ICANCER RESEARCH 56. 4430-4437. October I, IW6| Correlations between S and G2 Arrest and the Cytotoxicity of Camptothecin in Human Colon Carcinoma Cells' Francois Goldwasser,2 Tsunehiro Shimizu, Joany Jackman, Yuko Hoki, Patrick M. O'Connor, Kurt W. Kohn, and Yves Pommier3 lÂihortiltn~\ of Molecular Pììtinnucolo^y. Division oj Basic Sciences, National Cancer Institute. N1H, Bctlicsilu. Man-land 20ÃOEW1-4255 ABSTRACT Previous cell line comparisons indicated that neither S-phase fraction nor topoisonu'rase I {topI) levels are sufficient to predict camptothecin (CRT) cytotoxicity (F. Goldwasser et al.. Cancer Res., 55: 2116-2121. 1995.). To identify new determinants for CPT activity, two mutant p53 human colon cancer cell lines, SW620 and KM 12, that were previously reported to have similar topi levels and differential sensitivity to CPT were studied. No difference in the kinetics of topi-mediated DNA single- strand hreaks or DNA synthesis inhibition were observed after l h expo sure to 1 /KMCPT. Pulse-labeling alkaline elution showed deficiency of damaged replicón elongation in the more sensitive SXV620 cells. Consis tently, flow cytometry analyses showed that KM12 was arrested in G2, whereas SW620 cells were irreversibly blocked in S phase. Aphidicolin protection was minimal in KM 12 and more pronounced in the more sensitive SYV620 cells. Thus, CPT appears to have two cytotoxic mecha nisms, one protectable by aphidicolin and present in SVV620 and the other not protectable by aphidicolin and common to both cell lines. SYV620 exhibited also a greater capacity to break through the I., checkpoint after DNA damage. Consistently, SVV620 cells failed to down-regulate cyclin B-cdc2 kinase activity, whereas KM 12 cells down-regulated cyclin B/cdc2 kinase activity within 30 min to 20% of control level after CPT treatment. Analysis of the 7 human colon carcinoma cell lines of the NCI Anticancer Drug Screen showed that defects in replicón elongation and (., break through capability correlate with sensitivity to CPT. Our results suggest that misrepair of damaged replicons and/or alterations in DNA damage checkpoints is critical to defining chemosensitivity to CPT-induced topl- cleavahle complexes and that CPT appears to have two cytotoxic mecha nisms, one protectable by aphidicolin, and the other not. INTRODUCTION CPT4 derivatives exhibit promising activity for the treatment of solid tumors, such as lung and colon cancers, two high-incidence diseases with poor prognosis (1—5).CPT and its derivatives are specific inhibitors of eukaryotic DNA topi (6, 7). a ubiquitous en zyme with key roles in DNA replication, transcription, and possibly recombination and repair (for recent review, see Ret. 8). Topi cata lyzes linking number changes of DNA in steps of one by breaking and resealing a single phosphodicster bond at a time, and CPTs covalently trap these top I-DNA intermediates, termed cleavable complexes, thereby converting topi into a cellular poison (7. 8). Collisions between advancing replication forks and trapped top I-cleavable com plexes are considered a critical step for CPT cytotoxicity (9-11) because they lead to DNA fork breakage and highly toxic DNA DSBs Received 1/18/96: accepted 7/29/6. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a fellowship from the French Ligue Nationale contre le Cancer and from the NCl/Huropean Organisation for Research and Treatment of Cancer (to F. G.). - Present address: Hôpital Paul Brousse. 948IX) Villejuif. France. 3 To whom requests for reprints should be addressed, at Laboratory of Molecular Pharmacology. Building 37. Room 5C25. National Cancer Institute. NIH. Bethesda. MD 20892-4255. Fax: (301)402-0752. 4 The abbreviations used are: CPT. camptothecin: topi, topoisomcrase I: DSB. double- strand break; SSB. single-strand break: NCI. National Cancer Institute; GI„„. 90'/r cell growth inhibition. (12. 13). This DNA-damaging mechanism, at least in some cell types, accounts for the maximum toxicity of CPT derivatives during S phase despite nearly constant topi protein levels throughout the cell cycle (II. 14). CPT cytotoxicity also depends on the potential of a cell to undergo apoptosis or respond adequately to DNA damage (11). Replication- dependent DNA strand breaks induce wild-type p53 protein (15), which may lead to arrest at the G,-S transition and prevention of "cell commitment and progression through S phase" ( 16). Arrest in G-, is a common cellular response to DNA damage ( 17-19); it is mediated by a negative feedback control; it is also referred to as a checkpoint response to DNA damage (20-22). Down-regulation of cyclin B/cdc2 activity after CPT exposure arrests cells in G, and presumably allows DNA repair before commitment into M phase (23). To identify determinants for CPT activity, we previously used the colon cancer cell lines of the NCI Anticancer Drug Screen as a "miniclinic" to identify cell lines that exhibit intrinsic sensitivity and resistance to CPTs ( 14). In contrast to hematopoietic cells (24). colon cancer cells do not undergo rapid apoptosis.5 Our results showed that the seven colon carcinoma cell lines of the NCI cell screen exhibit a range of CPT sensitivity that is better correlated with the frequency of cleav able complexes than with topi levels ( 14). Two of these lines. SW620 and KM 11. both with mutant p53 genes'1 responded very differently to CPT despite similar levels of topi expression and top I-cleavable complexes (14). The aim of the present study was to identify determinants for CPT activity located downstream from topi-cleavable complexes, using these two colon cell lines, SW620 and KM 12. and to correlate these determinants and cytotoxicity using the overall panel of colon cell lines from the NCI Anticancer Drug Screen. Our results suggest that CPT cytotoxicity is related to two types of defects: (a) during S phase, accumulation of damaged replicons and failure to complete elongation of replicating DNA; and (/>) during G-,. deficiency to down-regulate cyclin B/cdc2 kinase and reduced G2 arrest. We also report an S phase-independent CPT-induced cytotoxicity. MATERIALS AND METHODS Cell Culture. SW620. KMI2. HCT1I6. HCTI5. HCC2998. HT29. and coli>2()5 correspond to the seven colon cancer cell lines used in the NCI Anticancer Drug Screen (25). They were obtained from Dr. Dominic Scudieri) (NCI, Frederick. MD). Cells were grown at 37°Cin 95% air/5% CO, in RPM1 1640 (Lite Technologies. Inc.. Grand Island. NY) containing 5r/f fetal bovine serum (Inlergen. Purchase, NY). 2 HIMglutamine (AB1, Columbia. MD). Drugs and Chemicals. CPT was a kind gift from Drs. Monroe E. Wall and Mansukh C. Wani (Research Triangle Institute. Research Triangle Park. NC). Nocoda/.ole was purchased from Sigma Chemical Co. (St. Louis, MO). Stock solutions of CPT and nocodazole were prepared in DMSO at 10 HIMand 10 mg/ml, respectively, and further diluted in deionized water immediately prior to each experiment. [y^PI ATP (4500 mCi/mmol) was purchased from NEN Research Products (DuPont NEN. Boston. MA). s F. Goldwasser and Y. Pommier, unpublished data. " P. M. O'Connor. I. Jackman. I. Bae. T. G. Myers. S. Fan. D A. Scudieri). A. Monks. E. A. Sausville, J. N. Weinstein. S. Friend. A. J. Fornace. Jr.. and K. W. Kohn. manuscript in preparation. 4430 on May 4, 2016. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from