Chlorozotocin-induced selection of autocrine and multidrug resistant variants from a rat rhabdomyosarcoma

Abstract

The ability of tumor cells to develop simultaneous resistance to structurally different cytotoxic drugs constitutes a major problem in cancer chemotherapy. We previously described that a nitrosourea, chlorozotocin (CZT), increased in vivo the pulmonary metastatic dissemination of a rat rhabdomyosarcoma and enhanced in vitro the cloning efficiency (CE) of the same tumor cell line. The selection procedure for chlorozotocin-selected cell lines (R1 to R9 lines), by in vitro CZT-treatment and cloning assay, indicated that 2.5% of the parental cell line was a CZT-resistant subpopulation. This subset acquired, in the course of the selection steps in semi solid medium, a multidrug-resistant phenotype. In a stem cell assay, the resistance of the R9 line was increased 10-fold for adriamycin (ADR) and up to 2 fold for cisplatinum. In comparison, C8 control line sensitivity to ADR did not change significantly. This resistance did not affect the non clonogenic population, as shown in a monolayer proliferation assay. Re-exposure of R (R1-R9) lines to CZT or initial CZT contact with C lines transiently but consistently increased the CE; however, clonogenicity remained stable, and this was also observed when deticene, another alkylating drug, was used instead of CZT. Moreover, the CZT-selected R9 line easily proliferated in serum-free medium in the presence of transferrin and insulin, whereas serum was necessary to maintain the growth of the parental cell line or C lines. Finally, we show that in vitro selection of variants, by both resistance to CZT and cloning, led to the isolation of a multi-drug resistant subpopulation, serum independent for its proliferation - two properties associated with progressive malignancy.