Ng C E, Cybulski S E, Bussey A M, Aubin R A, Raaphorst G P
Ottawa Regional Cancer Centre, Ont., Canada.
Anticancer Res. 1998 Jul-Aug;18(4C):3119-26.
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.
在本研究中，我们着手确定耐辐射的（Sk-Mel-3）和辐射敏感的（HT-144）人黑色素瘤细胞系之间，以及每个细胞系中处于不同生长阶段的培养物之间对喜树碱（CPT）的差异敏感性，是否与CPT的细胞转运内源性差异、或与DNA拓扑异构酶I（Topo I）的催化活性及含量有关。我们比较了HT-144和Sk-Mel-3细胞在指数生长期或平台期（即汇合期）的培养物。通过克隆形成存活试验确定细胞对CPT的反应。使用[³H]CPT测定药物积累和流出。根据从细胞制备的核提取物使超螺旋DNA质粒松弛的能力来确定Topo I催化活性。细胞的核提取物也用于通过蛋白质印迹法测定Topo I含量。相对于平台期培养物，两种细胞系的指数期培养物对CPT的敏感性显著增强，这与[³H]CPT在一种细胞系（即HT-144）而非另一种细胞系中的积累增强有关。因此，HT-144指数期培养物相对于Sk-Mel-3指数期培养物的较高敏感性至少可以部分基于相对较高的积累来解释。然而，积累较高并非HT-144平台期培养物比Sk-Mel-3平台期培养物相对更敏感的原因。尽管从这两种细胞系的指数期和平台期培养物中提取的Topo I的内源性催化活性之间没有显著差异（在p < 0.05水平），但与Sk-Mel-3细胞相比，HT-144细胞有显示出更高内源性Topo I催化活性的趋势。相反，当比较处于相似生长阶段的培养物时，两种细胞系的指数期培养物相对于平台期培养物的Topo I含量更高，并且HT-144相对于Sk-Mel-3的Topo I含量更高。在这两种细胞系之间，以及在每个细胞系的指数期和平台期培养物之间，体外观察到的对CPT敏感性的相对差异，与Topo I含量的相关性最佳，而非与Topo I催化活性或[³H]CPT转运的相关性最佳。
