Induction of Apoptosis by Deregulated Expression of DNA Topoisomerase Ila1

[CANCER RESEARCH 58, 4519-4524. October 15. 1998] Advances in Brief

J. Peter McPherson2 and Gerald J. Goldenberg3

Departments of Pharmacology IJ. P. M., G. J. C.Â¡,Medicine Â¡G.J. G.Â¡.limi lilten/eparlmenlal Division of Oncology Â¡G.J. G.I. University of Toronto, Toronto. Ontario. M5S IAX Ciinudii

Abstract isoform (3-6). The apparent inhibition of clonal expansion of stably transfected cells by deregulated topo II activity may be due to induc DNA topoisomerase II (topo II) is an essential nuclear enzyme required tion of programmed cell death in recipient cells. Accordingly, studies for chromatin condensation and chromosome segregation during mitosis. were undertaken to determine whether forced expression or deregu Forced overexpression of topo Ila was found to cause morphological changes in recipient cells associated with apoptosis. This induction of lation of topo II expression triggers apoptotic cell death. apoptosis required nuclear localization of topo Ila, yet was independent of Apoptosis or programmed cell death is an essential physiological the DNA cleavage-religation activity of the enzyme. Apoptosis mediated process required for development, homeostasis. and protection by the by topo Ha deregulation was blocked by overexpression of crmA, a immune system (reviewed in Ref. 7). Furthermore, attenuation of specific inhibitor of certain caspases, but not by bcl-2. topo Ila-induced apoptotic potential is associated with cancer progression and resist apoptosis was also blocked by overexpression of a dominant-acting mu ance to cancer chemotherapy. Cytoplasmic shrinkage, chromatin con tant of stress-activated protein kinase kinase (SEK1/MKK4) but not by densation, and fragmentation of DNA are widely accepted distin the overexpression of its normal counterpart. Furthermore, apoptosis was guishing features of apoptotic cells. In contrast, necrotic cells blocked by coexpression of a dominant-negative form of the cyclin- dependent kinase cdk2 but not by dominant-negative cdc2. These results typically display cytoplasmic swelling and lysis of the cell membrane provide a rationale for the tight regulation of topo Ha levels through the and do not exhibit the condensed and fragmented chromatin associ cell cycle in that deregulation of topo Ila expression results in apoptotic ated with apoptosis. The present study demonstrates that mammalian cell death. cells with deregulated topo Ila display characteristic features associ ated with apoptosis. The propensity of topo Ila to trigger cell death was found to be independent of the enzyme's DNA cleavage-religa Introduction tion activity, yet required nuclear localization of the topo Ila protein. The nuclear enzyme DNA topo II4 has been shown to be a struc Topo Ila-mediated cell death was attenuated by inhibiting caspase tural component of chromosomes that is required for condensation of activity and stress-activated protein kinase signal transduction. Fur chromatin at metaphase and segregation of intertwined chromosomes thermore, cell death triggered by topo Ila was found to be attenuated prior to anaphase (reviewed in Ref. l). Several important classes of by coexpression of dominant-negative cdk2 but not cdc2, suggesting antineoplastic agents interfere with the catalytic activity of topo II by that progression through the G,-S transition but not the G,-M transi trapping the enzyme in a covalently bound form with its DNA substrate. The resulting "cleavable complexes" are believed to be tion of the cell cycle is required for apoptosis induced by topo Ila. converted to permanent DNA lesions that ultimately elicit cell death. Materials and Methods Mammalian cells contain two genetically distinct isoforms (a and ÃŸ).Many cell lines selected for resistance to these antineoplastic Expression Plasmids. The coding sequence of human topo Ila cDNA was agents have been found to have decreased expression of, or allelic amplified from pBShTOP2 (kindly provided by Dr. James Wang, Harvard mutations in, the topo Ila gene (reviewed in Ref. 2). Complementa University, Cambridge, MA) using Vent polymerase (New England Biolabs) tion studies using drug-resistant cell lines stably transfected with topo and was subcloned into the Noll and Apa\ sites of pRc/CMV (Invitrogen) to II a or ÃŸcDNAs(3-6) have been problematic in that: (a) considerable create the expression vector pRc/CMVhTIIa. A hernagglutinin epitope was added at the COOH terminus of topo Ila cDNA using a two-stage PCR-based selection pressure against correct integration of topo II cDNA in strategy to create plasmid pRc/CMVhTIIaHA. A similar method was used to recipient cells has been observed; and (b) cells that demonstrate add a hemagglutinin epitope and a stop codon after residue 1263 of topo Ila unrearranged integration of the transfected topo II cDNA often dem to create the plasmid pRc/CMVhTIIa(AC!263)HA. Plasmid pRc/ onstrate only a minimal or transient overexpression of the transfected CMVhTIIaHA was altered by site-directed mutagenesis (Transformer Site- directed mutagenesis kit: Clontech) to substitute the active Tyr residue at Received 6/11/98: accepted 9/1/98. position 804 for Phe. The open reading frames of all topo Ila expression The costs of publication of this article were defrayed in part by the payment of page constructs were verified by sequencing using gene-specific primers. The charges. This article must therefore he hereby marked oilvertiscnu-nt in accordance with CMV/3 plasmid expressing lacZ was obtained from Clontech. Bcl-2 cDNA was 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Grant NCI-7439 from the National Cancer Institute of obtained as a 1.9-kb EcoRl fragment from the SFFV-bcl2 expression vector Canada and Grant MT-13158 from [he Medical Research Council of Canada. J. P. M. was (kindly provided by Dr. Stanley J. Korsmeyer. Washington University School supported by an Ontario Graduate Scholarship. " Present address: Molecular Pharmacology and Therapeutics Program. Memorial of Medicine, St. Louis, MO) and was subcloned into pcDNA3 (Invitrogen). Sloan-Kettering Cancer Center. 1275 York Avenue. New York. NY 10021. The pcDNA3/cnnA expression plasmid was kindly provided by Dr. V. M. 3 To whom requests for reprints should be addressed, at Interdepartmental Division of Dixit (University of Michigan, Ann Arbor. MI). Expression plasmids HA/ Oncology. University of Toronto. Medical Sciences Building. Room 2113, I King's SEK/HA and HA/SEK-AL in pcDNA3 were kindly provided by Dr. Brent College Circle. Toronto, Ontario. M5S IA8 Canada. Phone: (416)978-3649; Fax: (4161971-2462: E-mail: [email protected]. Zanke and Dr. James Woodgett (Ontario Cancer Institute. Toronto. Ontario. 4 The abbreviations used are: topo Ila. DNA topoisomerase Ila; X-gal. 5-bromo-4- Canada). Expression plasmids encoding hemagglutinin epitope-tagged domi chloro-3-indolyl-/3-galactosidase; TRITC. tetramethvlrhodamine isothiocyanate: DAPI. nant-negative and wild-type cdk2 and cdc2 were kindly provided by Dr. 4',6'-diamidine-2'-phenylindole dihydrochloride: CMV. cytomegalovirus: HA. hemag- Sander van den Heuvel (Massachusetts General Hospital, Boston. MA). glutinin; crmA, cytokine response modifier A: SEK1/MKK4, stress-activated protein kinase extracellular response kinase kinase-1/mitogen-activated protein kinase kinase 4; Cell Lines and Transfections. HeLa. MCF7. and HER 293 cell lines used SAPK/JNK. stress-activated protein kinase/Jun NH,-terminal kinase: edk. cyclin-depend- in this study were cultured under standard conditions. Prior to transfections. ent kinase. cells were seeded in six-well culture dishes at a density of 2.5 X 10s cells/well. 4519

Cells were then transfected with expression vectors using Lipofectin (Life Technologies. Inc.) according to the manufacturer's directions. 60 A9i1lii X-gal Staining and Indirect Immunofluorescence. At various times after addition of Lipofectin-DNA complexes to the cells, adherent cells were fixed in y/c formaldehyde in PBS and stained with X-gal. For indirect immunofluorescence. cells were transfected as described above, except that prior to transfection, cells were seeded on sterilized glass coverslips. Adherent cells were fixed in 47r paraformaldehyde in PBS for 10 min at room temperature, rinsed with several washes of PBS. and incubated for 30 min at 37Â°Cwith 1% donkey scrum (Jackson ImmunoResearch Laboratories. Inc.) and 0.1% Triton 10 X-KK) in PBS. After rinsing cells with PBS, cells were sequentially incubated with either a 1:2(X)dilution of rabbit anti-ÃŸ-galactosidase antibody (5 Prime â€”¿* 0 11 3 Prime. Inc.). followed by donkey anti-rabbit antibody labeled with TRITC iBÃCJ1rrrrrr (Jackson ImmunoResearch Laboratories) diluted 1:500, or a 1:200 dilution of I2CA5 antibody (Boehringer-Mannheim) followed by TRITC-labeled donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories) diluted 1:500. Prior to mounting, cells were incubated with 0.2 jug/ml of DAPI in PBS to visuali/.e chrotnatm. Dual detection of DNA fragmentation and ÃŸ-galactosidase was performed as described above, except that prior to blocking, DNA O. strand breaks were labeled with FITC-dUTP using the in xiiu cell death 20 detection kit (Boehringer Mannheim). Coverslips were mounted on slides using glycerol or Mowiol (Calbiochem) and visualized for red fluorescence 10 (TRITC) at 570 nm, green fluorescence (FITC) at 525 nm, and DAPI at 420 nm using a Zeiss Axioskop fluorescent microscope. 0 I Results

Deregulation of topo Ila Induces Apoptosis in Mammalian 40 Cells. Three highly transfectable human cell lines (MCF7. HeLa, and HEK 293 cells) were used in these studies because these cells have been shown previously to display hallmark features of apoptosis (cytoplasmic shrinkage, chromatin condensation, and DNA fragmen tation) when stimulated by various apoptogenic signals. To determine whether topo 11Â«was capable of inducing cell death, preliminary 10 studies in these cells were performed using an X-gal staining method in which a gene of interest is cotransfected with the LacZ expression il plasmid. Transfected cells are then detected using in situ X-gal stain 6 12 18 24 48 60 0 0.1 0.5 2.5 5.0 ing and scored for apoptotic morphology (round, intensely staining Time after transfection (h) pRc/CMVhTIIa cells loosely attached to the dish). Accordingly. MCF7, HeLa, and transfected HEK 293 cells were transfected with either topo Ila cDNA under Fig. 1. Time and dose dependency ol apoptosis upon Iranslection with topo Ila. A-C. control of the CMV promoter (pRc/CMVhTIIa) or vector alone time course of percentage of transfected cells demonstrating apoptosis (cytoplasmic (pRc/CMV) along with LacZ expression vector (pCMVÃŸ)at a 5:1 shrinkage alter X-gal staining) in MCF7 (Ai. HeLa (B). and HEK 293 (O cells after ratio. The percentage of transfected cells that displayed cytoplasmic transfection with I ^g of CMV/j and either 5 /ig of Rc/CMV (D) or pRc/CMV-hTIIa (â€¢). D-F, dose-response study of percentage of transfected MCF7 (/)). HeLa (E). and HHK shrinkage, as an index of apoptosis, was initially measured as a 293 (F} cells demonstrating apoptosis 48 h after transfection with 1 fig of CMVÃŸand 0. function of time after transfection (Fig. 1, A-C). 0.1, 0.5. 1.0. 2.5, or 5.0 jig of pRc/CMV-hTIIa. For experiments in D-F. total amount of The percentage of HeLa, MCF7, and HEK 293 cells transiently DNA transfected was kept constant by adding 5.0. 4.9. 4.5. 4.0, 2.5. and 0 /ig. respec tively, of Rc/CMV vector without insert. All results shown are the means of at least three transfected with topo lia that became condensed, rounded, and independent experiments; bar*. SK. To exclude experimental bias, several experiments loosely attached was observed to increase in a time-dependent man were scored blindly for apoptosis. ner. A low baseline frequency of apoptosis was evident in cells transfected with pRc/CMV and pCMVÃŸ,which is typical of cells subjected to transient transfection. At 6 h after transfection of MCF7 cells transfected with topo Ila were apoptotic compared with 10% of cells with human topo Ila expression vector pRc/CMV-hTIIa and vector-transfected cells. pCMV/3. the percentage of transfected cells showing apoptotic mor A dose-response study was undertaken at 48 h, which appeared to phology did not vary from cells transfected with pRc/CMV and be the peak effect in the above time courses (Fig. 1. D-F). Induction pCMVÃŸ(Fig. 1<4).However, a dramatic increase in apoptosis was of apoptosis followed a dose-response relationship, increasing pro observed between 12 and 48 h after transfection with topo Ila. At gressively with increasing amounts of pRc/CMVhTIIa. 48 h. 449Ã•of topo Ila-transfected cells displayed apoptotic morphol Because hallmarks of apoptosis include chromatin condensation ogy compared with 14% of vector-transfected cells. Because cells lose and DNA fragmentation in addition to cytoplasmic shrinkage as their ability to adhere in advanced stages of cell death, the data are measured by X-gal staining, experiments were undertaken to deter likely to be an underestimation of cell death. HeLa cells and HEK 293 mine whether cells transfected with topo Ila also underwent these cells were also found to undergo a similar time-dependent increase in morphological changes. MCF7 cells were cotransfected with either apoptosis after transfection with topo Ila (Fig. 1, B and C), suggesting pRc/CMV and LacZ vector or pRc/CMVhTIIa and LacZ vector and that this effect was not cell type specific. At 48 h, 45% of topo examined 48 h after transfection, following indirect immunofluores- Ila-transfected HeLa cells were apoptotic, compared with 21% of cence staining using rabbit anti-ÃŸ-galactosidase antibody and TRITC- vector-transfected cells. At 60 h after transfection, 52% of HEK 293 conjugated donkey anti-rabbit secondary antibody, followed by stain- 4520

Fig. 2. Cells transfected with topo Ila display cytoplasmic shrinkage, chromatin condensation, and DNA fragmentation. MCF7 cells were cotransfected with 1 /xg of CMVÃŸand 5 /ng of either pRc/CMV (A and ÃŸ)or pRC/CMV-hTIIa (C-F). Forty-eight h after transfection, cells were fixed and stained for indirect immunofluorescence using an anti-ÃŸ-galactosidase antibody (A, C, and Â£),and DNA was either counterstained using DAP1 (B and Â£>)or DNA fragmentation was detected using Tdt- mediated end-labeling of DNA ends using FITC-dUTP (F~>.Cytoplasmic shrinkage of cells after topo Ila trans fection (C) was accompanied by chromatin condensation (D), in contrast with the normal cellular (A) and nuclear (B) morphology of vector-transfected cells. Representa tive illustration of topo Ila-transfected cell showing cytoplasmic shrinkage (E) and concordant DNA fragmenta tion (F).

ing of cellular DNA using DAPI (Fig. 2, A-D). The percentage Recent studies have demonstrated the presence of nuclear localiza (mean Â± SE) of cells displaying chromatin condensation was tion signals in the COOH terminus of human topo Ila (9, 10). Human 76.7 Â± 5.7% after transfection with topo Ila compared with topo Ila truncated after amino acid residue 1263 has been shown 21.3 Â±1.2% in vector-transfected cells in three independent experi previously to be catalytically active in vitro, yet is not capable of ments. Simultaneous fluorescent detection of LacZ expression using complementation in topo Il-deficient S. cerevisiae because of defec anti-ÃŸ-galactosidase antibody plus TRITC-conjugated donkey anti- tive nuclear localization of the human enzyme (9). Transfection and rabbit IgG and DNA strand breaks with FITC-dUTP further demon detection of topo IlaHA truncated after amino acid residue 1263 (topo strated that the death of cells overexpressing topo Ila occurred by an IIaAC1263HA) demonstrated abundant expression of truncated topo apoptotic mechanism (Fig. 2, E and F). IlaHA in the cytoplasm as opposed to the nucleus, as expected (Fig. Induction of Ã€poptosis by topo Ila Requires Nuclear Activity 3E). Truncated topo Ila localized to the cytoplasm did not induce of the Enzyme Independent of DNA Cleavage-Religation. To un apoptosis in transfected cells, as can be seen from the intact interphase ambiguously determine that apoptosis was coincident with the expres nuclei of transfected cells (Fig. 3F). sion of transfected topo Ilo, the pRc/CMVhTIIa expression vector Contribution of Signaling Involving Caspases and Stress-acti was modified such that a HA epitope was inserted at the COOH vated Protein Kinases to topo Ila-mediated Apoptosis. To char terminus of topo Ila (pRc/CMVhTIIaHA), thus facilitating detection acterize the molecular events involved in topo Ila-mediated apoptosis, using the anti-HA antibody 12CA5. At 48 h after transfection, topo the contribution of apoptotic signaling pathways that result in the Ila-HA was only detectable in cells that had underwent apoptosis, as activation of caspases and stress-activated protein kinases was exam detected by cytoplasmic shrinkage and apoptotic chromatin conden ined. Apoptosis mediated by deregulated topo Ila was observed to be sation (Fig. 3, A and B). dramatically inhibited by coexpression of crmA (Fig. 4, A and B), an A tyrosine residue essential for the decatenating activity of topo II inhibitor of certain members of the cysteine-dependent protease fam has been mapped to residue position 783 in Saccharomyces cerevisiae ily known as caspases (reviewed in Ref. 11). Quantitation of apoptotic (8), and the functionally equivalent tyrosine in human topo Ila has frequency upon X-gal staining of transfected cells revealed that crmA been mapped to residue 804 (9). To determine whether catalytic coexpression resulted in a dramatic decrease in percentage of cells activity associated with this residue is required for induction of showing apoptotic morphology (10.3 Â±0.4%), compared with cells apoptosis, cells were transfected with the topo Ila expression vector transfected with topo Ila and empty pcDNA3 vector (28.9 Â±1.0%). pRc/CMVhTIIa(Y804F)HA. Substitution of the tyrosine residue for In marked contrast, coexpression of Bcl-2 was marginally protective phenylalanine had no apparent effect on the ability of transfected topo against topo II-induced apoptosis (Fig. 4, C and D) in that Ila to induce apoptosis (Fig. 3, C and D). 26.3 Â±0.3% of transfected cells were found to be apoptotic when 4521

Fig. 3. Induction of apoplosis by topo Ha re quires nuclear activity of the enzyme independent of DNA cleavage-rcligation activity. MCF7 cells were transfected with 5 ;ig of either pRc/CMV- hTHirHA (A and fl>. pRc/CMV-hTlla(Y804F>HA (C and Dl or pRc/CMV-hTHÂ«ACI263HA (Â£and F). topo Ila was detected using I2CA5 antibody and TRTTC-conjugate donkey anti-mouse IgG (A. C. E}. and DNA was counterslained using DAPI (B, 1), and F).

quantified by X-gal staining. Activation of SAPKs by MEKK-1 is staining revealed that 42.0 Â±2.3% of cells transfected with wild-type mediated through activation of the SAPK/JNK kinase SEK1/MKK4 cdk2 and topo Ila exhibited apoptotic morphology, a similar fre (12). A dominant-interfering SEK1 mutant (SEK-AL) that is deficient quency to that seen when cells were transfected with topo Ila and an in sites of phosphorylation by MEKK acts as a dominantly active equivalent amount of expression vector minus the cdk insert inhibitor of wild-type SEK1 and has been shown to be capable of (41.7 Â± 3.9%). In marked contrast, only 16.3 Â± 1.3% of cells blocking SAPK activation (12. 13). When coexpressed with topo Ila, transfected with dominant-negative cdk2 and topo Ila exhibited ap SEK-AL but not wild-type SEK (Fig. 4, Â£-//) was found to inhibit optotic morphology. Cotransfection with dominant-negative cdc2 did topo Iltt-induced cell death. When scored for cellular morphology not confer a similar protective effect from topo Ila-induced cell death after X-gal staining, 10.0 Â±2.2% of cells transfected with topo Ila upon evaluation by indirect immunofluorescence (Fig. 4, K and L). and SEK-AL exhibited apoptotic morphology, compared with Quantitation of transfected cells upon X-gal staining revealed that 26.1 Â±0.3% of cells transfected with topo Ila and wild-type SEK. 38.7 Â±0.9% of cells transfected with dominant-negative cdc2 and Cell Cycle Component of topo Hot-mediated Apoptosis. Cellu topo Ila were apoptotic, compared with 40.7 Â±1.2% of cells trans lar topo II activity has been shown to fluctuate as a function of cell fected with wild-type cdc2 and topo Ila. cycle position in that activity is maximal during the G-, to M transition (14, 15). To explore whether apoptosis mediated by deregulated topo Ila has a cell cycle component, cells were cotransfected with domi Discussion nant-negative mutants of cdks that are required for progression Forced expression of topo Ila in three different cell lines was found through distinct transition points of the cell cycle. cdk2 is required for progression through the G, to S transition of the cell cycle, whereas to result in an increased propensity of transfected cells to undergo activity of cdc2 is required for progression through the G-, to M apoptosis, as indicated by cytoplasmic shrinkage, nuclear condensa transition. Overexpression of dominant-negative, kinase-inactive tion, and DNA fragmentation. The capacity for topo Ila to commit forms of cdk2 and cdc2 in mammalian cells has been shown to induce cells to undergo apoptosis is at least in part able to explain why such cell cycle arrest within the G, and G-.-M phases of the cell cycle, repeated attempts to isolate stable transfected lines overexpressing respectively (16). When MCF7 cells were cotransfected with domi topo II have met with limited success. This propensity to engage in nant-negative cdk2, transfected cells were protected from the apop cell death may also explain why the resulting cell lines that have been totic effect of topo Ila as evaluated by indirect immunofluorescence characterized that express transfected topo II display a seemingly (Fig. 4, / and /). Scoring of transfected cells after detection by X-gal disproportionate sensitivity to topo II antineoplastic agents compared 4522

Fig. 4. Abrogation of lopo lla-induced apoptotic morphology by coexpression of cnnA. SEK-AL, and dominant-negative cdk2. MCF7 cells were Irans- fected with 1 ng of CMV/3,3 jxgof pRc/CMVhTIIa. and 9 /ig of either pcDNA3/crmA (A and B). pcDNA3/bcl2 (C and DÃ¬.pcDNA3/sek(Â£and F). pcDNA3/sekAL (G and H). pCMVBam/cdk2-dn (/ and /). or pCMVBam/cdc2-dn (K and Â£).Forty-eight h after transfection, transfected cells were identified by detection of ÃŸ-galactosidase(A,C, E, G, I, and AO. and DNA was counterstained with DAPI (ÃŸ,D.F. H. J. and L).

with the relatively modest increases in topo II levels and activity and activation of caspases by different death-promoting signals has (3-6). been shown to be a common essential component of apoptotic signal Presently, the exact mechanism whereby topo Ila commits cells to transduction (11). Furthermore, activation of the stress-activated ki- an apoptotic program is unknown. A proposed mechanism based on nase signal transduction pathway has been shown to be mediated by our findings is presented in Fig. 5. Previous studies in vitro have caspases (19). The cowpox viral protein crmA has been shown to be demonstrated the ability of unchecked topo Ila catalytic activity to a selective inhibitor of caspa.se members in that crmA preferentially cause position-specific DNA cleavage in the presence of certain forms inhibits the proximal caspases such as caspase-1 and caspase-8 but not of DNA damage (17). However, the DNA cleavage-religation activity the distal caspase-3, caspase-6, or caspase-7 (11). Overexpression of of the enzyme mediated through tyrosine 804 was found not to be crmA has been shown to inhibit apoptosis signaled by cytokines (11). required to elicit apoptosis in transfected cells. A recent study has However, crmA overexpression does not protect cells from apoptosis postulated that topo II facilitates chromatin condensation by a mech induced by ionizing radiation or staurosporine, suggesting the exist anism independent of the enzyme's catalytic activity (18). The obser ence of distinct apoptotic signaling pathways that are differentially vation that topo Ila-mediated apoptosis does not require catalytic inhibited by crmA (11). Bcl-2, in contrast to crmA, has been shown to activity mediated by tyrosine 804 suggests that cell death may be due be protective against staurosporine-induced apoptosis (11). Apoptotic to inappropriate timing of chromatin condensation by topo Ila. These cell death induced by ionizing radiation has been shown to require findings suggest that cell death by deregulated expression of topo Ila ceramide mediated-activation of SAPK/JNK signaling (20), yet unlike is due to inappropriate nuclear events mediated by topo Ila that result apoptosis induced by topo Ila, is not inhibited by crmA (11). The in apoptosis, rather than a gross insult on cell viability during over- inhibition of topo Ila-mediated cell death by SEK-AL and crmA, but expression. not by Bcl-2, indicates that the onset of cell death is mediated by The activation of caspases and stress-activated protein kinases were distinct apoptotic signal transduction pathways. identified as apoptotic signal transduction events that are activated The finding that topo Ila overexpression results in apoptosis, along upon deregulation of topo Ila. The sequential recruitment, cleavage, with previous studies demonstrating the cell-cycle dependent nature

premature Unscheduled chromatin increase in Â» Â» Â» APOPTOSIS condensation Topo Ha (before G2 phase)

cdk2-dn CrmA SEK-AL Fig. 5. Proposed model of apoptosis mediated by deregulated expression of topo Ila. ICE, interleukin iÃŸconvertingenzyme. 4523

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1998 American Association for Cancer Research. TOPO IIA-INDUCED APOPTOSIS of topo lia expression, suggests that inappropriate expression of this 7. Thompson. C. B. Apoptosis in the pathogenesis and treatment of disease. Science enzyme perturbs the temporal order of events that topo II mediates (Washington DC) 267: 1456-1462, 1995. during cell division. Inhibition of topo Ila-mediated apoptosis by 8. Worland, S. T., and Wang, J. C. Inducible overexpression, purification, and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae. I. Biol. dominant-negative cdk2 but not dominant-negative cdc2 suggests that Chem., 264: 4412-4416, 1989. cell death is triggered after progression through the G,-S transition but 9. Jensen, S., Andersen, A. H.. Kjeldsen, E., Biersack, H., Olsen, E. H. N., Andersen, before the G2-M transition points of the cell cycle. Cell death resulting T. B.. 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J. Peter McPherson and Gerald J. Goldenberg

Cancer Res 1998;58:4519-4524.

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