin-1b. J Biol Chem 1989; 264: 5323–5326. cyclins A and B1. Exp Cell Res 1995; 220: 226–231. 655 Nicholson DW, Hornberry N. Caspases: killer proteases. Trends 630 Cvitkovic E, Izzo J. The current and future plase of vinorelbine Biochem Sci 1997; 22: 299–306. in cancer therapy. Drugs 1992; 44 (Suppl 4): 36–45. 656 Walker NPC, Taladian RV, Brady KD, Dang LC, Bump NJ, Ferenz 631 Weinstat-Saslow D, Merino MJ, Manrow RE, Lawrence JA, Bluth CR, Franklin S, Ghayur T, Hackett MC, Hammill LD, Herzog L, RF, Wittenbel KDA, Simpson JF, Page DL, Steeg PS. Over- Hugunin M, Houy W, Mankovich JA, McGuiness L, Orlewicz E, expression of cyclin D mRNA distinguishes invasive and in situ Paskind M, Pratt CA, Reis P, Summani A, Terranova M, Welch breast carcinomas from non-malignant lesions. Nature Med JP, Xiong L, Moller A, Tracey DE, Kamen R, Wong WW. Crystal 1995; 1: 1257–1260. structure of the cysteine protease interleukin-1b-converting 632 Wang YL, Uhara H, Yamazaki Y, Nikaido T, Saida T. Immunohis- enzyme, a (p20/p10)2 homodimer. Cell 1994; 78: 343–352. tochemical detection of Cdk4 and p16INK4 proteins in cutaneous 657 Wilson KP, Black J-AF, Thomson JA, Kim EE, Griffith JP, Navia malignant melanoma. Br J Der 1996; 134: 269–275. MA, Murcko MA, Chambers SP, Aldape RA, Raybuck SA, Liv- 633 Bartkova J, Lukas J, Guldberg P, Alsner J, Kirkin AF, Zeuthen J, ingston DJ. Structure and mechanism of interleukin-1b con- Bartek J. The p16-cyclin D/Cdk4-Rb pathway as a functional unit verting enzyme. Nature 1994; 370: 270–275. frequently altered in melanoma pathogenesis. Cancer Res 1996; 658 Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involve- 56: 5475–5483. ment of MACH, a novel MORT1/FADD-interacting protease, in 634 Bursch W, Oberhammer F, Schulte-Hermann R. Cell death by Fas/Apo-1- and TNF receptor-induced cell death. Cell 1996; 85: apoptosis and its protective role against disease. TIPS 1992; 13: 803–815. 245–251. 659 Fernandes-Alnemri T, Armstrong RC, Krebs J, Srinivasula SM, 635 Wyllie AH. Cell death: the significance of apoptosis. Int Rev Wang L, Bullrich F, Fritz LC, Trapani JA, Tomaselli KJ, Litwack Cytol 1980; 68: 251–307. G, Alnemri ES. In vitro activation of CPP32 and Mch3 by Mch4, 636 Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological a novel apoptotic cysteine protease containing two FADD-like phenomenon with wide-ranging implications in tissue kinetics. domains. Proc Natl Acad Sci USA 1996; 93: 7464–7469. Br J Cancer 1972; 26: 239–257. 660 Muzio M, Chinnaiyan AM, Kischkel FC, O’Rourke K, Shevch- 637 Gorman AM, McGowan A, O’Neill C, Cotter T. Oxidative stress enko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz R, Mann and apoptosis in neurodegeneration. J Neurol Sci 1996; 139: M, Kreammer PH, Peter ME, Dixit VM. FLICE, a novel FADD- 45–52. homologous ICE/CED-3-like protease, is recruited to the CD95 638 Thompson HJ, Strange R, Schedin PJ. Apoptosis in the genesis (Fas/Apo-1) death-inducing signaling complex. Cell 1996; 85: and prevention of cancer. Cancer Epidem Biomark Preven 1992; 817–827. 1: 597–602. 661 Vincenz C, Dixit VM. Fas-associated death domain protein 639 Thompson CB. Apoptosis in the pathogenesis and treatment of interleukin-1b-converting enzyme 2 (FLICE2), an ICE/Ced-3 hom- disease. Science 1995; 267: 1456–1462. ologue, is proximally involved in CD95- and p55-mediated 640 Majno G, Joris I. Apoptosis, oncosis and necrosis. Am J Pathol death signaling. J Biol Chem 1997; 272: 6578–6583. 1995; 146: 3–15. 662 Boldin MP, Varfolomeev EE, Pancer Z, Mett IL, Camonis JH, Wal- 641 Schwartzman RA, Cidlowski JA. Apoptosis: the biochemistry and lach D. A novel protein that interacts with the death domain of molecular biology of programmed cell death. Endocrine Rev Fas/Apo-1 contains a sequence motif related to the death 1993; 14: 133–151. domain. J Biol Chem 1995; 270: 7795–7798. 642 Arrends M, Wyllie A. Apoptosis: mechanisms and roles in pathol- 663 Chinnaiyan AM, O’Rourke K, Lane BR, Dixit VM. Interaction of ogy. Int Rev Expl Path 1991; 32: 223–251. CED-4 with CED-3 and CED-9: a molecular framework for cell 643 Clem RJ, Miller LK. Control of programmed cell death by the death. Science 1997; 275: 1122–1126. baculovirus genes p35 and iap. Mol Cell Biol 1994; 14: 5212– 664 Zou H, Henzel W, Liu X, Lutschg A, Wang X. Apaf-1, a human 5222. protein homologous to C. elegans CED-4, participates in cyto- 644 Duke DC, Chervenak R, Cohen JJ. Endogenous endonuclease- chrome c-dependent activation of caspase-3. Cell 1997; 90: induced DNA fragmentation: an early event in cell mediated 405–413.

cytolysis. Proc Natl Acad Sci USA 1983; 80: 6361–6365. 665 Pan G, O’Rourke K, Dixit VM. Caspase-9, Bcl-xL, and Apaf-1 645 Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is asso- form a ternary complex. J Biol Chem 1998; 273: 5841–5845. ciated with endogenous nuclease activation. Nature 1980; 284: 666 Hofmann K, Bucher P, Tschopp J. The CARD domain: a new 555–556. apoptotic signalling motif. Trends Biochem Sci 1997; 22: 155– 646 Wyllie AH, Morris RG, Smith AL, Dunlop D. Chromatin cleavage 156. in apoptosis: association with condensed chromatin morphology 667 Irmler M, Hofman D, Tschopp J. Direct physical interaction and dependence on macromolecular synthesis. J Pathol 1984; between the Caenorhabditis elegans ‘death proteins’ CED-3 and 142: 67–77. CED-4. FEBBS Lett 1997; 406: 189–190. 647 Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Hen- 668 Nagata S. Apoptosis by death factor. Cell 1997; 88: 355–365. son PM. Exposure of phosphatidyl serine on the surface of apop- 669 Nagata S. Apoptosis: telling cells their time is up. Curr Biol 1996; totic lymphocytes triggers specific recognition and removal by 6: 1241–1243. macrophages. J Immunol 1992; 148: 2207–2216. 670 Liu X, Zou H, Slaughter C, Wang X. DFF, a heterodimeric protein 648 Savill J. Recognition and phagocytosis of cells undergoing that functions downstream of caspase-3 to trigger DNA fragmen- apoptosis. Br Med Bull 1997; 53: 491–508. tation during apoptosis. Cell 1997; 89: 175–184. 649 Cryns V, Yuan J. Proteases to die for. Genes Dev 1998; 12: 671 Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, 1551–1570. Alnemri ES, Wang X. Cytochrome c and dATP-dependent forma- 650 Cryns V, Yuan J. The cutting edge: caspases in apoptosis and tion of Apaf-1/caspase-9 complex initiates an apoptotic protease disease. In: Z Zakeri, J Tilly, R Lockshin (eds). When Cells Die: cascade. Cell 1997; 91: 479–489. A Comprehensive Evaluation of Apoptosis and Programmed Cell 672 Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Death John Wiley: New York, 1998, pp 177–210. Nagata S. A caspase-activated DNase that degrades DNA during 651 Allen RT, Cluck MW, Agrawal DK. Mechanisms controlling apoptosis, and its inhibitor ICAD. Nature 1998; 391: 43–50. cellular suicide: role of Bcl-2 and caspases. Cell Mol Life Sci 673 Montague JW, Hughes Jr FM, Cidlowski JA. Native recombinant 1998; 54: 427–445. cyclophilins A, B, and C degrade DNA independently of peptid- Review WL Blalock et al 1162 ylpropyl cis-trans-isomerase activity. J Biol Chem 1997; 272: 693 Kuida K, Zheng TS, Na S, Kuan C-Y, Yang D, Karasuyama H, 6677–6684. Rakic P, Flavell RA. Decreased apoptosis in the brain and prema- 674 Emoto Y, Manome Y, Meinhardt G, Kisaki H, Kharbanda S, Rob- ture lethality in CPP32-deficient mice. Nature 1996; 384: 368– ertson M, Ghayur T, Wong WW, Kamen R, Weischselbaum RR, 372. Kufe D. Proteolytic activation of protein kinase C␦ by an ICE- 694 Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apop- like protease in apoptotic cells. EMBO J 1995; 14: 6148–6156. totic program in cell-free extracts: requirement for dATP and 675 Ghayur T, Hugunin M, Talanian RV, Ratnofsky S, Quinlan C, cytochrome c. Cell 1996; 86: 147–157. Emoto Y, Pandey P, Datta R, Huang Y, Kharbanda S, Allen H, 695 Bergeron L, Perez GI, McDonald G, Shi L, Sun Y, Jurisicova A, Kamen R, Wong W, Kufe D. Proteolytic activation of protein kin- Varmuza S, Latham KE, Flaws JA, Salter JCM, Hara H, Moskowitz ase C␦ by an ICE/CED 3-like protease induces characteristics of MA, Li E, Greenberg AH, Tilly J, Yuan J. Defects in regulation apoptosis. J Exp Med 1996; 184: 2399–2404. of apoptosis in caspase-2-deficient mice. Genes Dev 1998; 12: 676 Cardone MH, Salvesen GS, Widmann C, Johnson G, Frisch SM. 1304–1314. The regulation of anoikis: MEKK-1 activation requires cleavage 696 Li P, Allen H, Banerjee S, Franklin S, Herzog L, Johnston C, by the caspases. Cell 1997; 90: 315–323. McDowell J, Paskind M, Rodman L, Salfeld J, Towne E, Tracey 677 Datta R, Kojima H, Yoshida K, Kufe D. Caspase-3-mediated D, Wardwell S, Wei F-Y, Wong W, Kamen R, Seshadri T. Mice cleavage of protein kinase C theta in induction of apoptosis. J deficient in IL-1b-converting enzyme are defective in production Biol Chem 1997; 272: 20317–20320. of mature IL-1b and resistant to endotoxic shock. Cell 1995; 80: 678 Lee N, MacDonald H, Reinhard C, Halenbeck R, Roulston A, 401–411. Shi T, Williams LT. Activation of hPAK65 by caspase cleavage 697 Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS- induces some of the morphological and biochemical changes of S, Flavell RA. Altered cytokine export and apoptosis in mice apoptosis. Proc Natl Acad Sci USA 1997; 94: 13642–13647. deficient in interleukin-1␤-converting enzyme. Science 1995; 679 Rudel T, Bokoch G. Membrane and morphological changes in 267: 2000–2002. apoptotic cells regulated by caspase-mediated activation of 698 Song Q, Burrows SR, Smith G, Lees-Miller SP, Kumar S, Chan PAK2. Science 1997; 276: 1571–1574. DW, Rapani JA, Alnemri E, Litwack G, Lu H, Moss DJ, Jackson 680 Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, S, Lavin MF. Interleukin-1b-converting enzyme-like protease McGarry TJ, Kirschner MW, Koths K, Kwiatkowski DJ, Williams cleaves DNA-dependent protein kinase in cytotoxic T cell killing. LT. Caspase-3-generated fragment of gelsolin: effector of mor- J Exp Med 1996; 184: 619–626. phological change in apoptosis. Science 1997; 278: 294–298. 699 Darmon AJ, Nicholson DW, Bleackley RC. Activation of the 681 Brancolini C, Benedetti M, Schneider C. Microfilament reorgani- apoptotic protease CPP32 by cytotoxic T cell-derived granzyme zation during apoptosis: the role of Gas2, a possible substrate B. Nature 1995; 377: 446–448. for ICE-like proteases. EMBO J 1995; 14: 5179–5190. 700 Quan LT, Tewari M, O’Rourke K, Dixit V, Snipas SJ, Poirier GG, 682 Lazebnik YA, Takahashi A, Moir RD, Goldman RD, Poirier GG, Ray C, Pickup DJ, Salvesen GS. Proteolytic activation of the cell Kaufman SH, Earnshaw WC. Studies of the lamin proteinase death protease Yama/CPP32 by granzyme B. Proc Natl Acad Sci reveal multiple parallel biochemical pathways during apoptotic USA 1996; 93: 1972–1976. execution. Proc Natl Acad Sci USA 1995; 92: 9042–9046. 701 Chinnaiyan AM, Hanna WL, Orth K, Duan H, Poirier GG, Froel- 683 Orth K, Chinnaiyan AM, Garg M, Froelich CJ, Dixit VM. The ich CJ, Dixit VM. Cytotoxic T cell-derived granzyme B activates CED-3/ICE-like protease Mch2 is activated during apoptosis and the apoptotic protease ICE-LAP3. Curr Biol 1996; 6: 897–899. cleaves the death substrate lamin A. J Biol Chem 1996; 271: 702 Gu Y, Sarnecki C, Fleming MA, Lippke JA, Bleackley RC, Su MS- 16443–16446. S. Processing and activation of CMH-1 by granzyme B. J Biol 684 Duan H, Orth K, Chinnaiyan AM, Poirier GG, Froelich GJ, He Chem 1996; 271: 10816–10820. W-W, Dixit VM. ICE-LAP6, a novel member of the ICE/Ced-3 703 Croal DE, DeMartino GN. Calcium-activated neutral protease gene family, is activated by the cytotoxic T cell protease gran- (calpain) system: structure, function, and regulation. Physiol Rev zyme B. J Biol Chem 1996; 271: 16720–16724. 1991; 71: 813–847. 685 Takahashi A, Alnemri ES, Lazebnik YA, Fernandes-Alnemri T, Lit- 704 Sarin TC, Adams DH, Henkart PA. Protease inhibitors selectively wack G, Moir RD, Goldman RD, Poirier GG, Kaufman SH, block T cell receptor-triggered programmed cell death in a Earnshaw WC. Cleavage of lamin A by Mch2a but not CPP32: murine T cell hybridoma and activated peripheral T cells. J Exp multiple interleukin 1b-converting enzyme-related proteases Med 1993; 178: 1693–1700. with distinct substrate recognition properties are active in 705 Squier MKT, Miller ACK, Malkinson AM, Cohen JJ. Calpain acti- apoptosis. Proc Natl Acad Sci USA 1996; 93: 8395–8400. vation in apoptosis. J Cell Physiol 1994; 159: 229–237. 686 Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in 706 Squier MKT, Cohen JJ. Calpain, an upstream regulator of thymo- CAD activation and DNA degradation during apoptosis. Nature cyte apoptosis. J Immunol 1997; 158: 3690–3697. 1998; 391: 96–99. 707 Edelstein CL, Wieder ED, Yaqoob MM, Gengaro PE, Burke TJ, 687 Cheng EHY, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Nemenoff RA, Schrier RW. The role of cysteine proteases in Ueno K, Hardwick JM. Conversion of Bcl-2 to a Bax-like death hypoxia-induced rat renal proximal tubular injury. Proc Natl effector by caspases. Science 1997; 278: 1966–1968. Acad Sci USA 1995; 92: 7662–7666. 688 Clem R, Cheng E, Karp C, Kirsch D, Ueno K, Takahashi A, Kastan 708 Saido TC, Sorimachi H, Suzuki K. Calpain: new perspectives in M, Griffin D, Earnshaw W, Veliuona M, Hardwick J. Modulation molecular diversity and physiological–pathological involvement.

of cell death by Bcl-xL through caspase interaction. Proc Natl FASEB J 1994; 8: 814–822. Acad Sci USA 1998; 95: 554–559. 709 Schwartz LM, Smith SW, Jones MEE, Osborne BA. Do all pro- 689 Grandgirard D, Studer E, Monney L, Belser T, Fellay I, Borner C, grammed cell deaths occur via apoptosis? Proc Natl Acad Sci Michel MR. Alphaviruses induce apoptosis in Bcl-2-overexpress- USA 1993; 90: 980–984. ing cells: evidence for a caspase-mediated, proteolytic inacti- 710 Olie RA, Durrieu F, Cornillon S, Loughran G, Gross J, Earnshaw vation of Bcl-2. EMBO J 1998; 17: 1268–1278. WC, Golstein P. Apparent caspase independence of programmed 690 Widmann C, Gibson S, Johnson GL. Caspase-dependent cleav- cell death in Dictylostelium. Curr Biol 1998; 8: 955–958. age of signaling proteins during apoptosis. J Biol Chem 1998; 711 Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization 273: 7141–7147. of BAX results in its translocation, mitochondrial dysfunction and 691 Martins LM, Kottke T, Mesner PW, Basi GS, Sinha S, Frigon N apoptosis. EMBO J 1998; 17: 3878–3885. Jr, Tatar E, Tung JS, Bryant K, Takahashi A, Svingen PA, Madden 712 McCarthy NJ, Whyte MK, Gilbert CS, Evan GI. Inhibition of Ced- BJ, McCormick DJ, Earnshaw WC, Kaufmann SH. Activation of 3/ICE-related proteases does not prevent cell death induced by multiple interleukin-1␤ converting enzyme homologues in cyto- oncogenes, DNA damage, or the Bcl-2 homologue Bak. J Cell sol and nuclei of HL-60 cells during etoposide-induced Biol 1997; 136: 215–227. apoptosis. J Biol Chem 1997; 272: 7421–7430. 713 Xiang J, Chao DT, Korsmeyer SJ. BAX-induced cell death may 692 Faleiro L, Kobayashi R, Fearnhead H, Lazebnick Y. Multiple not require interleukin 1 beta-converting enzyme-like proteases. species pf CPP32 and Mch2 are the major active caspases Proc Natl Acad Sci USA 1996; 93: 14559–14563. present in apoptotic cells. EMBO J 1997; 16: 2271–2281. 714 Goltsev YV, Kovalenko AV, Arnold E, Varfolomeev EE, Brodian- Review WL Blalock et al 1163 ski VM, Wallach D. CASH, a novel caspase homolog with death 734 Tenneti L, D’Emilia DM, Lipton SA. Suppression of neuronal effector domains. J Biol Chem 1997; 272: 19641–19644. apoptosis by S-nitrosylation of caspases. Neurosci Lett 1997; 715 Han DKM, Chaudhary PM, Wright ME, Friedman C, Trask BJ, 236: 139–142. Riedel RT, Baskin DG, Schwartz SM, Hood L. MRIT, a novel 735 Ray CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, Salv- death-effector domain-containing protein, interacts with casp- esen GS, Pickup DJ. Viral inhibition of inﬂammation: cowpox ases and Bcl-XL and initiates cell death. Proc Natl Acad Sci USA virus encodes an inhibitor of the interleukin-1b-converting 1997; 94: 11333–11338. enzyme. Cell 1992; 69: 597–604. 716 Inohara N, Koseki T, Hu Y, Chen S, Nunez G. CLARP, a death 736 Gagliardini V, Fernandez PA, Lee RK, Drexler HC, Rotello RJ, effector domain-containing protein interacts with caspase-8 and Fishman MC, Yuan J. Prevention of vertebrate neuronal death by regulates apoptosis. Proc Natl Acad Sci USA 1997; 94: the crmA gene. Science 1994; 263: 826–828. 10717–10722. 737 Tewari M, Dixit VM. Fas- and tumor necrosis factor-induced 717 Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner apoptosis is inhibited by the poxvirus crmA gene product. J Biol V, Bodmer J-L, Schroter M, Burns K, Mattmann C, Rimoldi D, Chem 1995; 270: 3255–3260. French L, Tschopp J. Inhibition of death receptor signals by cellu- 738 Tewari M, Quan LT, O’Rourke K, Desnoyers S, Zeng Z, Beidler lar FLIP. Nature 1997; 388: 190–195. DR, Poirier GG, Salvesen GS, Dixit VM. Yama/CPP32b, a mam- 718 MacFarlane M, Ahmad M, Srinivasula S, Fernandes-Alnemri T, malian homolog of CED3, is a crmA-inhibitable protease that Cohen G, Alnemri E. Identiﬁcation and molecular cloning of two cleaves the death substrate poly(ADP-ribose) polymerase. Cell novel receptors for the cytotoxic ligand TRAIL. J Biol Chem 1997; 1995; 81: 801–809. 272: 25417–25420. 739 Miura M, Friedlander RM, Yuan Y. Tumor necrosis factor- 719 Marsters SA, Pitti RM, Donahue CJ, Ruppert S, Bauer KD, Asken- induced apoptosis is mediated by a CrmA-sensitive cell death azi A. Activation of apoptosis by Apo-2 ligand is independent of pathway. Proc Natl Acad Sci USA 1995; 92: 8318–8322. FADD but blocked by CrmA. Curr Biol 1996; 6: 750–752. 740 Tewari M, Telford WG, Miller RA, Dixit VM. Crm A, poxvirus- 720 Marsters SA, Sheridan JP, Pitti RM, Huang A, Skubatch M, Bald- encoded serpin, inhibits cytotoxic T-lymphocyte-mediated win D, Gurney A, Goddard AD, Godowski P, Ashkenazi A. A apoptosis. J Biol Chem 1995; 270: 22705–22708. novel receptor for Apo2L/TRAIL contains a truncated death 741 Bertin J, Mendrysa SM, LaCount DJ, Gaur S, Krebs JF, Armstrong domain. Curr Biol 1997; 7: 1003–1006. RC, Tomaselli KJ, Friesen PD. Apoptotic suppression by baculo- 721 Schneider P, Bodmer J, Thome M, Hofmann K, Holler N, virus P35 involves cleavage by and inhibition of a virus-induced Tschopp J. Characterization of two receptors for TRAIL. FEBS Lett CED-3/ICE-like protease. J Virol 1996; 70: 6251–6259. 1997; 416: 329–334. 742 Xue D, Horvitz HR. Inhibition of the Caenorhabditis elegans cell- 722 Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Bald- death protease CED-3 by a CED-3 cleavage site in baculovirus win D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard p35 protein. Nature 1995; 377: 248–251. AD, Godowski P, Ashkenazi A. Control of TRAIL-induced 743 Antoku K, Liu Z, Johnson DE. IL-3 withdrawal activates a CrmA- apoptosis by a family of signaling and decoy receptors. Science insensitive poly(ADP-ribose) polymerase cleavage enzyme in 1997; 277: 818–821. factor-deprived myeloid progenitor cells. Leukemia 1998; 12: 723 Degli-Esposti MA, Dougall WC, Smolak PJ, Waugh JY, Smith CA, 682–689. Goodwin RG. The novel receptor TRAIL-R4 induces NF-␬B and 744 Antoku K, Liu Z, Johnson DE. Inhibition of caspase proteases by protects against TRAIL mediated apoptosis, yet retains an incom- CrmA enhances the resistance of human leukemia cells to mul- plete death domain. Immunity 1997; 7: 813–820. tiple chemotherapeutic agents. Leukemia 1997; 11: 1665–1672. 724 Pan G, Ni J, Wei Y-F, Yu G-l, Gentz R, Dixit VM. An antagonist 745 Bakhshi A, Jensen JP, Goldman P, Wright JJ, McBride OW, decoy receptor and a death domain-containing receptor for Epstein AL, Korsmeyer SJ. Cloning the chromosomal breakpoint

TRAIL. Science 1997; 277: 111–113. of t(14;18) human lymphomas: clustering around JH on chromo- 725 Uren AG, Coulson EJ, Vaux DL. Conservation of baculovirus some 14 and near a transcriptional unit on 18. Cell 1985; 41: inhibitor of apoptosis repeat proteins (BIRPs) in viruses, nema- 899–906. todes, vertebrates and yeasts. Trends Biochem Sci 1998; 23: 746 Cleary ML, Sklar J. Nucleotide sequence of a t(14;18) chromo- 159–162. somal breakpoint in follicular lymphoma and demonstration of 726 Duckett CS, Nava VE, Gedrich RW, Clem RJ, Van Dongen JL, a breakpoint cluster region near a transcriptionally active locus Gilﬁllan MC, Shiels H, Hardwick JM, Thompson CB. A con- on chromosome 18. Proc Natl Acad Sci USA 1985; 82: 7439– served family of cellular genes related to the baculovirus iap gene 7443. and encoding apoptosis inhibitors. EMBO J 1996; 15: 2685– 747 Hockenbery DM, Nunez G, Milliman C, Schreiber RD, Korsme- 2694. yer SJ. Bcl-2 is an inner mitochondrial membrane protein that 727 Liston P, Roy N, Tamai K, Lefebvre C, Baird S, Cherton-Horvat blocks programmed cell death. Nature 1990; 348: 334–336. G, Farahani R, McLean M, Ikekda JE, MacKenzie A, Korneluk 748 Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in RG. Suppression of apoptosis in mammalian cells by NAIP and vivo with a conserved homolog, Bax, that accelerates pro- a related family of IAP genes. Nature 1996; 379: 349–353. grammed cell death. Cell 1993; 74: 609–619. 728 Uren AG, Pakusch M, Hawkins CJ, Puls KL, Vaux DL. Cloning 749 Vaux DL, Cory S, Adams JM. Bcl-2 promotes the survival of and expression of apoptosis inhibitory homologs that function haemopoietic cells and cooperates with c-myc to immortalize to inhibit apoptosis and/or bind tumor necrosis factor receptor- pre-B cells. Nature 1988; 335: 440–442. associated factors. Proc Natl Acad Sci USA 1996; 93: 4974– 750 Nunez G, London L, Hockenbery D, Alexander M, McKearn JP, 4978. Korsmeyer SJ. Deregulated Bcl-2 gene expression selectively pro- 729 Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, longs survival of growth factor-deprived hemopoietic cell lines. survivin, expressed in cancer and lymphoma. Nature Med 1997; J Immunol 1990; 144: 3602–3610. 3: 917–921. 751 Yang E, Jiping Z, Jockel J, Boise LH, Thompson CB, Korsmeyer

730 Duckett CS, Li F, Wang Y, Tomaselli KJ, Thompson CB, Arm- SJ. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces strong RC. Human IAP-like protein regulates programmed cell Bax and promotes cell death. Cell 1995; 80: 285–291.

death downstream of Bcl-xL and cytochrome c. Mol Cell Biol 752 Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the 1998; 18: 608–615. BCL2 family and cell death Blood 1996; 88: 386–401. 731 Kim Y-M, Talanian RV, Billiar TR. Nitric oxide inhibits apoptosis 753 Kroemer G. The proto-oncogene Bcl-2 and its role in regulating by preventing increases in caspase-3-like activity via two distinct apoptosis. Nature Med 1997; 3: 614–620. mechanisms. J Biol Chem 1997; 272: 31138–31148. 754 Mignotte B, Vayssiere JL. Mitochondria and apoptosis. Eur J 732 Li J, Billiar TR, Talanian RV, Kim YM. Nitric oxide reversibly Biochem 1998; 252: 1–15. inhibits seven members of the caspase family via S-nitrosylation. 755 Zamzami N, Brenner C, Marzo I, Susin SA, Kroemer G. Subcellu- Biochem Biophys Res Commun 1997; 240: 419–424. lar and submitochondrial mode of action of Bcl-2-like oncoprote- 733 Mohr S, Zech B, Lapetina EG, Brune B. Inhibiton of caspase-3 ins. Oncogene 1998; 16: 2265–2282. by S-nitrosylation and oxidation caused by nitric oxide. Biochem 756 Troppmair J, Rapp UR. Apoptosis regulation by Raf, Bcl-2 and Biophys Res Commun 1997; 238: 387–391. R-Ras. Rec Res Can Res 1997; 143: 245–249. Review WL Blalock et al 1164 757 Hickman JA, Fisher TC, Milner AE, Gregory CD, Jackman AJ, CED-3 processing and CED-3-induced apoptosis. Curr Biol 1997; Aherne GN, Hartley JA, Sive C. Bcl-2 modulation of apoptosis 7: 455–460. induced by anticancer drugs: resistance to thymidylate stress is 781 Huang DCS, Adams JM, Cory S. The conserved N-terminal BH4 independent of classical resistance pathways. Cancer Res 1993; domain of Bcl-2 homologues is essential for inhibition of 53: 3321–3326. apoptosis and interaction with CED-4. EMBO J 1998; 17: 758 Young LS, Eliopoulos AG, Kerr DJ, Herod J, Hodgkins L, Krajew- 1029–1039. ski S, Reed JC. The control of apoptosis and drug resistance in 782 Hu Y, Benedict MA, Wu D, Inohara N, Nunez G. Bcl-XL interacts ovarian cancer: inﬂuence of p53 and Bcl-2. Oncogene 1995; 11: with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation. 1217–1228. Proc Natl Acad Sci USA 1998; 95: 4386–4391. 759 Green DR, Reed C. Mitochondria and apoptosis. Science 1998; 783 Strasser A, Harris AW, Huang DCS, Krammer PH, Cory S. Bcl- 281: 1309–1312. 2 and Fas/APO-1 regulate distinct pathways to lymphocyte 760 Thornberry N, Lazebnik Y. Caspases: enemies within. Science apoptosis. EMBO J 1995; 14: 6136–6147. 1998; 281: 1312–1316. 784 Newton K, Harris AW, Bath ML, Smith KGC, Strasser A. A domi- 761 Evan G, Littlewood T. A matter of life and cell death. Science nant interfering mutant of FADD/MORT1 enhances deletion of 1998; 281: 1317–1322. autoreactive thymocytes and inhibits proliferation of mature T 762 Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell sur- lymphocytes. EMBO J 1998; 17: 706–718. vival. Science 1998; 281: 1322–1326. 785 Smith KGC, Strasser A, Vaux DL. CrmA expression in T lympho- 763 Reed JC, Miyashita T, Takayama S, Wang HG, Sato T, Krajewski cytes of transgenic mice inhibits CD95 (Fas/APO-1)-transduced S, Aime-Sempe C, Brodrug S, Kitada S, Hanada M. BCL-2 family apoptosis, but does not cause lymphadenopathy or autoimmune proteins: regulators of cell death involved in the pathogenesis disease. EMBO J 1996; 15: 5167–5176. of cancer and resistance to therapy. J Cell Biochem 1996; 60: 786 Petit PX, Susin S-A, Zamzami N, Mignotte B, Kroemer G. Mito- 23–32. chondria and programmed cell death: back to the future. FEBS 764 Newmeyer DD, Kluck RM, Bossy-Wetzel E, Green DR. The Lett 1996; 396: 7–13. release of cytochrome c from mitochondria: a primary site for 787 Zamzami N, Susin SA, Marchetti P, Hirsh T, Gomez-Monterrey Bcl-2 regulation of apoptosis. Science 1997; 275: 1132–1136. I, Castedo M, Guido K. Mitochondrial control of nuclear 765 Wang X, Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, apoptosis. J Exp Med 1996; 183: 1533–1544. Peng T, Jones DP. Prevention of apoptosis by Bcl-2: release of 788 Zoratti M, Szabo I. The mitochondrial permeability transition. cytochrome c from mitochondria blocked. Science 1997; 275: Biochim Biophys Acta 1995; 1241: 139–176. 1129–1132. 789 Minn AJ, Velez P, Schendel SL, Liang H, Muchmore SW, Fesik

766 Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, SW, Fill M, Thompson CB. Bcl-xL forms an ion channel in syn- Turka LA, Mao X, Nunez G, Thompson CB. Bcl-x,abcl-2-related thetic lipid membranes. Nature 1997; 385: 353–357. gene that functions as a dominant regulator of cell death. Cell 790 Antonsson B, Conti F, Ciavatta A, Montessuit S, Lewis S, Mar- 1993; 74: 597–608. tinou I, Bernasconi L, Bernard A, Mermod J-J, Mazzei G, Maund- 767 Reed JC, Zha H, Aime-Sempe C, Takayama S, Wang HG. Struc- rell K, Gambale F, Sadoul R, Martinou J-C. Inhibition of Bax ture-function analysis of Bcl-2 family protein, regulators of pro- channel-forming activity by Bcl-2. Science 1997; 277: 370–372. grammed cell death. Adv Exp Med Biol 1996; 406: 99–112. 791 Schendel SL, Xie Z, Montal MO, Matsuyama S, Montal M, Reed 768 Thompson CB, Gajewski TF. Apoptosis meets signal transduc- JC. Channel formation by antiapoptotic protein Bcl-2. Proc Natl tion: elimination of a Bad inﬂuence. Cell 1996; 87: 589–592. Acad Sci USA 1997; 94: 5113–5118. 769 Conradt B, Horvitz HR. The C. elegans protein EGL-1 is required 792 Schlesinger PH, Gross A, Yin XM, Yamamoto K, Saito M, Waks- for programmed cell death and interacts with the Bcl-2-like pro- man G, Korsmeyer SJ. Comparison of the ion channel character- tein CED-9. Cell 1998; 93: 519–529. istics of proapoptotic BAX and antiapoptotic BCL-2. Proc Natl 770 Chittenden T, Flemington C, Houghton AB, Ebb RG, Gallo GJ, Acad Sci USA 1997; 94: 11357–11362. Elangovan B, Chinnadurai G, Lutz RJ. A conserved domain in 793 Lam M, Bhat MB, Nunez G, Ma J, Distelhorst CW. Regulation Bak, distinct from BH1 and BH2, mediates cell death and protein of Bcl-xl channel activity by calcium. J Biol Chem 1998; 273: binding functions. EMBO J 1995; 14: 5589–5596. 17307–17310. 771 Kelekar A, Thompson CB. Bcl-2 family proteins: the role of the 794 Deveraux QL, Takahashi R, Salvesen GS, Reed JC. X-linked IAP BH3 domain in apoptosis. Trends Cell Biol 1998; 8: 324–330. is a direct inhibitor of cell death proteases. Nature 1997; 388: 772 Wang K, Yin X-M, Chao DT, Milliman CL, Korsmeyer SJ. BID: 300–304. a novel BH3 domain-only death agonist. Genes Dev 1996; 10: 795 Rosse T, Olivier R, Monney L, Rager M, Conus S, Fellay I, Jansen 2859–2869. B, Borner C. Bcl-2 prolongs cell survival after Bax-induced 773 Inohara N, Ding L, Chen S, Nunez G. Harakiri, a novel regulator release of cytochrome c. Nature 1998; 393: 496–499. of cell death, encodes a protein that activates apoptosis and inter- 796 Kharbanda S, Pandey P, Schoﬁeld L, Israels S, Roncinske R, Yosh- acts selectively with survival-promoting proteins Bcl-2 and Bcl- ida K, Bharti A, Yuan ZM, Saxena S, Weichselbaum R, Nalin C,

X(L). EMBO J 1997; 16: 1686–1694. Kufe DW. Role for Bcl-xL as an inhibitor of cytosolic cytochrome 774 O’Connor L, Strasser A, O’Reilly LA, Hausmann G, Adams JM, c accumulation in DNA-damage induced apoptosis. Proc Natl Cory S, Huang DC. Bim: a novel member of the Bcl-2 family Acad Sci USA 1997; 94: 6939–6942. that promotes apoptosis. EMBO J 1998; 17: 384–395. 797 Wang HG, Rapp UR, Reed JC. Bcl-2 targets the protein kinase 775 Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM. FADD, a Raf-1 to mitochondria. Cell 1996; 87: 629–638. novel death domain-containing protein, interacts with the death 798 Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phos- domain of Fas and initiates apoptosis. Cell 1995; 81: 505–512. phorylation of death agonist Bad in response to survival factor 776 Chaudhary D, O’Rourke K, Chinnaiyan AM, Dixit VM. The death results in binding to 14–3-3 not Bcl-xL. Cell 1996; 87: 619–628. inhibitory molecules CED-9 and CED-4L use a common mech- 799 Craddock BL, Orchiston EA, Hinton HJ, Welham MJ. Dis- anism to inhibit the CED-3 death protease. J Biol Chem 1998; sociation of apoptosis from proliferation, protein kinase B acti- 273: 17708–17712. vation, and Bad phosphorylation in interleukin-3-mediated phos- 777 Spector MS, Desnoyers S, Hoeppner DJ, Hengartner MO. Inter- phoinositide 3-kinase signaling. J Biol Chem 1999; 274: action between the C. elegans cell-death regulators CED-9 and 10633–10640. CED-4. Nature 1997; 385: 653–656. 800 Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcrip- 778 Wu D, Wallen HD, Nunez G. Interaction and regulation of sub- tional activator of the human bax gene. Cell 1995; 80: 293–299. cellular localization of CED-4 by CED-9. Science 1997; 275: 801 White E, Chiou S-K, Rao J. Bcl-2 blocks p53-dependent 1126–1129. apoptosis. Mol Cell Biol 1994; 14: 2556–2563. 779 James C, Gschmeissner S, Fraser A, Evan GI. CED-4 induces 802 Levine AJ. The tumor suppressor genes. Ann Rev Biochem 1994; chromatin condensation in Schizosaccharomyces pombe and is 62: 623–651. inhibited by direct physical association with CED-9. Curr Biol 803 Desoize B. Anticancer drug resistance and the inhibition of 1997; 7: 246–252. apoptosis. Anticancer Res 1994; 14: 2291–2294. 780 Seshagiri S, Miller LK. Caenorhabditis elegans CED-4 stimulates 804 Fujita N, Tsuro T. Involvement of Bcl-2 cleavage in the acceler- Review WL Blalock et al 1165 ation of VP-16-induced U937 cell apoptosis. Biochem Biophys 825 Dexter TM, Garland J, Scott D, Scolnick E, Metcalf D. Growth Res Comm 1998; 246: 484–488. of factor-dependent hemopoietic precursor cell lines. J Exp Med 805 Klucar J, Al-Rubeai M. G2 cell cycle arrest and apoptosis are 1982; 152: 1036–1047. induced in Burkitt’s lymphoma cells by the anticancer agent ora- 826 McKearn JP, McCubrey JA, Fagg B. Enrichment of hematopoietic cin. FEBS Lett 1997; 400: 127–130. precursor cells and cloning of multipotential B lymphocyte pre- 806 Haldar S, Chintapilli J, Croce CM. Taxol induces Bcl-2 phos- cursors. Proc Natl Acad Sci USA 1985; 82: 7414–7418. phorylation and death of prostate cancer cells. Cancer Res 1996; 827 Palacios R, Steinmetz M. IL-3 dependent mouse clones that 56: 1253–1255. express B-220 surface antigens, contain Ig genes in germline con- 807 Fisher TC, Milner AE, Gregory CD, Jackman AL, Aherne W, Hart- figuration, and generate B-lymphocytes in vivo. Cell 1995; 41: ley JA, Dive C, Hickman JA. Bcl-2 modulation of apoptosis 727–734. induced by anticancer drugs: resistance to thymidylate stress is 828 Greenberger JS, Sakakeeny MA, Humphries RK, Eaves CJ, Eckner independent of classical resistance pathways. Cancer Res 1993; RJ. Demonstration of permanent factor-dependent multipotential 53: 3321–3326. (erythroid/neutrophil/basophil) hematopoietic progenitor cell 808 Miyashita T, Reed JC. Bcl-2 oncoprotein blocks chemotherapy- lines. Proc Natl Acad Sci USA 1983; 80: 2931–2935. induced apoptosis in a human leukemia cell line. Blood 1993; 829 Rovera G, Valtieri M, Mavilio F, Reddy EP. Effect of Abelson 81: 151–157. murine leukemia virus on granulocytic differentiation and 809 Reed JC. Bcl-2: prevention of apoptosis as a mechanism of drug interleukin-3 dependence of a murine progenitor cell line. Onco- resistance Hematology/Oncology Clin North Am 1995; 9: gene 1987; 1: 29–35. 451–473. 830 McCubrey JA, Steelman LS, Viral models for understanding 810 Gottlieb E, Hoffover R, von Ru¨den T, Wagner EF, Oren M. chronic myelogenous leukemia. In: AB Deisseroth, RB Down-regulation of wild-type p53 interferes with apoptosis of Arlinghaus (eds). Hematology Volume 13, Chronic Myelogenous IL-3-dependent hematopoietic cells following IL-3 withdrawal. Leukemia Marcel Dekker: New York, 1991, pp 137–152. EMBO J 1994; 13: 1368–1374. 831 Cook WD, Metcalf D, Nicola NA, Burgess AW, Walker F. Malig- 811 Piche A, Grim J, Rancourt C, Gomez-Navarro J, Reed JC, Curiel nant transformation of a growth factor-dependent myeloid cell DT. Modulation of Bcl-2 protein levels by an intracellular anti- line by Abelson virus without evidence of an autocrine mech- Bcl-2 single-chain antibody increases drug-induced cytotoxicity anism. Cell 1985; 41: 677–683. in the breast cancer cell line MCF-7. Cancer Res 1998; 58: 832 Pierce JH, Di Fiore PP, Aaronson SA, Potter M, Pumphrey J, Scott 2134–2140. A, Ihle JN. Neoplastic transformation of mast cells by Abelson- 812 Dorai T, Olsson CA, Katz AE, Buttyan. Development of a ham- MuLV: abrogation of IL-3 dependence by a nonautocrine mech- merhead ribozyme against bcl-2. I. preliminary evaluation of a anism. Cell 1985; 41: 685–693. potential gene therapeutic agent for hormone-refractory human 833 Mathey-Prevot B, Nabel G, Palacios R, Baltimore D. Abelson prostate cancer. Prostate 1997; 32: 246–258. virus abrogation of interleukin-3 dependence in a lymphoid cell 813 Dorai T, Goluboff ET, Olsson CA, Buttyan R. Development of line. Mol Cell Biol 1986; 6: 4133–4135. a hammerhead ribozyme against Bcl-2. II Ribozyme treatment 834 Kipreos ET, Wang JY. Reversible dependence on growth factor sensitizes hormone-resistant prostate cancer cells to apoptotic interleukin-3 in myeloid cells expressing temperature sensitive v- agents. Anticancer Res 1997; 17: 3307–3312. abl oncogene. Oncogene Res 1988; 2: 277–284. 814 Wang H-G, Miyashita T, Takayama S, Sato T, Torigoe T, Krajew- 835 Pierce JH, Ruggiero M, Fleming TP, DiFiore PP, Greenberger JS, ski S, Tanaka S, Lawrence Hovey I, Troppmair J, Rapp UR, Reed Varticovski L, Schlessinger J, Rovera G, Aaronson SA. Signal JC. Apoptosis regulation by interaction of Bcl-2 protein and Raf- transduction through the EGF receptor transfected intp IL-3- 1 kinase. Oncogene 1994; 9: 2751–2756. dependent hematopoietic cells. Science 1988; 239: 628–631. 815 Reed JC, Wang HG, Takayama S, Rapp UR. Bcl-2 interacting 836 Shounan Y, Miller M, Symonds G. Transformation of FDC-P1 protein, Bag-1, binds to and activates the kinase Raf-1. Proc Natl cells to IL-3 independence by a murine retrovirus containing v- Acad Sci USA 1996; 93: 7063–7068. erb-B. Exp Hematol 1995; 23: 492–499. 816 Packham G, Brimmell M, Cleveland JL. Mammalian cells express 837 Miller M, Kennewell A, Takayama Y, Bruskin A, Bishop JM, John- two differently localized Bag-1 isoforms generated by alternative son G, Symonds G. Transformation of early erythroid precursor translation initiation. Biochem J 1997; 328: 807–813. cells (BFU-E) by a recombinant murine retrovirus containing v- 817 Sekiya M, Adachi M, Takayama S, Reed JC, Imai K. IFN-gamma erb-B. Oncogene 1990; 5: 1125–1131. upregulates anti-apoptotic gene expression and inhibits apoptosis 838 Wheeler EF, Askew D, May S, Ihle JN, Sheer CJ. The v-fms onco- in IL-3-dependent hematopoietic cells. Biochem Biophys Res gene induces factor-independent growth and transformation of Comm 1997; 239: 401–406. the interleukin-3-dependent myeloid cell line FDC-P1. Mol Cell 818 Clevenger CV, Thickman K, Ngo W, Chang WP, Takayama S, Biol 1987; 7: 1673–1680. Reed JC. Role of Bag-1 in the survival and proliferation of the 839 Rohrschneider LR, Metcalf D. Induction of macrophage colony- cytokine-dependent lymphocyte lines, Ba/F3 and Nb2. Mol Endo 1997; 11: 608–618. stimulating factor-dependent growth and differentiation after 819 Lin EY, Orlorsky A, Wang HG, Reed JC, Prystowsky MB. A1, a introduction of the murine c-fms gene into FDC-P1 cells. Mol Bcl-2 family member, prolongs cell survival and permits myeloid Cell Biol 1989; 9: 5081–5092. differentiation. Blood 1996; 87: 983–992. 840 Anderson SM, Carol DM, Lee FD. Abrogation of IL-3 dependent 820 Bischoff JR, Fernandez-Sarabia MJ. Bcl-2 associates with the Ras- growth requires a functional v-src gene product: evidence for an related protein R-Ras p23. Nature 1993; 366: 274–275. autocrine growth cycle. Oncogene 1990; 5: 317–325. 821 Chen CY, Faller DV. Phosphorylation of Bcl-2 protein and associ- 841 Overell RW, Watson JD, Gallis B, Weisser KE, Cosman D, ation with p21Ras in Ras-induced apoptosis. J Biol Chem 1996; Widmer MB. Nature and specificity of lymphokine indepen- 271: 2376–2379. dence induced by a selectable retroviral vector expressing v-src. 822 Kitamura T, Tange T, TeRasawa T, Chiba S, Kuwaki T, Miyagawa Mol Cell Biol 1987; 7: 3394–3401. K, Piao Y-F, Miyazono K, Urabe A, Takaku F. Establishment and 842 McCubrey JA, Smith SR, Algate PA, de Vente JE, White MK, Steel- characterization of a unique human cell line that proliferates man LS. Retroviral infection can abrogate the factor dependency dependently on GM-CSF, IL-3 or erythropoietin. J Cell Physiol of hematopoietic cells by autocrine and non-autocrine mech- 1989; 140: 323–334. anisms depending on the presence of a functional viral oncog- 823 Avanzi A, Lista P, Giovinazzo B, Miniero R, Saglio G, Benneton ene. Oncogene 1993; 8: 2905–2915. G, Coda R, Cattoretti B, Pegoraro L. Selective growth response 843 Meckling-Gill KA, Yee SP, Schrader JW, Pawson T. A retrovirus to IL-3 of a human leukaemic cell line with megakaryoblastic encoding the v-fps protein-tyrosine kinase induces factor-inde- features. Br J Haematol 1988; 69: 355–359. pendent growth and tumorigenicity in FDC-P1 cells. Biochim 824 Quentmeier H, Zaborski M, Drexler HG. Effector of thrombopo- Biophys Acta 1992; 1137: 65–72. ietin, interleukin-3 and the kinase inhibitor K-252a on growth 844 McCubrey JA, Steelman LS, Wang X-Y, Algate PA, Hoyle PE, and polyploidization of the megakaryocytic cell line M-07e. Leu- White C, Davidian EW, Prevost KD, Robbins P, Mylott D, White kemia 1998; 12: 1603–1611. MK. Differential effects of viral and cellular oncogenes on the Review WL Blalock et al 1166 growth factor-dependency of hematopoietic cells. Int J Oncol growth by retroviral transduction and expression of p210bcr/abl. 1995; 7: 295–310. Blood 1992; 80: 1788–1797. 845 McCubrey JA, Steelman LS, Wang XY, Davidian EW, Hoyle PE, 857 Rubnitz JE, Pui C-H, Downing JR. The role of TEL fusion genes White CR, Prevost KD, Prevost KD, Algate PA, Robbins P, Mylott in pediatric leukemias. Leukemia 1999; 13: 6–13. D, White MK. Autocrine growth factor secretion after transform- 858 Okuda K, Golub TR, Gilliland DG, Griffin JD. p210BCR/ABL, ation of human cytokine-dependent cells by viral and cellular p190/BCR/ABL, and TEL/ABL activate similar signal transduction oncogenes. Int J Oncol 1995; 7: 573–586. pathways in hematopoietic cells. Oncogene 1996; 13: 11147– 846 Hibi S, Lohler J, Friel J, Stocking C, Ostertag W. Induction of 11152. monocytic differentiation and tumorigenicity by v-Ha-ras is dif- 859 Golub TR, Goga A, Barker GF, Afar DE, McLaughlin J, Bohlander ferentiation-arrested hematopoietic cells. Blood 1993; 81: SK, Rowley JD, Witte ON, Gilliland DG. Oligomerization of the 1841–1848. abl tyrosine kinase by the Ets protein TEL in human leukemia. 847 Mandanas RA, Leiboiwitz DS, Gharehaghi K, Tauchi T, Burgess Mol Cell Biol 1996; 16: 4107–4116. GS, Miyazawa K, Jayarem HN, Boswell HS. Role of 21Ras in 860 Lacronique V, Boureux A, Valle VD, Poirel H, Quang CT, Mau- p210 bcr-abl transformation of murine myeloid cells. Blood chauffe M, Berthou C, Lessard M, Berger R, Ghysdael J, Bernard 1993; 82: 1838–1847. OA. A Tel-Jak2 fusion protein with constitutive kinase activity in 848 Uemura N, Ozawa K, Tojo A, Takahashi K, Okano A, Karasuy- human leukemia. Science 1997; 278: 1309–1312. ama H, Tani K, Asano S. Acquisition of interleukin-3 indepen- 861 Gubina E, Rinaudo MS, Szallasi Z, Blumberg PM, Mufson RA. dence in FDC-P2 cells after transfection with the activated c-H- Overexpression of protein kinase C isoform epsilon but not delta ras gene using a bovine papillomavirus-based plasmid vector. in human interleukin-3-dependent cells suppresses apoptosis and Blood 1992; 80: 3198–3204. induces bcl-2 expression. Blood 1998; 91: 823–829. 849 Daley GQ, Baltimore D. Transformation of an interleukin-3 862 Rinaudo MS, Su K, Falk LA, Halder S, Mufson RA. Human dependent hematopoietic cell line by the chronic myelogenous interleukin-3 receptor modulates Bcl-2 mRNA and protein levels leukemia-specific p210 BCR-ABL protein. Proc Natl Acad Sci through protein kinase C in TF-1 cells. Blood 1995; 86: 80–88. USA 1988; 85: 9312–9315. 863 Mufson RA. The role of serine/threonine phosphorylation in hem- 850 Hariharan IK, Adams JM, Cory S. bcr-abl oncogene renders atopoietic cytokine receptor signal transduction. FASEB J 1997; 11: 37–44. myeloid cell line factor independent: potential autocrine mech- 864 Perkins GR, Marshall CJ, Collins MKL. The role of MAP kinase anism in chronic myeloid leukemia. Oncogene Res 1988; 3: in interleukin-3 stimulation of proliferation. Blood. 1996; 87: 387–399. 3669–3675. 851 Spooncer E, Fairbairn L, Cowling GJ, Dexter TM, Whetton AD, 865 Hoyle PE, Steelman LS, McCubrey JA. Autocrine transformation Owen-Lynch PJ. Biological consequences of p60v-abl protein of human hematopoietic cells after transfection with an activated tyrosine kinase activity in a primitive, multipotent haemopoietic granulocyte/macrophage colony stimulating factor gene. Cytokin cell line. Leukemia 1994; 8: 620–630. Cell Mol Ther 1997; 3: 159–168. 852 Kabarowski JH, Allen PB, Wiedemann LM. A temperature sensi- 866 Reilly I, Kozlowski R, Russell NH. Heterogeneous mechanisms tive p210 BCR-ABL mutant defines the primary consequences of of autocrine growth of AML blast cells. Br J Haematol 1989; 72: BCR-ABL tyrosine kinase expression in growth factor dependent 363–369. cell lines. EMBO J 1994; 13: 5887–5895. 867 Rogers SY, Bradbury D, Kozlowski R, Russel NH. Evidence for 853 Sirard C, Laneuville P, Dick JE. Expression of bcr-abl abrogates internal autocrine regulation of growth in acute myeloblastic leu- factor-dependent growth of human hematopoietic M07E cells by kemia cells. Exp Hematol 1994; 22: 593–598. an autocrine mechanism. Blood 1994; 83: 1575–1585. 868 Young DC, Griffin JD. Autocrine secretion of GM-CSF in acute 854 Takahashi M, Furukawa T, Tanaka I, Ohsawa Y, Nikkuni K, Oaki myeloblastic leukemia. Blood 1986; 68: 1178–1181. A, Goto T, Hahimoto T, Kishi K, Koike T. Transfection of the 869 Sewing A, Wiseman B, Lloyd AC, Land H. High-intensity Raf bcr/abl oncogene into factor-dependent cells by electroporation: causes cell cycle arrest mediated by p21Cip1. Mol Cell Biol acquisition of autonomous proliferation. Hematol Oncol 1994; 1997; 17: 5588–5597. 12: 53–60. 870 Ito T, Deng X, Carr B, May WS. Bcl-2 phosphorylation required 855 Laneuville P, Heisterkamp N, Groffen J. Expression of the chronic for anti-apoptosis function. J Biol Chem 1997; 272: 11671– myelogenous leukemia-associated p20bcr/abl oncoprotein in a 11673. murine IL-3 dependent myeloid cell line. Oncogene 1991; 6: 871 Deng X, Ito T, Carr B, Mumby M, May WS. Reversible phos- 275–282. phorylation of Bcl2 following interleukin 3 or bryostatin 1 is 856 Laneuville P, Sun G, Timm M, Vekemans M. Clonal evolution mediated by direct interaction with protein phosphatase 2A. J in a myeloid cell line transformed to interleukin-3 independent Biol Chem 1998; 273: 34157–34163.