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

Clinical Pharmacology Branch [C. R. F., S. P. I., K. H. C., M. E. G.], Medicine Branch [C-S. K-S., J. W-P.], and Pediatric Branch [N. R., M. A. I.], National Cancer Institute, NIH, Bethesda, Maryland 20892, and Sloan-Kettering Institute for Cancer Research, Walker Laboratory, Rye, New York 10580 [P. W. M.]

An MCF-7 human breast cancer cell line was selected which was 200-fold more resistant to Adriamycin than the wild type cell line. This Adriamycin-resistant (Adr^R) 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^R cell line showed the presence of homogeneously 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 50- to 100-fold in the Adr^R cell line and which covered a total of over 140 kilobases were isolated. In addition, Adr^R 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 gt10 cDNA library made from human Adr^R cell line mRNA and human cDNA sequences homologous to the P-glycoprotein gene were isolated. Hybridization studies with the cloned human cDNA (pADR1) showed that the Adr^R MCF-7 cell line contained a 60-fold amplification of this DNA sequence and that polyadenylated mRNA from the Adr^R cell line contained 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 Adriamycin-resistant 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^R 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^R cells.

¹ Supported by a Pharmacology Research Associate Training Program fellowship from the National Institutes of General Medical Sciences.

² To whom requests for reprints should be addressed, at NIH, Building 10, Room 6N119, Bethesda, MD 20892.

Received 12/31/86. Revised 5/22/87. Accepted 7/ 1/87.

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