ETOPOSIDE-INDUCED CELL-CYCLE DELAY AND ARREST-DEPENDENT MODULATION OF DNA TOPOISOMERASE-II IN SMALL-CELL LUNG-CANCER CELLS

As an approach to the rational design of combination chemotherapy involving the anti-cancer DNA topoisomerase II poison etoposide (VP-16), we have studied the dynamic changes occurring in small-cell lung cancer (SCLC) cell populations during protracted VP-16 exposure. Cytometric methods were used to analyse changes in target enzyme availability and cell cycle progression in a SCLC cell line, mutant for the tumour-suppressor gene p53 and defective in the ability to arrest at the G(1)/S phase boundary. At concentrations up to 0.25 mu M VP-16, cells became arrested in G(2) by 24 h exposure, whereas at concentrations 0.25-2 mu M Gz arrest was preceded by a dose-dependent early S-phase delay, confirmed by bromodeoxyuridine incorporation. Recovery potential was determined by stathmokinetic analysis and was studied further in aphidicolin-synchronised cultures released from G(1)/S and subsequently exposed to VP-16 in early S-phase. Cells not experiencing a VP-16-induced S-phase delay entered G(2) delay dependent upon the continued presence of VP-16. These cells could progress to mitosis during a 6-24 h period after drug removal. Cells experiencing an early S-phase delay remained in long-term G(2) arrest with greatly reducing ability to enter mitosis up to 24 h after removal of VP-16. Irreversible G(2) arrest was delimited by the induction of significant levels of DNA cleavage or fragmentation, not associated with overt apoptosis, in the majority of cells. Western blotting of whole-cell preparations showed increases in topoisomerase II levels (up to 4-fold) attributable to cell cycle redistribution, while nuclei from cells recovering from S-phase delay showed enhanced immunoreactivity with an anti-topoisomerase II alpha antibody. The results imply that traverse of G(1)/S and early S-phase in the presence of a specific topoisomerase II poison gives rise to progressive low-level trapping of topoisomerase II alpha, enhanced topoisomerase II alpha availability and the subsequent irreversible arrest in G(2) of cells showing limited DNA fragmentation. We suggest that protracted, low-dose chemotherapeutic regimens incorporating VP-16 are preferentially active towards cells attempting G(1)/S transition and have the potential for increasing the subsequent action of other topoisomerase II-targeted agents through target enzyme modulation. Combination modalities which prevent such dynamic changes occurring would act to reduce the effectiveness of the VP-16 component.