DNA topoisomerase I content of a pair of human melanoma cell lines with very different radiosensitivities correlates with their in vitro sensitivities to camptothecin.
In this study, we set out to determine if the differential sensitivities to CPT between a radioresistant (Sk-Mel-3) and a radiosensitive (HT-144) human melanoma cell line, and also between cultures with a different growth phase in each cell line, were related to endogenous differences in cellular transport of CPT or to DNA topo I catalytic activities and content. Cultures of HT-144 and Sk-Mel-3 cells in both the exponential, or plateau (i.e. confluent), phase of growth were compared. Cellular response to CPT was determined by clonogenic survival assay. Drug accumulation and efflux were determined using [3H]CPT. Topo I catalytic activity was determined from the ability of nuclear extracts prepared from the cells to relax supercoiled DNA plasmid. Nuclear extracts of the cells were also used to determine topo I content by western blotting. The significantly enhanced sensitivity of exponential-phase, relative to plateau-phase, cultures of both cell lines was related to an enhanced accumulation of [3H]CPT in one (i.e. HT-144), but not the other, cell line. Thus the higher sensitivity of exponential-phase cultures of HT-144 relative to those of Sk-Mel-3 can at least be partially accounted for on the basis of a relatively higher accumulation. However, a higher accumulation was not the reason why plateau-phase cultures of HT-144 were relatively more sensitive than those of Sk-Mel-3. Although there were no significant differences (at the p < 0.05 level) between the endogenous catalytic activities of topo I extracted from exponential- and plateau phase-cultures of both these cell lines, there was a trend for HT-144 cells to show higher endogenous topo I catalytic activities compared to Sk-Mel-3 cells. In contrast, topo I content was higher in exponential- relative to plateau phase-cultures of both cell lines, and in HT-144 relative to Sk-Mel-3 when cultures in a similar growth phase were compared. The relative differences in sensitivity to CPT observed in vitro between these two cell lines, and also between exponential- and plateau phase-cultures of each cell line, correlates best with topo I content rather than topo I catalytic activity or [3H]CPT transport.
Ottawa Regional Cancer Centre, Ont., Canada. Cheng_Ng@cancercare.on.ca
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Ng
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Cybulski
A M
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Bussey
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Aubin
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Raaphorst
D000328:Adult; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001692:Biological Transport; D002166:Camptothecin / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004354:Drug Screening Assays, Antitumor; D005260:Female; D006801:Humans; D008297:Male; D008545:Melanoma / Q000188:drug therapy* / Q000201:enzymology* / Q000532:radiotherapy; D011836:Radiation Tolerance / Q000502:physiology*; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin