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 sensitiviti es to CPT between a radioresistant (Sk-Mel-3) and a radiosensitive (HT -144) human melanoma cell line, and also between cultures with a diffe r ent growth phase in each cell line, were related to endogenous diffe rences in cellular transport of CPT or to DNA topo I catalytic activit ies and content. Cultures of HT-144 and Sk-Mel-3 cells in both the exp onential, 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 [H-3]CPT. Topo I ca talytic activity was determined from the ability of nuclear extracts p repared from the cells to relax supercoiled DNA plasmid. Nuclear extra cts 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 these cell lines was rel ated 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-p hase 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 accumulati on. However, a higher accumulation was not the reason why plateau-phas e cultures of HT-144 were relatively more sensitive than those of Sk-M el-3. Although there were no significant differences (at the p<0.05 le vel) between the endogenous catalytic activities of topo I extracted f rom exponential- and plateau phase-cultures of both these cell lines, there was a trend for HT- 144 cells to show higher endogenous topo I c atalytic activities compared to Sk-Mel-3 cells. In contrast, topo I co ntent was higher in exponential- relative to plateau phase- cultures o f both cell lines, and in HT- 144 relative to Sk-Mel-3 when cultures i n a similar growth phase were compared. The relative differences in se nsitivity to CPT observed in vitro between these two cell lines, and a lso between exponential- and plateau phase-cultures of each cell line, correlates best with topo I content rather than topo I catalytic acti vity or [H-3]CPT transport.