CORRELATIONS BETWEEN S AND G(2) ARREST AND THE CYTOTOXICITY OF CAMPTOTHECIN IN HUMAN COLON-CARCINOMA CELLS

Previous cell line comparisons indicated that neither S-phase fraction nor topoisomerase I (top1) levels are sufficient to predict camptothe cin (CPT) cytotoxicity (F. Goldwasser et al., Cancer Res., 55: 2116-21 21, 1995.). To identify new determinants for CPT activity, two mutant p53 human colon cancer cell lines, SW620 and KM12, that were previousl y reported to have similar top1 levels and differential sensitivity to CPT were studied. No difference in the kinetics of top1-mediated DNA single-strand breaks or DNA synthesis inhibition were observed after 1 h exposure to 1 mu M CPT. Pulse-labeling alkaline elution show;ed def iciency of damaged replicon elongation in the more sensitive SW620 cel ls. Consistently, flow cytometry analyses showed that KM12 was arreste d in G(2), whereas SW620 cells were irreversibly blocked in S phase. A phidicolin protection was minimal in KM12 and more pronounced in the m ore sensitive SW620 cells. Thus, CPT appears to have two cytotoxic mec hanisms, one protectable by aphidicolin and present in SW620 and the o ther not protectable by aphidicolin and common to both cell fines. SW6 20 exhibited also a greater capacity to break through the G(2) checkpo int after DNA damage. Consistently, SW620 cells failed to down-regulat e cyclin B-cdc2 kinase activity, whereas KM12 cells down-regulated cyc lin 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 replicon elongat ion and G(2) breakthrough capability correlate with sensitivity to CPT . Our results suggest that misrepair of damaged replicons and/or alter ations in DNA damage checkpoints is critical to defining chemosensitiv ity to CPT-induced top1-cleavable complexes and that CPT appears to ha l e tno cytotoxic mechanisms, one protectable by aphidicolin, and the other not.