Alkylator resistance in human B lymphoid cell lines: (2). Increased levels of topoisomerase II expression and function in a melphalan-resistant B-CLL cell line.
Previous studies have demonstrated alkylating (melphalan) resistance in the B-CLL derived WSU-CLL cell line as compared to WIL2 B lymphocytic cells. Nuclear extracts from WSU-CLL cells demonstrate a highly significant increase in DNA topoisomerase II activity as compared to WIL2 cells. Western blot analysis showed the level of topoisomerase II proteins expressed in WSU-CLL cells to be increased as compared to WIL2 cells. WSU-CLL cells were 5.24-fold more sensitive than WIL2 cells to the cytotoxic effect of the topoisomerase II inhibitor doxorubicin. No difference in topoisomerase I activity or of the level of topoisomerase I protein expression was observed comparing the two cell lines. The sensitivity to the cytotoxic effects of topoisomerase I inhibitor, camptothecin, did not differ in WSU-CLL and WIL2 cell lines. Pre-incubation with doxorubicin significantly increased melphalan induced interstrand-DNA-crosslink formation and cytotoxicity in WSU-CLL cells as compared to WIL2 cells. The affinity of topoisomerase II for WSU-CLL UV-irradiated-crosslinked DNA was increased 2.84-fold as compared to that of WSU-CLL native DNA. The affinity of topoisomerase II for both UV-irradiated (crosslinked) and for native DNA was significantly decreased after doxorubicin-pretreatment. Measurement of DNA polymerase beta and DNA polymerase beta revealed significant elevations in DNA polymerase beta (58.82 +/- 3.67 units/mg protein in WSU-CLL cells, as compared to 27.82 +/- 4.39 units/mg protein in WIL 2 cells; p < 0.01) but not DNA polymerase beta (0.82 +/- 0.11 units/mg protein in WSU-CLL cells, compared to 0.74 +/- 0.09 units/mg protein in WIL2, p > 0.05). However, exposure to aphidicolin (an inhibitor of DNA polymerase a) failed to increase melphalan induced cytotoxicity suggesting that although DNA polymerase a activity was increased in WSU-CLL cells the mechanisms of resistance does not involve this specific DNA repair pathway. Elevated topoisomerase II activity and the increased affinity of topoisomerase II for crosslinked DNA in melphalan resistant cells appears to be the major factor responsible for alkylator resistance by changing DNA topology and thereby facilitating DNA repair.
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Paterson Institute for Cancer Research, Christie Hospital, Manchester, U.K. qpu@pier.man.ac.uk
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D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D016590:Aphidicolin / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D004250:DNA Topoisomerases, Type II / Q000502:physiology*; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D008558:Melphalan / Q000494:pharmacology*
D018906:Antineoplastic Agents, Alkylating; D016590:Aphidicolin; D004317:Doxorubicin; D004247:DNA; D004250:DNA Topoisomerases, Type II; D008558:Melphalan