Further Characterization of Bleomycin‐resistant HeLa Cells and Analysis of Resistance Mechanism

Bleomycin (BLM)‐resistant HeLa cells (HeLa‐BLM(r)), which have been subcultured for more than 150 passages during over 2 years in the presence of 1 μg/ml of BLM and stably possess a 20‐fold‐increased BLM‐resistance in vitro, were further characterized. The nude mouse tumors produced by HeLa‐BLM(r) were significantly less sensitive (P < 0.005–0.01) to BLM administration than those produced by HeLa cells, and the cells primarily cultured from nude mouse tumors of HeLa‐BLM(r) and transplanted serially 5 times in the absence of BLM also exhibited a similar degree of BLM resistance to that of HeLa‐BLM(r) cultured in BLM‐containing medium. The BLM‐resistance mechanism of HeLa‐BLM(r) was partially analyzed. The cells showed about 40% decreased accumulation and 2–3 times reduced retention of [(3)H]peplomycin, a novel BLM analog, as compared to HeLa cells, but the BLM‐hydrolase activity was at almost the same level as that of HeLa cells when determined by HPLC. Furthermore, alkaline sucrose gradient analysis of cellular DNA after BLM treatment revealed that the damaged DNA was more efficiently repaired in HeLa‐BLM(r) than in HeLa cells. These results suggest that decreased drug accumulation and retention, and elevated DNA repair activity are the main mechanisms of BLM resistance in HeLa‐BLMr.