In vitro selection and characterization of a bleomycin-resistant subline of B16 melanoma

A subline of B16 melanoma cells which is 10-fold resistant to bleomycin (BLM) was developed by exposure of the parental cell line to sequential increases in BLM concentration. This resistance to BLM is stable for over 30 passages in drug-free medium. Neither double minute chromosomes nor homogeneously staining regions were evident in karyotypes of the resistant cells. The subline, B16/BLM-R1, was slightly radioresistant, with a D0 ratio of 1.4 compared to the parental cells. No cross-resistance was observed to a number of cytotoxic drugs, including doxorubicin, melphalan, cisplatin, carmustine, dactinomycin, mitomycin C, and vinblastine. However, slight cross-resistance (2-fold) was noted with etoposide. Marked resistance to BLM also was demonstrated in vivo in mice bearing B16/BLM-R1 implanted s.c. Possible mechanisms of BLM resistance in these cells were explored through examination of the degree of drug inactivation by BLM hydrolase and measurement of single- and double-strand DNA scission, as well as repair of single strand breaks by the alkaline elution technique. The specific activity of BLM hydrolase was 70% higher in the resistant subline, commensurate with a 50% increase in protein content in these cells. Because this is insufficient to account for the 10-fold resistance, BLM hydrolase activity does not appear to be a major determinant of resistance in B16/BLM-R1. The overall number of single and double strand breaks in DNA produced by bleomycin treatment did not differ in the sensitive and resistant cells. The cross-resistance with ionizing radiation and etoposide suggests an enhanced capability of B16/BLM-R1 cells to withstand or repair single strand breaks in DNA. However, this was not evident by measuring repair of single strand scission by alkaline elution.