Fludarabine-Mediated Repair Inhibition of Cisplatin-Induced DNA Lesions in Human Chronic Myelogenous Leukemia-Blast Crisis K562 Cells: Induction of Synergistic Cytotoxicity Independent of Reversal of Apoptosis Resistance

Abstract

We demonstrated previously that the nucleoside of fludarabine (F-ara-A), a clinically effective agent against chronic lymphocytic leukemia and low-grade lymphoma, produces synergistic cytotoxicity against cisplatin-resistant CP2.0 human colon tumor cells when administered in combination with cisplatin. The purpose of this study was 2-fold: (i) to determine whether the synergy occurs in K562 human chronic myelogenous leukemia cells, which, unlike CP2.0 cells, are relatively resistant to drug-induced apoptosis because they express P210(bcr-abl) and (ii) to study the underlying mechanism for the synergy if the enhancement of cytotoxicity occurs in K562 cells. When K562 cells were treated with fludarabine nucleoside and cisplatin as single agents for 4 hr, IC50 values for fludarabine and cisplatin were 3.33 and 2.28 microM, respectively, as measured by a clonogenic survival assay. The simultaneous treatment of K562 cells with the two agents resulted in synergistic cell killing as determined by median-effect analysis. Such synergistic cell killing by combined cisplatin and fludarabine could not be detected in repair-deficient human xeroderma pigmentosum cell lines. Within the range of cytotoxic concentrations, fludarabine (2.5-15 microM) and cisplatin (3-30 microM) as single agents produced no detectable internucleosomal DNA fragmentation as revealed by gel electrophoresis, nor did the combination of the two drugs induce apoptotic DNA degradation. The effects of fludarabine on the repair of cisplatin-induced DNA adducts and interstrand cross-links in K562 cells were analyzed to determine their correlation with the cytotoxic synergy. The interstrand cross-links were measured by the ethidium bromide binding fluorescence assay and quantitative Southern blotting technique. Repair of the intrastrand adducts was detected with whole-cell extracts using a cisplatin-damaged plasmid as the substrate for the in vitro repair assay. Fludarabine at clinically achievable concentrations (1.5-4.5 microM fludarabine nucleoside; 20-100 microM fludarabine triphosphate) inhibited the repair of the DNA lesions induced by cisplatin in a dose-dependent fashion in K562 cells but not in xeroderma pigmentosum cells. Cotreatment with fludarabine preferentially increased the number of interstrand cross-links induced by cisplatin in actively transcribed genes in K562 cells. These data demonstrate the DNA-repair-inhibitory effect of fludarabine and suggest that this effect may contribute to the synergistic cytotoxicity of the fludarabine/cisplatin combination that resulted in decreased clonogenic survival of apoptosis-resistant K562 cells.