Abstract
Pt-r4是中國倉鼠卵巢細胞 (Chinese hamster ovary cells, CHO-K1)長期培養在順雙氨雙氯鉑 (cisplatin) 培養基中，發展成對順雙氨雙氯鉑藥物具有兩倍抗性之細胞株。順雙氨雙氯鉑抑制Pt-r4細胞50%的群落形成之劑量 (IC50) 為14μM，而母細胞CHO-K1欲達此效果的劑量則為7 μM。一般認為，順雙氨雙氯鉑藥物抗性細胞對於氯化鎘亦具同樣抗性，反之亦然。但對順雙氨雙氯鉑具抗性之Pt-r4細胞卻對氯化鎘是一敏感細胞株，氯化鎘抑制50%細胞群落形成之劑量為2.5 μM；對於CHO-K1，其抑制50%細胞群落形成之氯化鎘劑量則需7.5 μM。分析此細胞中金屬硫蛋白(metallothionein, MT)、麩胱甘胜 (glutathione, GSH)、以及麩胱甘胜酵素 (glutathione S-transferase, GST)，此兩種細胞株之間的差異經比較，於統計上均未能有顯著之分別。但由細胞對於氯化鎘的吸收排除試驗結果顯示，氯化鎘在Pt-r4細胞的累積量隨著時間或劑量的增加，均比母細胞CHO-K1的累積量多。然而氯化鎘自兩細胞株中排除速率並無差別，因此造成氯化鎘累積量在Pt-r4細胞增加的原因，並不是由於氯化鎘排除量過少所導致。此外，細胞暴露在氯化鎘培養基2小時、立即以鹼性溶液沖提，並未觀察到氯化鎘所造成小片斷的單股DNA斷裂；然而將細胞以氯化鎘處理後，再置於不含氯化鎘的的培養基四小時後，在Pt-r4細胞可見顯著小片斷的單股DNA斷裂；CHO-K1經同樣步驟處理，並未發現小片斷的單股DNA斷裂現象。Pt-r4細胞對氯化鎘敏感的原因由實驗結果可歸納於兩個原因：(1).氯化鎘在Pt-r4細胞累積量較CHO-K1細胞高。(2).氯化鎘造成Pt-r4細胞延遲性小片斷的單股DNA斷裂，而CHO-K1細胞無論有無四小時不含藥物後續培養，均未觀察到小片斷單股DNA斷裂的現象。Cisplatin-resistant cells, Pt-r4, were developed throughchronic exposure of Chinese hamster ovary (CHO-K1) cells byincreasing concentrations of cisplatin. The IC50 of clonogenicsurvival after 2-h cisplatin treatment was 14 and 7 μM forresistant Pt-r4 and CHO-K1 cells, respectively. Mostinterestingly, Pt-r4 cells were more sensitive to cadmiumchloride than CHO-K1 cells. The IC50 of clonogenic survivalafter 2-h cadmium chloride treatment was 2.5 and 7.5 μM for Pt-r4 and CHO-K1 cells, respectively. The levels of cadmiumdetoxification molecules including metallothionein,glutathione, and glutathione S-transferase were not changed inPt-r4 cells. However, Pt-r4 cells exhibited enhanced cadmiumaccumulation in both time course and dose dependent studies.Further, the enhancement of cadmium accumulation in Pt-r4 cellscould not be ascribed to the decrease of efflux. In theaspect of cadmium induced DNA damages, the DNA single-strandbreaks were not observed by alkaline elution assay immediatelyafter cadmium exposure in Pt-r4 and CHO-K1 cells. However, theextent of DNA damages was significantly increased when thecadmium-treated Pt-r4 cells were cultured in drug-free mediumfor another 4 h. In summary, these results indicated that theincreased sensitivity of Pt-r4 cells to cadmium chlorideinvolved two factors: (1) Cellular cadmium chlorideaccumulation was increased. (2) Cadmium enhanced delayed DNAlesions in Pt-r4 cells.