THE ROLE OF CELL-CYCLE REGULATION AND APOPTOSIS TRIGGERING IN DETERMINING THE SENSITIVITY OF LEUKEMIC-CELLS TO TOPOISOMERASE-I AND TOPOISOMERASE-II INHIBITORS

Topoisomerase (topo) inhibitors induce enzyme-linked DNA breaks. Resul ting DNA damage can lead to cell cycle arrest and/or cell death by apo ptosis. The sensitivity of five human leukemic cell lines to topo I (c amptothecin or CPT) and topo II (etoposide or VP-16) inhibitors varied widely (100-fold for CPT and 30-fold for VP-16). Three cell lines wer e more sensitive (BV173, HL60, U937) and two cell lines were resistant (K562, KCL22) to both drugs. None of these cell lines were selected f or drug resistance and overexpressed mdr1 gene. Their sensitivity was not related to their doubling time nor to cell cycle repartition. The initial DNA damage (cleavable complexes) induced by topo I and II inhi bitors was measured as DNA-protein crosslinks (DPC) using alkaline elu tion. Neither DPC level induced by 30-min treatment with CPT or VP-16 nor the levels of topo 1, topo II alpha and topo II beta mRNA were rel ated to sensitivity. Electron microscopy and DNA fragmentation measure d by filter elution and agarose gel electrophoresis demonstrated that apoptosis was induced by both drugs in the five cell lines. The kineti cs of DNA fragmentation was related to cell sensitivity. At drug conce ntrations higher than IC50, DNA fragmentation increased very rapidly i n the three sensitive, compared with the two resistant, cell lines. Co ntinuous exposure to both drugs induced cell cycle arrest in either G2 or S phase that was related both to cell sensitivity and drug concent ration. Comparison between cell lines indicated that the ability of ce lls to arrest cell cycle in G2 or S phase was related to their drug se nsitivity and increased with cell resistance. In a given cell line, ce ll cycle progression was observed to be progressively inhibited by inc reasing drug concentrations. Treatment of synchronized cells demonstra ted that highly cytotoxic drug concentration induced a complete inhibi tion of cell cycle progression. Altogether, these data suggest that th e ability of leukemic cell lines to regulate cell cycle progression an d to trigger apoptosis is more indicative of their sensitivity to topo isomerase poisons than cleavable complexes induced by these drugs.