EFFECTS OF INHIBITORS OF TOPOISOMERASES-I AND TOPOISOMERASES-II ON DNA METHYLATION AND DNA-SYNTHESIS IN HUMAN COLONIC ADENOCARCINOMA CELLS-INVITRO

Exposure of human and animal cells to inhibitors of topoisomerase I or II has recently been shown to alter gene expression and induce differentiation in a number of experimental systems. We have previously shown that nalidixic acid and novobiocin, inhibitors of topoisomerase II, induce DNA hypomethylation. Since DNA hypomethylation is frequently associated with transcriptional activation, we wished to further explore the relationship between inhibition of DNA topoisomerases and enzymatic DNA methylation. When HT-29 human colonic adenocarcinoma cells were exposed to the specific topoisomerase II inhibitor teniposide (VM-26), a dose-dependent hypomethylation of DNA was observed during the window of drug treatment. Exposure to the topoisomerase I inhibitor camptothecin (CPT) produced a small but not statistically significant trend toward DNA hypomethylation. CPT-treated cells were found to have up to 19 fold increased levels of topoisomerase II protein, which may have compensated for decreased levels of non-drug-bound topoisomerase I. Both VM-26 and CPT were found to increase [H-3]thymidine incorporation into DNA when administered in low dose (0.5 muM VM-26; 8 nM CPT). Combination of VM-26 and CPT (0.5 muM and 8 nM, respectively) produced DNA hypomethyltion, a synergistic increase in [H-3]thymidine incorporation, and an increasing number of cells entering a higher DNA ploidy cycle. Since VM-26 interferes with the DNA strand breakage-reunion reaction by stabilizing a topoisomerase II-relaxed DNA complex, our results suggest that DNA existing in this form may be a poor substrate for DNA methylase. Topoisomerase inhibitor-induced DNA hypomethylation may offer a possible explanation for the induction of differentiation observed upon exposure to this family of drugs. Altered topoisomerase activity occurring during the process of tumor progression may also provide a link between the induction of polyploidy, DNA hypomethylation and aberrant gene expression frequently observed in tumor cells.