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Adriamycin-resistant Human Breast Cancer Cells Isolation of Amplified and Overexpressed DNA Sequences from

An MCF-7 human breast cancer cell line was selected which was 200fold more resistant to Adriamycin than the wild type cell line. This Adriamycin-resistant (Adr") cell line exhibited a multidrug-resistant phenotype and was cross-resistant to a wide range of antineoplastic agents including Vinca alkaloids, anthracyclines, and epipodophyllotoxins. Cytogenetic analysis of the Adr" cell line showed the presence of homoge neously staining regions on several chromosomes which were not present in the parental cell line. Using the technique of in-gel renaturation, DNA sequences which were amplified 50to 100-fold in the Adr" cell line and which covered a total of over 140 kilobases were isolated. In addition, Adr" cells were found to contain amplified and overexpressed sequences which were homologous to hamster P-glycoprotein gene sequences. A hamster cDNA P-glycoprotein gene probe was used to screen a XgtlO cDNA library made from human Adr" cell line mRNA and human cDNA sequences homologous to the P-glycoprotein gene were isolated. Hybrid ization studies with the cloned human cDNA (pADRl) showed that the Adr* MCF-7 cell line contained a 60-fold amplification of this DNA sequence and that polyadenylated mRNA from the Adr" cell line con tained a 4.8-kilobase transcript which was overexpressed 45-fold. There was a direct correlation between DNA and RNA copy number of this sequence and level of resistance among several MCF-7 Adriamycinresistant cell lines. In situ hybridization studies demonstrated that the human P-glycoprotein gene sequence was found on chromosome 7q21.1 in normal human lymphocytes and that amplified DNA sequences isolated from the Adr" MCF-7 cells by the in-gel hybridization technique were linked to the human P-glycoprotein sequences in the homogeneously staining regions in the Adr" cells.