title: Induction of alkylator (melphalan) resistance in HL60 cells is accompanied by increased levels of topoisomerase II expression and function.
abstract: 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.
authors.affiliation: Division of Clinical Hematology and Medical Oncology, Department of Medicine, University of the Witwatersrand Medical School, Parktown, South Africa. 014pu@chiron.wits.ac.za
authors.forename: Q Q
authors.initials: QQ
authors.lastname: Pu
authors.forename: W R
authors.initials: WR
authors.lastname: Bezwoda
mesh_terms: 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
chemical_list: 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