High-Intensity Raf Signals Convert Mitotic Cell Cycling Into Cellular Growth'

(CANCERRESEARCH58. 1636-1640. April 15. 19981 Advances in Brief

High-intensity Raf Signals Convert Mitotic Cell Cycling into Cellular Growth'

Eugen Kerkhoff and Ulf R. Rapp2

lnstitutfÃ¼rmedizinischeStrahlenkundeund Zellforschung(MSZ). Universityof Wurzburg,97078 Wiirzburg, Germany

Abstract oncogernc c-Raf-1-estrogen receptor fusion protein (c-Raf-1-BxB ERTM). Activation of the exogenous kinase induces high and sustained The selectionof NIH 3T3 cellsexpressinga hydroxytamoxifen-induci c-Raf signals and inhibits DNA synthesis and mitosis. Despite the ble c-Raf-1-estrogen receptor fusion protein (c-Raf-1-BxB-ER@) in the inhibition of cell cycle progression, cell growth remains unaffected, absence or presence of the inducer results in dramatic differences in the expression levels of the fusion protein. Hydroxytamoxifen-mediated con leading to cells with a DNA content of G1 cells and a cell size of stitutive activation of the Rafsignal favors the selection ofcells expressing G2-M-phase cells. Therefore, high-intensity Raf signals uncouple the low levels of c-R.af-1-BxB-ER@. Cells selected in the absence of hy regulation of cell growth from DNA replication and mitosis. droxytamoxifen express up to 20 tImes higher levels of the inducible Raf kinase. Activation ofthe oncogenic Rafkinase in cells expressing low levels Materials and Methods leads to a weak activation of the Raf/Mek/Erk cascade and the induction of S phase In confluentcells.The activationof cellsexpressinghigh levels Expression Vector. The pBabe-puro-BxB-ER@ vector was constructed by ofthe kinaseleadsto a strongpersIstentsignalandinhibitsDNAsynthesis releasing sequences encoding the c-Raf-l-BxB-ER@ protein from the BJ4- andmitOsisinproliferatingcells.TheinhibitionofDNAsynthesisandcell BxB-ER@ vector (7) by EcoRI digestion (2.2-kb fragment) and inserting them dIvision is presumably due to the elevated expression ofthe cyclin-depend into the unique EcoRI recognition site of the pBabe-puro vector (13). @@ ent kinaseinhibitorp21@', cellsexposedto ionizingradiation. Cell Culture and Transfections. NIH 313 and N-BxB-ER'@ cells were Despite the inhibition of DNA synthesis and mitOSIS, the constltutive grown in DMEM (Life Technologies, Inc.) supplemented with glucose (Life activity of the Raf signaling pathway is still able to initiate cell growth. Technologies, Inc.), L-glutamine (Life Technologies, Inc.), 10% FCS (Hy Activationofthe high-intensityRafsignalin arrestedserum-starvedcells clone), 100 units/mi penicillin (Life Technologies,Inc.), and 100 @&g/mi induces cell growth up to a size corresponding to that of M-phase cells in streptomycin (Life Technologies, Inc.). The cells were incubated at 37Â°C,5% the absenceof DNAsynthesis.High-intensityRafsignalsin proliferating CO2.and90%humidity.FortheinductionofcellswithOHT,asolutionof 1 cells consistently lead to an accumulation of cells with the size of M-phase mM OHT in ethanol was added to the medium to a final concentration of 200 cellsandthe DNAcontentof G1cellsor G2-M-phasecells.Therefore,the flM (5000 X dilution). For puromycin selection, the cells were grown in the activation of Raf kinase is sufficient to drive cell growth, even in the presence of 6 @tg/mipuromycin (Sigma). The cells were transfected with a presence of high levels of the cydlin-dependent kinase inhibitor p21@1. high-efficiency liposome transfection method, using LipofectAMiNE reagent supplied by Life Technologies, Inc. (7). Introduction Immunoblot and Antibodies. Immunobbot experiments were performed as described previously (7). The following antibodies were used: (a) HL7, The Ras/Raf signal transduction cascade is an important mediator mouseestrogenreceptor-sensitiverabbitpolycbonalantibody(a gift from A. in the regulation of cell proliferation and differentiation. Experiments Sewing and H. Land); (b) C-19, cyclin A-sensitive affmity-purified rabbit in the rat pheochromocytoma cell line PC12 led to the discovery that polyclonal antibody (Santa Cruz); (c) C-19, p2l@@'-scnsifive affmity-purified the intensity and duration of the signal determine proliferation or rabbit pobyclonal antibody (Santa Cruz); (d) M-2, cdk2-sensitive affinity neuronal differentiation and cell cycle arrest (1). The extended activity purified rabbit pobycbonal antibody (Santa Cruz); (e) C-22, cdk4-sensitive affinity-purified rabbit polycbonal antibody (Santa Cruz); (f) phospho Eat of the RasfMeklErk cascade in this respect favors cellular differenti sensitive affinity-purified rabbit polyclonab antibody (New England Biolabs); ation. Constitutive activation of the Ras/Raf cascade in immortalized and (g) C-l6, Erkl-sensitive affinity-purified rabbit polycbonalantibody (Santa fibroblast cell lines can induce either cell cycle progression or cell Cruz). cycle arrest in the G@or G2-M phases (2â€”9).Ithas long been proposed Immunocomplex Kinase Assay. The kinase assays were performed as that high activity of the Ras signal is responsible for the growth arrest, described previously (7), with the following specifications. Cells were lysed because the expression of an activated Ras oncoprotein is only toler with a buffer containing 50 nmi HEPES (pH 7.5), 150 mMNaG, 1 mMEDTA, ated at very low levels in the absence of a second cooperating 1 mMEGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 2 @&g/mileupeptin,2 oncogene (2, 3). Experiments using dominant negative mutants of p53 lLg/mlaprotinin, 1 m@iphenylmethylsulfonybfluoride, 10mMsodium fluoride, protein have led to the conclusion that p53-related activity is an and 0.2 mM sodium orthovanadate. For immunodetection, the following anti important mediator of Ras-induced cell cycle arrest (10). The p21'@'@ bodies were used: (a) M-2, cdk2-sensitive affinity-purified rabbit polycbonal antibody (Santa Cruz); and (b) C-22, cdk4-sensitive affinity-purified rabbit cdk3 inhibitor is induced by p53 or p73, a recently discovered p53 polycbonalantibody (Santa Cruz). As substrates for the kinase reaction, 1 g@g homobogue (11, 12). Fibroblast cells isolated from p2l@'1-deficient of histone Hi (Boebringer) or 6 g.@gofGST-Rb (an 769-921; a kind gift from mice cannot be arrested by high-intensity Raf signals, indicating that R. Lilischkis) were used for each reaction. the cdk inhibitor is essential for Ras/Raf-mediated cell cycle arrest (9). Flow CytOmetry Analysis. The cells were fixed in ethanol, stained with We have established cell lines that express high levels of an inducible propidium iodide (Sigma), and analyzed with a FACSCalibur flow cytometer (BectonDickinson;Ref.7). Received 2/25/98; accepted 3/2/98. Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage Results charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. High expression of constitutively active forms of the Ran or Raf I Supported by the Wilhelm Sander-Stiftung and the SFB 456. 2 To whom requests for reprints should be addressed, at Institut fÃ¼r medizinische oncoproteins has been shown to block cell proliferation (2, 3, 8, 9). To StrahlenkundeundZellforsehung.UniversityofWtirzburg,VersbacherStrasse5,97078 analyze the effects of high-intensity Raf signals on the cell cycle, it is Wurzburg, Germany. Phone: 49-931-201-5140; Fax: 49-931-201-3835. therefore essential to establish conditional cell lines expressing an 3 The abbreviations used are: cdk, cycin-dependent kinase; OHT, 4-hydroxytamox ifen; as. amino acid. inducible form of the oncoprotein. Fusion of the oncogernc c-Raf-l 1636

Raf SIGNALS AND CELL GROWTH

BxB deletion mutant to the hormone-binding domain of the estrogen N-BxB-ER@cells receptor (c-Raf-1-BxB-ER@) provides a 4 OHT-inducible kinase (7). On treatment with the inducer, the fusion protein is constitutively active. We transfected NIH 3T3 cells with a eukaryotic expression vector driving the constitutive expression of the c@Raf@l@BxB@ERTM kinase and a puromycin resistance protein (pBabe-puro-BxB-ER@). F)*d.1+@' fi4el+CIfI 0.1+0111 Selection for puromycin-resistant colonies in the presence or absence O@O30 224 30 2 24 0 10 30 2 24 of OHT resultsin dramaticdifferences in the expression levels of the

oncogenic Raf kinase. Clonal cell lines selected while the kinase was â€”@----@----- @@@@@@@1@â€” â€”cyclinA active (N-BxB-ER@-FA clones) express around 20-fold less of the c-Raf-1-BxB-ER@ protein than clones selected in the absence of 01ff (N@BxB@ERTM@Clclones),in whichthe kinaseis inactive(Fig. 1). This is in agreement with the finding that high levels of activity of the oncogenic Raf kinase areinhibitoryfor cell proliferation.The fact @ that all four isolated FA clones express comparable low levels of the .@â€”â€”- â€”Ii =pbosphoErkl,2 inducible c-Raf-1 kinase suggests that there is a sharp tolerance threshold up to which proliferation is possible. The addition of 01ff to all clones expressing high levels of the :::__@__@_@_ â€”ii: .-Erkl,2 inducible Raf kinase causes a cell cycle arrest in the G1 and G2-M phases (Fig. 4B; data not shown). Within 24 h, the cells go out of S Fig. 2. Activation of the c-Rrf-l-BxB-ER@ protein in N@BxB@ERTM@Cl@lcellsby OHT leads to the induction of a strong and persistent Raf signal as measured by Erk phospho phase and accumulate with a 2N and 4N DNA content. All additional rylation and a rapid induction of overexpression of the cdk inhibitor [email protected] analyses were performed with the cell clones N-BxB-ER@-Cl-l and ERâ€•@-FA-landCl-I cells were grown to confluence (0) and subsequently stimulated with 10% serum (FA-l) or OHT (FA-l, Cl-f) for 10 mm, 30 mm, 2 h, and 24 h. Total protein FA-l, which representa high- andlow-expressingclone. Activationof lysates were separated by SDS-PAGE and analyzed by immunoblotting. Antibodies the Raf kinase triggersa kinase cascade, leading to the phosphoryla specific for cyclin A, p2ldhI@@,phosphoErkl,2 and Erkl,2 were used. tion of a central tyrosine residue of the Erk kinases (14). To analyze the strength of the Raf signal in the different cell clones, we compared the serum-mediatedErk phosphorylationwith the Erk phosphoryla tion found after OHT treatment of N-BxB-ER@-Cl-l and Fa-l cells by N@BxB@[email protected] using an Erk phosphotyrosine-specific antibody (Fig. 2). The addition of serumto arrestedconfluentN-BxB-ER@-FA-l cells in the absence highserum,subconfluent of 01ff led to a transient phosphorylation of the Erk kinases, peaking within 10 rain after the stimulation (Fig. 2). The addition of OHT to arrested confluent N-BxB-ER@-FA-l cells expressing only low levels of the c-Raf-l-BxB-ER@ protein led to a modest increase in Erk phosphorylation within 30 mm, and the signal increased slightly 0 1 3 6 24 OHT induction, hours duringthe following 24 h (Fig. 2). The Erk phosphorylationseen in these cells, however, is much lower than the serum-mediated peak â€” histone HI activity. N-BxB-ER@-Cl-l cells express high levels of the inducible [email protected] in the absenceof OHT, these cells @ already exhibit a slightly increased level of Erk phosphorylation, -cdk2 indicating a slight leakiness of the inductionsystem (Fig. 2). Within 10 mm after OHT stimulation,the inducible Raf kinase mediated a strong and persistentincrease in Erkphosphorylation(Fig. 2). Corn pared to the peak Erk phosphorylation by serum stimulation, the â€” -= = â€”@ â€”GST-Rb

a M a â€” cdk4 N 41 2 3 4 1 2 3 Fig. 3. High-intensity Rat signals lead to a down-regulation of cdk2 and cdk4 kinase @@ @.:â€¢@: ; . :@â€”â€˜â€”;@â€” O.Rd.1@&lB@&M activity. Subeonfluent N-BxB-ER@ cells cultured under high-serum conditions (0) were induced with 01ff for 1,3, 6, and 24 h. The cells were lysed, and the cdk2 orcdk4 kinases were immunoprecipitated by the use of specific antibodies and protein A agarose. The @@@ â€”@â€” â€” @â€”â€” â€” cdb4 activity of the precipitated kinases was analyzed by incubating the cdk2 precipitates with histone HI and incubating the cdk4 precipitates with GST-Rb (as 769-921) as substrates in the presence of [y-32P]ATP.The proteins were separated by SDS-PAGE and blotted Fig. 1. Activation of the c-Raf-l-BxB-ER@ protein during the selection for clonal cells onto a nitrocellulose membrane. The phosphorylation of the substrates was analyzed by leads to a highly reduced expression of the oncogenic Rat kinase. NIH 3T3 cells were autoradiography, and the amount of the precipitated kinases was analyzed by immuno transfectedwitha eukaryoticexpressionvectorcarryingsequencesencodinga OHT detection. inducible oncogenic c-Raf-l/estrogen receptor fusion protein (c-Raf-l-BxB-ER@) and a puromycin resistance gene (pBabe-puro-BxB-ER@). After transfection, puromycin re sistance colonies were isolated in the absence of OHT (Cl clones) and in the presence of 01ff (FA.l-3 clones). The FA.1-3 clones were grown to confluence before the puromycin 01ff-mediated phosphorylation in N-BxB-ER@-Cl-l cells is not very was added. Clone FA.4 has been described previously (7) and was isolated by soft agar cloning in the presence of OHT. Total cellular protein lysates of the different clones and much higher. Whereas the serum-induced phosphorylation was tran NIH3T3cells(N)wereseparatedbySDS-PAGEandanalyzedbyimmunoblotting.The sient and declined within 2 h, the Erk phosphorylation induced by the clones selected in the absence of OHT (Cl) express markedly higher levels of the c-Raf-l-BxB-ER@' protein compared to clones selected in the presence of OHT (FA). As oncogenic Raf kinase remained high for 24 h. a controlforequalproteinloading,theexpressionofcdk4hasbeenanalyzed. The expression of cycin A protein is down-regulated in cells 1637

Raf SIGNALS AND CELL GROWTH

A C N-BxB-ER@-Cl-1cells N-a@B-&'@-a-1cells @w @rum highserum, subconfluent,-OHT @ D@om a

01 81.6% @ S 7.5% @11 -ONT 02/M 10.7 % @ all c.Iis 0 j1@frâ€¢@1@j0 0 R@ @ ssc@e, O@s005

S 1.8% Gi 41.5% 01 85.0% !j i 1 !Lrj4@ rj 02/M 13.0 % + OHT 29 Ii @@ S 46.9% RI fraction 0 0 G2IM11.@% 0â€¢ 1000 0 tom 0ffI@_ 0 tan @ sec@9N D@om a

2N 4N GI 86.0% S 1.8% + 01ff 48 h @ DNA content R2 traction 02/PA 12.0 S !011.4,I..I11*l,...j !0k@__A@ 0 woo 0 â€˜ tom 0 1000 R@ 2N 4N side scatter DNA content side scatter B D N..BXB@ER@M@CI@1

@ N-a@B-ER@-a-1@&h -vn@ 86Ã¨con@, *@ highserum,subconflu.nt

D@001 @.001

Gi 41.6% @I a . OHT @@ @2lM !OLJ@P,l*L. !@

0 1000 0 1000 -OHT FL2@ ssc@_

+ OHT 24 h

@ L:@â€˜@Li1T1Li0 toss 0 1000 R@

@@ -JL

+OHT48h +OHT48h @ @:/MIO@ !ei,[email protected] 0 1000 0 tom FL2@ 219 4N @ @@L_@ll A lii DNA content side scatter

Fig. 4. High-intensity Raf signals induce cell growth and inhibit DNA synthesis and mitosis. A, proliferating N-BxB-ER@-Cl-l cells were ethanol-fixed, stained with propidium iodide, and analyzed for their DNA content and side scatter properties by flow cytometry. The smaller G@phasecells (2NDNA content, Rifraction) exhibit lower side scatter values than the larger G2-M-phase cells (4NDNA content, R2fraction). B, subeonfluent proliferating (10% serum) N-BxB-ER@'-Cl-lcells (â€”0117)wereinduced with OHT for 24 and 48 h. The cells were ethanol-fixed, stained with propidium iodide, and analyzed for their DNA content and side scatter properties by flow cytometry. OHT treatment leads to an accumulation of large cells with high side scatter values and a 2N and 4N DNA content. C, N-BxB-ER@'-Cl-l cells were serum-starved for 24 h (â€”OHT)and subsequently induced with OHT for 29 and 48 h. The cells were ethanol-fixed, stained with propidium iodide, and analyzed for their DNA content and side scatter properties by flow cytometry. Activation ofthe Raf kinase in these cells does not induce S-phase entry but strongly induces cell growth, as shown by the accumulation ofcells with high side scatter values. D, N-BxB-ER@-Cl-l cells proliferating under high-serum conditions in the absence of OHT and cells induced for 48 h with 01ff were trypsinized and analyzed by phase-contrast microscopy to document the increase in cell size of the OHT-treated cells.

arrested by confluence (Fig. 2) and is strongly induced when the cells induce entry into the cell cycle and cell proliferation (7, 8, 9). For the reenter the S phase (15). Cyclin A protein expression is therefore an low-expressing clone FA-4 (Fig. 1), we have previously shown that indicator for the induction of S phase and cell proliferation. The activation of the c-Raf-l-BxB-ER@ protein in these cells is sufficient addition of serum or OHT to N-BxB-ER@-FA-l cells induces the to induce cell proliferation in cells arrested by serum starvation or expression of the cyclin A protein within 24 h (Fig. 2). This is confluence (7). In the high-expressing N@BxB@ERTM@Cl@lcells,we consistent with the finding that low and constitutive Raf signals detected the expression of the cyclin A protein even under cell density 1638

conditions, whereas the wild-type NIH 3T3 or N@BXB@ERTM@FAcells analysis of the DNA content of the cell population revealed that the were arrested(Fig. 2). This is presumablydue to the slight leakiness cells did not initiate DNA replication. The number of S-phase cells of the inductionsystem, enabling these cells to grow to much higher was decreased to background levels (1.8%) by the action of high cell densities. In contrast to N-BxB-ER@-FA-l cells, the treatment of intensity Raf signals. These data show that even under low-serum N-BxB-ER@-Cl-l cells with OHTcauseda strongdown-regulationof conditions, the activation of Raf kinase is sufficient to initiate cell the expression of the cyclin A protein (Fig. 2), underlining the growth. However, because of the inhibition of the cdks by increased antiproliferativecharacterof the high-intensityRaf signals. p2lÂ°@@levels,the cells were unable to replicate their DNA or undergo High-intensity Raf signals have been previously shown to induce mitosis. overexpression ofthe cdk inhibitor p21'1â€•t(8,9, 16). In N@BXB@ERTM@ Cl-i cells, we detecteda strongincreasein p2lC@expressionbetween Discussion 30 and 120 mm (2 h) of OHT induction (Fig. 2). The induction of the p2l@'1 protein is comparable to the induction seen after ionizing Cell growth, DNA replication, and cell division are characteristics radiation (data not shown). p2lc@ belongs to the class of cdk inhib of the mitotic cell cycle. Here we have shown that high-intensity Raf signals separate cellular growth from DNA replication and mitosis. itors. We have therefore analyzed the activity of the cdk2 and cdk4 The inhibition of DNA synthesis and cell division is most likely due idnases after 01ff stimulation of N-BxB-ER@-Cl-l cells. The activity ofthe kinases was determinedwith the help of in vitrocomplex kinase to the Raf-induced overexpression of the p2lÂ°@protein, leading to a down-regulation of cdk activity. The cdks therefore seem to be es assays using histone Hl as a substrate for cdk2 activity and GST-Rb sential for DNA replication and mitosis, but dispensible for the (aa 769-921) as a substrate for cdk4 activity. Although the kinetics differ, the activity of both kinases was down-regulated after OHT regulation of cell growth. Activation of the Myc oncoprotein in early treatment (Fig. 3). The cdk2 activity had already declined to very low G1 cells leads to a premature induction of cdk and E2F activity (19). levels by 1 h afterOHT addition.The decline of the cdk4 activity was Interestingly, these processes do not lead to a premature entry into S delayed and occurredbetween 6 and 24 h. The basis for the different phase. The onset of DNA replication remains delayed until the cells kinetics of down-regulation is unclear. One reason could be that in have reached a certain cell size. Taking these results and the results obtained in our studies together, it becomes clear that for the initiation contrast to cydlin/cdk2 complexes, which seem to be stable by them selves, the cyclin/cdk4 complexes need the p2l'1'@oranothermember of DNA replication, both cell growth and cdk activity are necessary, of the corresponding family of cdk inhibitors as an assembly factor and that each of the two processes can be regulated independently. An (17,18). interesting open question is the targets of Raf signaling in the induc High-intensity Raf signals impose a scenario on cells in which the tion of cell growth. The phenotype seen in N-BxB-ER@ cells may resemble the situation of terminal cell differentiation, in which cells components of the cell cycle machinery are silenced by the overex pression of the cdk inhibitor p21CIP1,and the Raf-triggered rnitogen (for example, neurons) show cell growth in the absence of DNA activated protein kinase cascade is constitutively active. Under these replication. In this respect, one may ask whether the induction of the conditions, the DNA synthesis as well as the cell division is inhibited, p2l'1â€•protein by the high-intensity Raf signals is a natural target of leading to an accumulation of cells with a 2N and 4N DNA content Raf signaling and hence an essential factor for Raf-mediated differ corresponding to a cell cycle arrest in the G1 and G2-M phases (Fig. entiation processes, or whether the induction of the p21C@Plprotein is 4B). By observing OHT-treated N-BxB-ER@-Cl-l cells with the help a stress response to artificially high Raf signals in our cell culture of a phase-contrastmicroscope, we discovered that the OHT-treated experiments. From a comparison of the serum-mediated Raf signal @ cells bad a much larger cell size than the untreated cells (Fig. 4D). The with the Raf signal reached in cells after OHT larger G2-M-phase cells exhibited higher side scatter values than the treatment (Fig. 2), we conclude that the cell cycle-inhibitory Raf smaller G@phase cells, allowing us to analyze the increase in cell size signals are not unphysiologically high. The observation that the with the help of a flow cytometer (Fig. 4A). Analyses of the side p2lÂ°@1â€•1proteinis elevated during nerve growth factor-mediated dif scatter values of a population of exponentially growing N-BxB-ER@Â° ferentiation of PC12 cells (20) may argue as well for a Raf/p2lc@ Cl-i cells untreated and treated with OHT for 24 and 48 h revealed a mechanism in differentiation. Experiments analyzing the still-un shift of the cell population to higher side scatter values. Although the known regulation mechanism of p2lciPl by Raf and the role of the DNA content of the cells did not change between 24 and 48 h (63% p2lÂ°@t@'1proteinin differentiation processes may help to clarify whether G1, 2% S, and 34% G2-M phases), nearly all cells had side scatter the high-intensity Raf signals described here can be considered to be values corresponding to the cell sizes of G2-M-phase cells after 48 h stress or differentiation signals. of treatment.These data show that the G5 cells have grown in the absence of DNA replication to a cell size of G2-M-phase cells. Acknowledgments Because the side scatter values do not rise higher than a threshold We thank A. Sewing and H. Land for providing the HL7 antiserum and R. value of the magnitude of the G2-M-phase cells, we conclude that the Lilischkis for providing the GST-Rb substrate. We thank S. Loffler for tech Raf signal is not able to induce cell growth in G2-M-phase cells. We nical assistance and S. Pfranger for excellent photographic reproduction. then asked whetherthe high-intensityRaf signal is sufficient by itself to directcell growth.Therefore,we serum-starvedN-BxB-ER@ cells References

in the absence of OHT. The low-serum conditions (0.05%) led to an 1. Oiu, M. S., and Green, S. H. PC12 cell neuronal differentiation is associated with accumulation of cells in the G1 phase of the cell cycle (81.6% G1, prolonged p2ir@@activityand consequent prolonged Erl activity. Neuron, 9: 705â€”717, 7.5% S, and 10.7% G2-M-phases; Fig. 4C). S-phase cells (7.5%) 1992. 2. Franza, R. B., Jr., Maruyama, K., Garrels, J. 1.,and Ruley, H. E. In vitro establishment indicate that even under low-serum conditions, the cells did not is not a sufficient prerequisite for transformation by activated Ras oncogenes. Cell, completely cease the cell cycle progression (Fig. 4C), which can be 44: 409â€”418,1986. explained by the weak Raf signal transmitted by the highly expressed 3. Hirakawa, T., and Ruley, E. H. Rescue of cells from ras oncogene-induced growth arrest by a second, complementing oncogene. Proc. Natl. Acad. Sci. USA, 85: c-Raf-l-BxB-ER@ protein even in the uninduced state (Fig. 2). As 1519â€”1523,1988. expected, the treatment of serum-starved cells with OHT induced a 4. Samuels, M. L., and McMahon, M. Inhibition of platelet-derived growth factor- and epidermal growth factor-mediated mitogenesis and signalling in 3T3 cells expressing synchronous shift of the population to higher cell sizes, documented ARaf-l:ER, an estradiol-regulated form of Raf-l. Mol. Cell. Biol., 14: 7855â€”7866, by increased side scatter values after 29 and 48 h. Simultaneous 1994. 1639

5. Pritchard, C. A., Samuels, M. L., Bosch, E., and McMabon, M. Conditionally Monoallelically expressed gene related to p53 at lp36, a region frequently deleted in oncogenic forms of the A-Raf and B-Raf protein kinase display different biolog neuroblastoma and other human cancers. Cell, 90: 809â€”819,1997. ical and biochemical properties in NIH 3T3 cells. Mol. Cell. Biol., 15: 6430â€” 13. Morgenstern, J. P., and Land, H. Advanced mammalian gene transfet high titre 6442, 1995. retroviral vectors with multiple drug selection markers and a complementary helper 6. Pumigila, K. M., and Decker, S. i. Cell cycle arrest mediated by the Mek/mitogen free packaging cell line. Nucleic Acids Res., 18: 3587â€”3596,1990. activated protein kinase pathway. Proc. Nail. Acad. Sci. USA, 94: 448â€”452, 14. Dawn, 0., Eisenmann-Tappe, I., Fries, H. W., Troppmair, I., and Rapp, U. R. The ins 1997. and outs of Rat kinases. Trends Biochem. Sci., 19: 474-480, 1994. 7. Kerkhoff, E., and Rapp, U. R. Induction of cell proliferation in quiescent NIH 3T3 15. Pines, J., and Hunter, T. Human cydlin A is adenovirus FAA-associatedprotein p60 cells by oncogenic c-Raf-l. Mol. Cell. Biol., 17: 2576â€”2586,1997. and behaves differently from cyclin B. Nature (Lond.), 346: 760â€”763,1990. 8. Sewing, A., Wiseman, B., Lloyd, A. C., and Land, H. High intensity Rafsignal causes 16. Lloyd, A. C., Obermtlller, F., Staddon, S., Barth, C. F., McMabon, M., and Land, H. cell cycle arrest mediated by p2Id1PI.Mol. Cell. Biol., 17: 5588â€”5597,1997. Cooperating oncogenes converge to regulate cydlin/cdk complexes. Genes Dcv., 11: 9. Woods, D., Parry, D., Cherwinski, H., Bosch, E., Lees, E., and McMahon, M. 663â€”677,1997. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity 17. LaBaer, J., Garrett, M. D., Stevenson, L F., Slingerland, J. M., Sandhu, C., Chou, with arrest mediated by p2l1P1. Mol. Cell. Biol., 17: 5598â€”5611, 1997. H. S., Fallacy, A., and Harlow, E. New functiOnalactivities for the p21 family of 10. Hicks, G. G., Egan. S. E., Greenberg, A. H., and Mowat, M. Mutant p53 suppressor CDK inhibitors. Genes Dev., 11: 847â€”862,1997. alleles release ras-induced cell cycle growth arrest. Mol. Cell. Biol., 11: 1344â€”1352, 18. Fisher, R. P. CDKS and cyclins in transition(s). Cmi. Opin. Genet. Dcv., 7: 32â€”38, 1991. 1997. 11. Maclord, K. F., Sherry, N., Hannon, G., Beach, D., Tokino, T., Kinzler, B., 19. Pusch, 0., Bemaschek, 0., Eilers, M., and Hengstschlager, M. Activation of c-Myc Vogelstein. B., and Jack, T. p53-dependent and independent expression of p21 uncouples DNA replication from activation of G1-cyclin-dependent kinases. Onco during cell growth, differentiation and DNA damage. Genes Dev., 9: 935â€”944, gene, 15: 649â€”656,1997. 1995. 20. Yan, G-Z., and Ziff, E. B. NGF regulates the PC12 cell cycle machinery through 12. Kagahad, M., Bonnet, H., Yang, A., Creancier, L., Biscan, J-C., Valent. A., Minty, A., specific inhibition of the Cdk k/oases and induction of cyclin Dl. J. Neurosci., 15: Chalon, P., Lelias, J-M., Dumont, X., Ferrara, P., McKeon, F., and Caput, D. 6200â€”6212,1995.

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Eugen Kerkhoff and Ulf R. Rapp

Cancer Res 1998;58:1636-1640.

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