Induction of alkylator (melphalan) resistance in HL60 cells is accompanied by increased levels of topoisomerase II expression and function.
Human leukemic HL60 cells were selected for resistance to alkylating agents by stepwise exposure to increasing concentrations of L-phenylalanine mustard (melphalan). The resulting resistant cell line (R-HL60) was 4-fold resistant (melphalan IC50 value, 27.84 +/- 4.2 microM) to melphalan compared with parental HL60 cells (melphalan IC50 value, 6.9 +/- 1.78 microM). Nuclear extracts from R-HL60 cells possess a approximately 4-fold increase in DNA topoisomerase II activity compared with parental HL60 cells. As determined using Western blot analysis, the level of topoisomerase IIalpha protein expressed in R-HL60 cells was approximately 3-fold that of parental HL60 cells. However, there were no differences observed in the level of topoisomerase IIbeta protein, in the topoisomerase I activity, or in the level of topoisomerase I protein expression comparing the two cell lines. R-HL60 cells were 5-fold more sensitive than parental HL60 cells to the cytotoxic effect of the topoisomerase II inhibitor doxorubicin. The sensitivity to the cytotoxic effects of the topoisomerase I inhibitor camptothecin did not differ in R-HL60 and parental HL60 cell lines. Preincubation with doxorubicin significantly increased melphalan-induced interstrand DNA cross-link formation and cytotoxicity in R-HL60 cells compared with the parental HL60 cells. The affinity of topoisomerase II for UV-irradiated cross-linked HL60 DNA was increased by approximately 2.5-fold compared with that of HL60 native DNA. The affinity of topoisomerase II for both UV-irradiated (cross-linked) and native DNA was significantly decreased after doxorubicin pretreatment. Elevated topoisomerase II activity and the increased affinity of topoisomerase II for cross-linked DNA in melphalan-resistant cells seems to contribute to alkylator resistance by changing DNA topology, thereby facilitating DNA repair.
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Division of Clinical Hematology and Medical Oncology, Department of Medicine, University of the Witwatersrand Medical School, Parktown, South Africa. 014pu@chiron.wits.ac.za
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D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018736:DNA Adducts; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis*; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D004789:Enzyme Activation; D015870:Gene Expression / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008558:Melphalan / Q000494:pharmacology*; D059005:Topoisomerase II Inhibitors
D018906:Antineoplastic Agents, Alkylating; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004273:DNA, Neoplasm; D059005:Topoisomerase II Inhibitors; D004317:Doxorubicin; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D008558:Melphalan