Topoisomerase-specific drug sensitivity in relation to cell cycle progression.

Abstract

. It also mediates the DNA cleavage activity and cytotoxicity of clinically important anticancer agents such as etoposide. We have examined the activity of topoisomerase H during the first cell cycle of quiescent BALB/c 3T3 cells following serum stimulation. Etoposide-mediated DNA break frequency in vivo was used as a parameter of topoisomerase II activity, and enzyme content was assayed by immunoblotting. Density-arrested A31 cells exhibited a much lower sensitivity to the effects of etoposide than did actively proliferating cells. Upon serum stimulation of the quiescent cells, however, there was a marked increase in drug sensitivity which began during S phase and reached its peak just before mitosis. Maximal drug sensitivity during this period was 2.5 times greater than that of log-phase cells. This increase in drug sensitivity was associated with an increase in intracellular topoisomerase II content as determined by immunoblotting. The induction of topoisomerase 11-mediated drug sensitivity was aborted within 1 h of exposure of cells to the protein synthesis inhibitor cycloheximide, but the DNA synthesis inhibitor aphidicolin had no effect. In contrast to the sensitivity of cells to drug-induced DNA cleavage, maximal cytotoxicity occurred during S phase. A 3-h exposure to cycloheximide before etoposide treatment resulted in nearly complete loss of cytotoxicity. Our findings indicate that topoisomerase II activity fluctuates with cell cycle progression, with peak activity occurring during the G2 phase. This increase in topoisomerase II is protein synthesis dependent and may reflect a high rate of enzyme turnover. The dissociation between maximal drug-induced DNA cleavage and cytotoxicity indicates that the topoisomerase-mediated DNA breaks may be necessary but are not sufficient for cytotoxicity and that other factors which are particularly expressed during S phase may be important as well.Type II DNA topoisomerases are ATP-dependent enzymes which catalyze the breakage and reunion of duplex DNA, allowing a second segment of DNA to pass through the break site (1, 23). They are required for the segregation of DNA following the conclusion of replication (4) and appear to be required at mitosis (12). During the breakagereunion cycle a covalent phosphotyrosyl bond between enzyme and DNA is formed at the 5' terminus of each strand. A number of commonly used anticancer agents such as intercalating agents and epipodophyllotoxins can stabilize this DNA protein intermediate, and convincing evidence exists that this interaction does mediate their cytotoxicity (15,16,25). A better understanding of the enzyme would be useful in improving the efficacy of these drugs. In addition, these topoisomerase-specific drugs have become useful probes in understanding the fundamental functions and control mechanisms of the enzyme.Topoisomerase II activity and content varies as a function of the proliferative status of the cell (5,19,29,30 Cell culture. BALB/c 3T3 (A31) were grown at 37°C in a monolayer in alpha minimal essential medium...

Characterisation of VP-16-induced DNA cleavage in oestrogen-stimulated human breast cancer cells

“…This is an important point if such observations are to serve as the basis of new strategies for improving the therapeutic index of clinical cancer therapy. The effects of cycloheximide and aphidicolin on VP-16-induced DNA cleavage in oestrogen-primed cells are in strong agreement with those reported by Chow & Ross (1987) for the effects of these inhibitors on enhanced cleavage seen in synchronised cultures released from quiescence. The inhibitory effect of cycloheximide documented in both reports indicates that the enhanced drug-induced DNA cleavage witnessed in activated cell cultures is dependent at some point on new protein synthesis.…”

“…Absolute levels of topoisomerase II have, moreover, recently been recognised to be higher in proliferating than in quiescent cells (Heck & Earnshaw, 1986). However, the report that enzyme levels (as determined by immunoblotting) rise only in S-and G2-phase cells following stimulation -even though enhancement of VP-16-induced DNA cleavage is unaffected by inhibition of DNA synthesis using aphidicolin (Chow & Ross, 1987) suggests that enzyme activation within GI-phase cells may accompany cellular activation, a model supported by the data presented here. Indeed, in vitro data have already implicated phosphorylation (Ackerman et al, 1985), poly(ADP-ribosyl)ation (Darby et al, 1985) and calciummediated pathways (Osheroff & Zechiedrich, 1987) …”

Genetic polymorphism and gene expression of microsomal epoxide hydrolase in non-small cell lung cancer

“…The procedure for immunoblotting has been described previously (22). Briefly, proteins were separated in a 10% SDS-polyacrylamide gel with 4.5% stacking gel.…”

[18F]2-Fluoro-2-deoxy-D-glucose incorporation by AGS gastric adenocarcinoma cells in vitro during response to epirubicin, cisplatin and 5-fluorouracil

“…Epirubicin, an anthracycline derivative of doxorubicin, exerts its anti-tumour effects via its action as a DNA intercalating agent and as an inhibitor of topoisomerase II (Cersosimo and Hong, 1986;Bartkowiak et al, 1992;Zoli et al, 2004). The arrest of AGS cells at higher concentrations of epirubicin may be related to peak activity of topoisomerases occurring during the G 2 phase (Chow and Ross, 1987). Exposure to cisplatin, an alkylating agent, results in the binding of cisplatin to DNA, forming cisplatin-DNA adducts which causes an alteration in the conformation of DNA leading to cell cycle arrest and apoptosis (Jordan and Carmo-Fonseca, 2000;Gonzalez et al, 2001;Wang et al, 2004).…”