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 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.
Smith
P J
PJ
MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Cambridge, UK.
Souès
S
S
Gottlieb
T
T
Falk
S J
SJ
Watson
J V
JV
Osborne
R J
RJ
Bleehen
N M
NM
D001973:Bromodeoxyuridine; D018288:Carcinoma, Small Cell / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000187:drug effects* / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology*; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects; D016158:Genes, p53; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D009154:Mutation; D016196:S Phase / Q000187:drug effects; D014407:Tumor Cells, Cultured
D005047:Etoposide; D004250:DNA Topoisomerases, Type II; D001973:Bromodeoxyuridine