Double minutes (DMS) and homogeneously staining regions (HSRS)

Resistance to antineoplastic drugs may develop through a variety of mechanisms. The unique mechanism for mutation to drug resistance is amplification of the gene coding for a target protein, leading to an elevated level of protein. The process of gene amplification in drug-resistant mammalian cells may occur within a single chromosome, producing an expanded homogeneously staining region (HSR) containing multiple copies of the gene coding for the target enzyme. The amplified genes may also be located in small pieces of DNA called double minutes (DMs). To study this question, we have chosen to examine the mechanisms associated with development of adriamycin resistance in the human breast cell line MCF-7. Adr(R) MCF-7 cells are 200 fold resistant to adriamycin and were produced by exposing the MCF-7 cells to increasing drug concentration through serial passages. These cells are also cross-resistrant to actinomycin D, VP-16, and vinblastine. Cytogenetic studies of the Adr(R) cell line showed the presence of HSRs on chromosome 11 and on a rearranged chromosome 7; neither were present in the parental cell lines. The HSRs became further lengthened with increasing drug concentrations. The presence of HSRs suggested that gene amplification may play a role in this resistant phenotype. Amplified DNA sequences (pA1) representing a total length of over 140 Kb were selected from Adr(R) cell DNA using in-gel hybridization techniques. Furthermore, a 3 Kb cDNA sequence pAdr1 was isolated from these cells by hybridization to each of the HSRs in the Adr(R) cells. In addition both probes hybridized to chromosome 7q21.2 in normal human lymphocytes. Both probes also exhibited some hybridization to chromosome 6pter. Thus the pAdr1 gene (a human p-glycoprotein or mdr-1 gene) resides on human chromosome 7q21.2, a region that also contains CF and C-met.