Abrogation of an S-Phase Checkpoint and Potentiation of Camptothecin

Home , S phase

[CANCER RESEARCH57, 4029-4035, september 15, 1997] Abrogation of an S-Phase Checkpoint and Potentiation of Camptothecin Cytotoxicity by 7-Hydroxystaurosporine (UCN-O1) in Human Cancer Cell Lines, Possibly Influenced by p53 Function Rong-Guang Shao, Chun-Xia Cao, Tsunehiro Shimizu, Patrick M. O'Connor, Kurt W. Kohn, and Yves Pommier' Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer lnstitute, NIH. Bethesda, Maryland 20892

ABSTRACT carcinoma cell lines (15, 16). This is probably related to the induction of DNA damage (replication fork collisions) in replicating DNA ( 11). 7-Hydroxystaurosporine (UCN-01) is a selective protein kinase C in. Thus, CPT can be considered as an S phase-specific DNA-damaging hibitor in clinical trial for cancer treatment, In this study, we found that agent (11). nanomolar concentrations of camptothecin (CPT), a topoisomerase I in hibitor, arrest or delay cell cycle progression during the S and G2 phases In the present study, we used human colon carcinoma p53-mutant (17) in p53 mutant human colon carcinoma HT29 cells and that UCN-O1 HT29 cells to investigate whether UCN-01 can affect cell cycle arrest abrogates the S-phase arrest or delay induced by CPT. Under these induced by CFT (15, 16). Our data demonstrate for the first time that conditions, CPT increased cyclm A levels and cycin A/cydin-dependent UCN-0l abrogates CPT-induced S-phase delay and enhances CVF cyto kinase 2 activity. UCN-01 prevented the Increase of cydlin A/cydin toxicity, suggesting the existence of a UCN-Ol-sensitive S-phase check dependent kinase 2 activity induced by CPT and enhanced Cdc2 kinase point in HT29 cells. These effects were analyzed further at the molecular activity. Replication protein A (RPA2) was hyperphosphorylated after level and in two other p53-wild-type and -mutant cell lines. CPT treatment, and this effect was also abrogated by UCN-O1. UCN-O1 potentiated the cytotoxicity of CPT and reduced by 6-fold the concentra MATERIALS AND METHODS lion of CPT required to kill 50% of the HT-29 cells, as determined by clonogemc assays. This effect was observed at concentrations of UCN-01 Drugs, Chemicals, and Antibodies. UCN-01 was provided by Dr. H. Na that alone were not cytotoxic and had no detectable effect on cell cycle kano (Kyowa Hakko Co., Tokyo, Japan) or the Drug Synthesis Chemistry Branch, progression. UCN-O1markedly potenfiated the cytotaxicity of CPT also m Division of Cancer Treatment, NC!. Aliquots were stored frozen at 10 nmi in HCT116/E6 and MCF-7/ADR cells defective for p53 function, whereas DMSO, and diluted further in water immediatelyprior to each experiment. significantly less potentiation was observed in p53-wild-type HCT116 and Anticyclin A, Bl, Dl, and Cdc2 monoclonal antibodies were purchased MCF-7 cells. These results suggest the existence of an S-phase checkpoint from Santa Cruz Biotechnology (Santa Cruz, CA). Anticyclin E monoclonal that delays replication and that may extend the time available for DNA antibody and anti-Cdk2 polyclonal antibody were purchased from PharMingen repair. Thus, pharmacological abrogation of CPT-induced S- and G2- (San Diego, CA). Anti-proliferating cell nuclear antigen, anti-p2l, and anti phase checkpoints by UCN-01 may provide an effective strategy for RPA2 monoclonal antibodies were from Oncogene Science, Inc. (Cambridge, enhancing the chemotherapeutic activity of CPT, particularly against MA). Antimouseimmunoglobulinhorseradishperoxidaseantibodywas pur p53-defective tumors. chased from Amersham Life Science (Arlington Heights, IL). [â€˜4C]Thymidine(53.6mCi/mmol), [methyl-3H]thymidine(80.9 Ci/mmol), and [y-32PIATP (4500 Ci/mmol) were purchased from New England Nuclear INTRODUCTION (Boston, MA). Cell Culture. Humancolon carcinomaHT29, HCTI16, and HCT1l6/E6 UCN-0l2 is presently in Phase I clinical trial. It inhibits the growth and breast cancer MCF-7 and MCF-7/ADR cells were obtained from the NCI of human and murine tumor cell lines in vitro and exhibits antitumor Anticancer Drug Screen (Developmental Therapeutics Program) and grown at activity in animal models (I , 2). UCN-Ol is a more selective protein 37Â°Cinthe presence of 5% CO2 in RPM! 1640 supplemented with 5% fetal kinase C inhibitor than staurosponne (3, 4), and cell cycle analyses bovine serum (Life Technologies, Inc.), 2 mM glutamine, 100 units/mI show that UCN-01 can induce G1-phase block (5â€”8). penicillin and 100 @g/m1streptomycin. HCT116 cells transfected with UCN-01 enhances the antitumor activity of mitomycin C both in control vector (HCT1 16) and vector containing overexpressed HPV-l6 E6 vitro and in vivo in oncogene-activated human and murine tumor cell (HCT1l6/E6) were grown in the same medium containing 0.5 mg/mI G418. lines (2). Recent studies indicate that UCN-Ol enhances cell killing by Flow Cytometry. Cell cycle assays were performed as described previ -y-irradiation both in CA46 and HT-29 cells and synergizes with ously (16). Briefly, cells were harvested and fixed in 70% ethanol. Before cisplatin to preferentially kill MCF-7 cells with defective p53 func analysis by flow cytometry, cells were washed with PBS, treated with I mg/ml RNase, and stained with 50 pg/mi propidium iodide for at least 30 mm. DNA tion. This enhancement has been related to an abrogation of the G2 content was determined by FACScan flow cytometry (SOBR model, 15,000 checkpoint and activation of cdc2 kinase activity (9). Bunch and cells per sample; Becton Dickinson Immunocytometry Systems, San Jose, Eastman (10) also reported that UCN-0l abrogates the G2 arrest CA). Shown are representativedata from individualexperimentsthat were induced by cisplatin and enhances cisplatin-induced cytotoxicity in repeated twice (Figs. 1, 3, and 9). Chinese hamster ovary cells. DNA Synthesis Assay. Cells were prelabeled with 0.005 @Ci/mlof CPT is a selective topoisomerase I inhibitor (1 1), and its derivatives [â€˜4Clthymidinefor 48 h at 37Â°C.The rate of DNA synthesis was measured by are clinically active against human cancers, such as colon and ovarian 10-mm pulses with 1 @Ci/ml of [methyl-3Hjthymidine. 3H incorporation was carcinomas (12â€”14).Previous studies indicated that CPT-induced stopped by washing cell cultures twice in ice-cold HBSS and then scraping cytotoxicity was correlated with S and G2 abnormalities in colon cells into 4 ml of ice-cold HBSS. One-mi aliquots were then precipitated with 100 p.1of 100% trichloroacetic acid in triplicate. Samples were vortexed, mixed, and centrifuged for 10 mm at 1200 rpm at 4Â°C.Theprecipitates were Received 5/7/97; accepted 7/I 7/97. then dissolved overnight at 37Â°Cin0.5 ml of 0.4 M NaOH. Samples were The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with counted by dual label liquid scintillation counting, and 3H values were nor 18 U.S.C. Section 1734 solely to indicate this fact. malized using â€˜4Ccounts.Inhibition of DNA synthesis was calculated as the @ To whomrequestsfor reprintsshouldbe addressed,at Laboratoryof Molecular ratio of 3H: â€˜4Cinthe treated samples over the 3H: â€˜4Cratio in the untreated Pharmacology, Building 37, Room 5C25, NIH, Bethesda, MD 20892-4255. Fax: (301) control samples. 402-0752. 2 The abbreviations used are: UCN-Ol, 7-hydroxystaurosporine; CPT, camptothecin; Immunoblotting. Cells were pelleted, washed once in PBS, and lysed at Cdk, cyclin-dependent kinase; RPA, replication protein A; Nd, National Cancer Institute. 4Â°Cas described previously (18). Protein detection was performed using a 4029

SDS-polyacrylamide gels (NOVEX, San Diego, CA) and electrophoresed at A 125 V for 2 h. For quantitation of cyclin/Cdk activity of immunoprecipitates, gels were dried, and histone Hl phosphorylation was measured using a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA). Shown are represent E ative data from an individual experiment that was repeated at least twice C GVM (Fig. 6). LA Clonogenic Assays. Cells were treated with CPT for 8 h. CPT was re IIcontrol I @t moved by rinsing the cultures in complete medium without drug, and UCN-Ol hL CPT, 8 hUCN-O1, 16 was added for the next 16 h. Following drug treatments, cells were washed in fresh medium and trypsinized. Two hundred to 500 cells were seeded in B triplicate in 1-25 tissue culture flasks with 5 ml of medium. Colonies were grown for 2 weeks and then washed with PBS, fixed with methanol, and E stained with methylene blue (0.04%). Cloning efficiencies of untreated cells C were 80% for HT29, 52% for HCTI 16,46% for HCT116/E6,51%for MCF-7, I and 29% for MCF-7/ADR. 0

@ I I @-ILI 5h lOh 16h RESULTS Timeafterremovalof CPT(no UCN-O1) Cell Cycle Effects of CPT in the Absence and Presence of UCN-01. We first investigatedwhether UCN-Ol affected the cell cycle distribution of CPT-treated HT29 cells. Flow cytometry analy C ses (Fig. 1) showed that treatment with low CPT concentration (10 E nM) for 8 h produced only minimal cell cycle perturbation (limited S C and G2-phase accumulation). After CPT removal, the cells accumu lated in both the S and 02 phases of the cell cycle. Addition of UCN-Ol immediately after CVF removal suppressed CPT-induced S-phase accumulation (Fig. 1). The treatment protocol outlined in Fig. Timeafterremovalof CPTandadditionof UCN-O1 2 was used subsequently unless otherwise indicated. Two possibilities may account for the suppression of CPT-induced S-phase G2-phase S-phase accumulation by UCN-Ol. One is that UCN-0I blocked cell D entry into S phase, and the other is that UCN-Ol overcame S-phase delay induced by CPT. To discriminate between these two possibili 65 ties, cells were treated with the mitotic inhibitor nocodazole immedi 20 8 55 ately after CPT treatment. The treatment protocol is shown in Fig. 2. Fig. 3 shows that CPT arrested HT29 cells in S and G2 in both the 45 10 absence and presence of nocodazole, indicating an S-phase arrest in 35 the first cell cycle following CPT treatment. When the CPT-treated +UCN-O1 -UCNO1 cells were treated with both UCN-Ol and nocodazole, a large fraction -8 a a 1@6 -8 0 8 16 of the cells were in G2-M (56%), indicating that a significant fraction Hours after CPT removal of cells was not blocked in G1 under these conditions. UCN-Ol alone (100tiM)didnotsignificantlyaffectcellcycleprogression.These Fig. I . Abrogation of CPT-induced S and G2 arrest/delay by UCN-Ol in HT29 cells. Cells were treated with 10 mi CPI for 8 h, after which cells were washed in fresh medium results indicate that UCN-Ol suppresses the S-phase accumulation and treated with 100 nM UCN-0l for 16 h. Cells were harvested and analyzed by flow induced by CPT in HT29 cells by enabling the cells to progress cytometry at various times. A, control, exponentially growing untreated cells; CPT, through the S phase. CPT-treated cells immediately after drug removal; UCN-0I. cells treated with UCN-Ol for 16 h. B and C, time course after CPT removal without (B) or with UCN-Ol added (C); D, DNA Synthesis Inhibition and Delayed S-Phase Progression quantitation of data of panels A, B, and C. Induced by CPT and Effects of UCN-01. We next tested whether the S-phase accumulation observed after CPT treatment was due to delayed S-phase progression or to delayed release from a G1 block. In protein assay kit according to the manufacturer's instructions (Bio-Rad, Her the latter case, this would be equivalent to an S-phase synchronization, cules, CA). Proteins were electrophoresed at 125 V on 12% SDS-polyacryl and DNA synthesis measured by thymidine incorporation would be amide gels and transferred electrophoretically to Immobilon membranes (Mil expected to be high. Fig. 4 shows that CPT treatment inhibited DNA lipore, Bedford, MA), for 2 h at 30 V. Membranes were blocked overnight in synthesis to approximately 60% of control, whereas the fraction of cells PBS-Tween containing 5% nonfat dried milk, probed for I h with primary with an S-phase DNA content was increased 2.2-fold above control. This antibody (0.1â€”1.0 @Wm1in PBS-Tween) and for 1 h with secondary antibody (1:1000 dilution), and visualized by enhanced chemiluminescence (DuPont NEN, Boston, MA) according to the manufacturer's instructions. Shown are representative data from individual experiments that were repeated at least 0 8 twice (Figs. 5 and 7). CPT (10nM) In Vitro Cyclin BlJCdc2 Hi Kinase Assay. Briefly, cells were washed once in cold PBS and lysed on ice as described previously (19). Total cell UCN-01(100nM) proteins were isolated, and 500 @gof cell proteins per sample were used for 0 16 immunoprecipitation. Immune complexes were resuspended in kinase buffer I â€”I [20 m@iTris-HC1,10nmiMgCl2(pH 7.5) containing 5 p.i@iunlabeledATP, and 24 hours 10 @Ci(-y-32PIATP]with3 ;@gofhistone Hl and incubated at 37Â°Cfor20 mm. Reactions were stopped by adding 3X SDS-gel loading buffer (NOVEX, San Fig. 2. Standard treatment protocol. Cells were treated with 10 nM CPT for 8 h, after Diego, CA) and boiling samples for 5 mm. Samples were loaded onto 12% which cells were washed in fresh medium and treated with 100 nsi UCN-Ol for 16 h. 4030

Control CPT CPT + Noc Noc

- UCN-O1 C

C, Fig. 3. Abrogation of CPT-induced S-phase arrest by UCN-Ol in G2@ HT29 cells. Cells were treated with 10 nti CPT for 8 h. Following CPT S treatment, cells were washed in fresh medium and treated with 100 nt@i & & UCN-Ol with (CPT + Noc, Noc) or without (Control, CP7) 0.4 @g/ml nocodazole for 16 h. Cells were harvested and analyzed for cell cycle distribution by flow cytometry. â€”UCN-Ol, no UCN-Ol; + UCN-01, in the presence of UCN-Ol. .1 aE + UCN-O1 C

E/Cdk activity. This was also the case for the combination of CPT and 250 I DNAsynthesis0.3 UCN-Ol (Fig. 6). These results indicate that the S-phase delay in :@200 @sphase duced by CPT is not related to an inhibition of cyclin A/Cdk2 activity C and that abrogation of the CPT-induced 02 and S arrest or delay by 0 .@ 150 UCN-Ol is associated with activation of cyclin Bl/Cdc2 activity and 0 1.0 1.1 suppression of cyclin A/Cdk2 activity. C -- @ .__@--@ I a) RPA is a mammalian single-stranded DNA binding factor essential 0

0@ 50

0 4'@\, ,@14cs @*is c@@Qs@' Cl Fig. 4. Effects of CPT and UCN-Ol on DNA synthesis and S-phase distribution in HT29 cells after drug treatment. Cells were treated as described in Fig. 2. DNA synthesis @ was measured by 10-mm pulses, as described in â€œMaterialsand Methods,â€•and S-phase cyclin A â€˜@-@ distribution was analyzed by flow cytometry. Numbers above columns, relative ratio of DNA synthesis:S-phase fraction. Data are means (bars, SD) of three independent exper iments. @ cyclin Bi â€” â€”@ demonstrates an S-phase delay (i.e., DNA replication delay) in CVT treated cells. UCN-01 alone did not markedly affect DNA synthesis or S-phase fraction. However, cells treated with UCN-01 after CPT treat ment tended to normalize their DNA synthesis and S-phase distribution. Molecular Changes Associated with the Cell Cyde Effects of cyclin Dl CFr In the Absence and Presence of UCN-O1. We next examined whether the cell cycle effects of CVF and UCN-01 were associated with modified expression ofcydlin and Cdk proteins by Western blotting (Fig. 5). Cydlin A protein was increased by CVF treatment. UCN-01 decreased Cdk2 =@=@H cyclin A levels and prevented the cyclin A increase induced by CVf. CPT did not affect Cdc2, Cdk2, cydlin Bl, or proliferating cell nuclear antigen protein levels. p21 was not detectable in p53-mutant HT29 cells in the absence or presence of drug (data not shown). @@@ Arrest of cells in 02 phase following DNA damage has been Cdc2 associated with suppression of Cdc2 kinase activity (20, 21). Consist ent with these earlier observations, we found that Cdc2 kinase activity in CPT-treated cells was not elevated in spite of the presence of an @ increased fraction of cells in G2. UCN-0l increased the Cdc2 kinase PCNA I TTi@ H activity 3- to 4-fold in CVF-treated cells (Fig. 6). By contrast, Cdk2 kinase activity was increased in CPT-treated cells and was decreased Fig. 5. Western blot analyses of HT29 cells treated with Ciâ€•!'and/or UCN-Ol . Cells by UCN-Ol treatment (Fig. 6). CPT and UCN-01 decreased cyclin were treated as described in the legend to Fig. 2 and examined 16 h after CPT removal. 4031

Cdc2 Cdk2 cyclin E

@@@@ @,HI I :â€”Tii ] â€”S.â€”

.@ 400 .250 Fig. 6. Effects of CPT and UCN-Ol on Cdk activ inca in HT29 cells. Cells were treated as described in h0râ€”i-200 Fig. 2, and kinase activities were measured 16 h after CPT removal. Immunoprecipitates were collected from cell extracts with anti-Cdc2. anti-Cdk2, and anti-cyclin @2001 1150 E antibody, respectively. Histone HI kinase activity was measured using a Phosphorlmager and was cx pressed relative to untreated control cells. Columns, quantitations of the typical experiments shown (top). Ii:LiJtil:@I 11

I @ , ,@ FFF F

potentiation was greater in the MCF-7/ADR cells. These results 4? -UCNO1 +UCNO1 demonstrate that UCN-Ol potentiates CPT-induced cytotoxicity in

C? 0 4 1 6 â€˜ â€˜4 1 2 16' hours after CPT removal three different p53-deficient cell lines. I:@â€”p@p@4RPA2 S-Phase Delay Induced by CPT in Synchronized HT29 Cells and Its Abrogation by UCN-O1. To further study the CPT-induced I@ S-phase arrest and its modulation by UCN-Ol, HT29 cells were Fig. 7. Western blot analyses of p34@A2 phosphorylation induced by CPT in the synchronized at the G1-S boundary by double-aphidicolin block. One absence (â€”)andpresence (+) of UCN-Ol. Cells were treated as described in Fig. 2, and h after removal of aphidicolin, CPT was added either alone or with samples were analyzed 16 h after CPT removal. UCN-Ol. Cells were analyzed for cell cycle distribution at various times (Fig. 9). The untreated cells were in mid-S phase by 4 h, late S phase by 7 h, and G2 phase by 10 h. CPT caused delayed S-phase for DNA replication, repair, and recombination. It is composed of progression with marked accumulation of cells in S phase at 10 h. three subunits of Mr 70,000, 34,000, and 11,000 (RPA1, RPA2, and UCN-01 by itself did not affect cell cycle progression. However, RPA3, respectively). Because RPA2 is a cyclin A substrate (22) and addition of UCN-Ol to CPT-treated cells restored cell cycle progres a key element of the replication complex, we studied its modifica sion as the cells were in 02 phase at 10 h (Fig. 9B). These results are tions. Fig. 7 shows that CPT induced RPA2 phosphorylation by 8 h consistent with an abrogation of the CPT-induced S-phase delay by and that RPA2 phosphorylation continued to increase following CPT UCN-01. removal. UCN-Ol suppressed RPA phosphorylation induced by CPT, demonstrating that abrogation of the S-phase delay by UCN-01 is associated with suppression of RPA phosphorylation, consistent with DISCUSSION the suppression of cyclin A. In the present study, we found that nanomolar concentrations of Potentiation of CPT Cytotoxicity by UCN-O1. The treatmentpro CF-F delayed cell cycle progression through S phase and that UCN-0l tocol (see Fig. 2) used to demonstrate the cell cycle effects of UCN-01 abrogated this S-phase delay and potentiated CPT cytotoxicity in and CFF was tested in cytotoxicity assays. We hypothesized that p53-mutant human colon carcinoma HT29 cells. These results suggest UCN-01 might enhance CPT cytotoxicity, because cells could not arrest that UCN-Ol is a potent abrogator of both the S and G2 checkpoints. in S phase. Under these conditions, UCN-Ol alone produced limited G2 checkpoint abrogation by UCN-01 was reported recently with two cytotoxicity (Fig. 8). However, UCN-0l potentiated CPT cytotoxicity up other DNA-damaging agents, cisplatin and ionizing radiation (9, 10). to 50-fold and reduced by 6-fold the concentration ofCVF required to kill 90% of the cells, as determined by clonogenic assays (Fig. 8). This effect was observed at concentrations of UCN-01 that alone were not cytotoxic. A B The above results raised the question whether such a potentiation of CPT cytotoxicity by UCN-Ol might be selective for the p53-mutant 100@ HT29 cells (9). We compared the effects of UCN-Ol on the cytotox 100 icity of CPT in human colon carcinoma HCT1 16 cells (p53 wild type) @â€˜ and in HCT1I6/E6 cells (p53 function disrupted by transfection with uc 10 the human papillomavirus type 16 E6 gene; Ref. 23). As for HT29, 0.1 â€˜Eo 1 )@ @LMUCN-Ol was not cytotoxic by itself in either cell line. However, â€¢0 the synergistic effect of UCN-Ol plus CPT was more pronounced in C the HCT1 l6IE6 than the HCT1 16 cells (Table 1). We also tested two 0 1 +UCN..O1@@@@ breast carcinoma cell lines, MCF-7 cells (p53 wild type), and MCF @@@<<â€œI''''â€•I 7/ADR cells (p53 mutant; Table l)@ and found that the UCN-Ol 0 10 30 100 0 100 1000 CPT (nM) UCN-OI(nM) 3 p. M. O'Connor, J. Jackman, I. Bae, T. 0. Myers, S. Fan, D. A. Scudiero, E. A. Sausville, J. N. Weinstein, S. Friend, A. J. Fornace, and K. W. Kohn. Characterization of Fig. 8. UCN-Ol potentiates CPT cytotoxicity in HT29 cells. Cells were treated as the p53 tumorsuppressorpathwayin cell linesof the NC!anticancerdrugscreenand described in Fig. 2. UCN-0l concentration was 100 nsi (A), and CPT concentration was correlations with the growth inhibitory potency of 123 anticancer agents, submitted for 10 flM(B).Cell survival was determined by clonogenic assays. Data are means (bars, SD) publication. of three independent experiments. 4032

ce/isaDrug TableI EffectofUCN-OIon CPT cytotoxicity in wild-type and p53-deficient HCTII6 and MCF-7 control)HCTII6MCF-7Wild-type cell survival (%

p53ADRCPTTreatmentClonogenic p53E6 (p53 deficient) Wild-type (3 nM) Â±2.0(3) Â±4.7 (3) 89.3 Â±6.2 (3) Â±5.6 (3) UCN-Ol (lOOnsi) 97.4Â±0.6(3) 96.2 Â±1.3(3) 96.0Â± 2.8(3) 99.1 Â±1.1 (3) CPT + UCN-Ol70.4 61.0 Â±1.2(3)76.5 30.1 Â±2.2 (3/' 82.5 Â±6.7 (2)77.5 51.7 Â±1.9(3)â€•

a Cells were treated with CPT and UCN-Ol as described in Fig. 2. Cell survival was determined by clonogenic assays. Values shown are the mean Â± SD. Numbers in parentheses indicate the number of independent experiments with triplicate determination.

b @,, < o.oos compared with wild type.

To our knowledge, this is the first report suggesting pharmacological EJCdk2 is essential for S-phase progression (28, 29). Cyclin A binds abrogation of an S-phase checkpoint by UCN-Ol in response to DNA both Cdk2 and Cdc2, giving two distinct cyclin A kinase activities, damage. one appearing in S phase and the other in G2 (29). Our data show that S-phase delay following DNA damage is associated with a block of abrogation of CPT-induced S-phase delay by UCN-Ol was associated replication initiation as well as suppression of DNA elongation (24, with a marked decrease of CPT-induced cyclin A/Cdk2 activation and 25). PassagethroughG1 into S phaseis regulatedby the activitiesof RPA2 phosphorylation. RPA2 is a known substrate of cyclin A/Cdk2 cycin D-, E-, and A-associated kinases. The accumulation of cyclin E with an essential function in DNA replication (22). These observa is highly periodic, peaking during late G@and declining during S tions indicate that CPT-induced S-phase delay is exerted logically phase. Thus, cyclin E is a key regulator of the G1-to-S phase transition downstream from cyclin A/Cdk2 kinase and RPA2 hyperphosphory (26, 27). The activation of cyclin A/Cdk2 following that of cyclin lation. It is possible that CPT-induced delay in the S phase allows the

A released control CPT UCN-01CPT+UCN-01

syn(APH) A4@tJAA@4k@kAtJ@d@_

lOh Fig. 9. Effectsof CPT and UCN-Olon cell cycleprogressionin aphidicolin-synchronized HT29 cells. Cells were synchronized in G1-S @4A@-@L @ phase using double aphidicolin block (16-h aphidicolin treatments with a 12-hinterval).Cells were releasedinto cycleby washingin fresh medium, and I h later they were treated with 10 nr@iCVrand/or 30 nsi UCN-Ol for the indicated times. A, cells were analyzed by flow cytom etry; B, quantitation of data in A. â€” syn control CPT @100t 1 100 100 2 80.j I 80-i @80 @@@ 401@I40@60-1 6oJ I :@II1IIJIIt@ @ @201 20-1w @ 0. 0 1I o ______@@@ 4 710 20 10 @ UCN-OI CPT+UCN-O1 @ @G1 as 60@I J@@60@I @G2/M 40@ â€¢ 40@

201 I 20-j I 0i@ o1@e 4710 4 7 10 hoursafterAPHrelease 4033

accumulation of abnormally high cyclin A levels. The reduced cyclin p53-mutated human epidermoid carcinoma A43l cells. Cancer Res., 57: 1495â€”1501, 1997. A/Cdk2 activity associated with UCN-Ol exposure may be secondary 6. Akinaga, S., Nomura, K., Gomi, K., and Okabe, M. Effect of UCN-0I, a selective to cells having progressed out of S phase. Alternatively, activation of inhibitor of protein kinase C, on the cell-cycle distribution of human epidermoid cyclin AJCdk2 kinase may directly affect DNA replication, given that carcinoma, A43l cells. Cancer Chemother. Pharmacol., 33: 273â€”280,1994. 7. Kawakami, K., Futami, H., Takahara, J., and Yamaguchi, K. UCN-Ol, 7-hydrox DNA polymerase a-primase phosphorylation by cyclin A/Cdk2 has yl-staurosporine. inhibits kinase activity of cyclin-dependent kinases and reduces recently been shown to inhibit the initiation of SV4O DNA replication the phosphorylation of the retinoblastoma susceptibility gene product in A549 in vitro (30). human lung cancer cell line. Biochem. Biophys. Res. Commun., 219: 778â€”783. 1996. CPT also arrests cells in G2, and we previously reported a corre 8. Seynaeve, C. M., Stetler-Stevenson, M., Sebers, S., Kaur, G., Sausville, E. A., and lation between G2 checkpoint deficiency and CPT cytotoxicity (15, Worland, P. J. Cell cycle arrest and growth inhibition by the protein kinase 16). Entry into mitosis is regulated by the formation and activation of antagonist UCN-Ol in human breast carcinoma cells. Cancer Ret., 53: 2081â€” 2086, 1993. the cyclin Bl/Cdc2 kinase complex, which requires p34cdc2 dephos 9. Wang, Q., Fan, S., Eastman, A., Worland, P. J., Sausville, E. A., and O'Connor, P. M. phorylation (31). DNA damage-induced 02 arrest is associated with UCN-Ol: a potent abrogator of 02 checkpoint function in cancer cells with disrupted p53. 3. NaIl. Cancer Inst., 88: 956â€”965, 1996. the accumulation of relatively inactive cyclin Bl/Cdc2 complexes (20, 10. Bunch, R. T., and Eastman, A. Enhancement of cisplatin-induced cytotoxicity by 32). Our data are consistentwith this, inasmuchasthey showlack of 7-hydroxystawosporine (UCN-Ol), a new G2-checkpoint inhibitor. Clin. Cancer Res.. cyclin Bl/Cdc2 kinase activation in the G2-arrested cells after CPT 2:791â€”797,1996. I 1. Pommier, Y. Eukaryotic DNA topoisomerase I: genome gate keeper and its intruders, treatment. Furthermore, we found that UCN-Ol markedly enhanced camptothecins. Serum. Oncol., 23: 1â€”10,1996. cyclin Bl/Cdc2 kinase activity following CPT treatment, which is 12. Potmesil, M. Camptothecins: from bench research to hospital wards. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1997 American Association for Cancer Research. Abrogation of an S-Phase Checkpoint and Potentiation of Camptothecin Cytotoxicity by 7-Hydroxystaurosporine (UCN-01) in Human Cancer Cell Lines, Possibly Influenced by p53 Function

Rong-Guang Shao, Chun-Xia Cao, Tsunehiro Shimizu, et al.

Cancer Res 1997;57:4029-4035.

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