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 G1/S phase boundary. At concentrations up to 0.25 microM VP-16, 
cells became arrested in G2 by 24 h exposure, whereas at concentrations 0.25-2 
microM G2 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 G1/S and subsequently exposed to VP-16 in early S-phase. 
Cells not experiencing a VP-16-induced S-phase delay entered G2 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 G2 arrest with greatly reducing ability to enter 
mitosis up to 24 h after removal of VP-16. Irreversible G2 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 G1/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 G2 
of cells showing limited DNA fragmentation. We suggest that protracted, low-dose 
chemotherapeutic regimens incorporating VP-16 are preferentially active towards 
cells attempting G1/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.