Mitoxantrone-DNA binding and the induction of topoisomerase II associated DNA damage in multi-drug resistant small cell lung cancer cells.
The cytotoxicity anti-tumour intercalating agents such as the anthraquinone mitoxantrone is thought to relate to DNA binding and the trapping of DNA topoisomerase II complexes on cellular DNA. We have studied the uptake, nuclear location, DNA binding mode and DNA damaging capacity of mitoxantrone in a small cell lung carcinoma cell line (NCI-H69) compared with an in vitro-derived variant subline (NCI-H69/LX4) that exhibits "classical" multi-drug resistance (MDR). Variant cells maintained under doxorubicin selection showed reduced RNA levels that returned to control values within 7 days of growth under non-selective conditions. Variant cells released from selection stress showed resistance to DNA cleavage by doxorubicin, mitoxantrone, 4'-epidoxorubicin, 4'-deoxy-doxorubicin but reduced resistance to aclacinomycin A and a 9-alkyl substituted anthracycline in broad agreement with the cross-resistance patterns for cytotoxicity. Mitoxantrone treated NCI-H69 cells were found to accumulate DNA-protein crosslinks during a 4 hr post-treatment incubation period whereas variant cells maintained depressed levels of crosslinking. There was no apparent abnormality in the availability or drug sensitivity of topoisomerase II assayed in crude nuclear extracts of NCI-H69/LX4 cells. Whole cell uptake of radiolabelled mitoxantrone was depressed (50%) in NCI-H69/LX4 compared with NCI-H69, whereas assessment of nuclear-bound drug in individual cells by a fluorescence quenching technique showed at least a 10-fold greater level of target protection. The quenching results provide evidence of a high affinity, saturable mode of drug binding, favoured at low drug concentrations, that correlated with DNA cleavage capacity. We propose that the cytotoxic action of mitoxantrone is dependent upon a restricted and persistent form of binding to DNA that favours the long-term or progressive trapping of topoisomerase II complexes.
Smith
P J
PJ
MRC Clinical Oncology, MRC Centre, Cambridge, U.K.
Morgan
S A
SA
Fox
M E
ME
Watson
J V
JV
D015250:Aclarubicin / Q000737:chemistry / Q000494:pharmacology; D018288:Carcinoma, Small Cell / Q000235:genetics* / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis; D004273:DNA, Neoplasm / Q000187:drug effects* / Q000378:metabolism; D004317:Doxorubicin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D004351:Drug Resistance; D004790:Enzyme Induction; D006801:Humans; D007364:Intercalating Agents; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism; D008942:Mitoxantrone / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects
D004273:DNA, Neoplasm; D007364:Intercalating Agents; D015250:Aclarubicin; D004317:Doxorubicin; D008942:Mitoxantrone; D004250:DNA Topoisomerases, Type II