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Amplification of the ATP-Binding Cassette 2 Transporter Gene Is Functionally Linked with Enhanced Efflux of Estramustine in Ovarian Carcinoma Cells¹

Departments of Pharmacology [N. M. L., J. T. B., L. B., K. D. T.] and Medical Oncology [M. G. B., G. D. K., D. W. B., J. R. T.], Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

An estramustine-resistant human ovarian carcinoma cell line, SKEM, was generated to explore resistance mechanisms associated with this agent. Cytogenetic analysis revealed that SKEM cells have a homogeneously staining region (hsr) at chromosome 9q34. Microdissection of the hsr, followed by fluorescence in situ hybridization to SKEM and normal metaphase spreads, confirmed that the amplified region was derived from sequences from 9q34. In situ hybridization with a probe specific for ABC2, a gene located at 9q34 that encodes an ATP-binding cassette 2 (ABC2) transporter, indicated that this gene is amplified 6-fold in the estramustine-resistant cells. Southern analysis confirmed that ABC2 was amplified in SKEM, and Northern analysis indicated that the ABC2 transcript was overexpressed 5-fold. The ABC1 gene located at 9q22–31 was not amplified in the resistant cells, and mRNA levels of several other ABC transporter genes were unaltered. Consistent with the concept that increased ABC2 expression contributes to the resistant phenotype, we observed that the rate of efflux of dansylated estramustine was increased in SKEM compared with control cells. In addition, antisense treatment directed toward ABC2 mRNA sensitized the resistant cells to estramustine. Together, these results suggest that amplification and overexpression of ABC2 contributes to estramustine resistance and provides the first indication of a potential cellular function for this product.

¹ This work was supported in part by NIH Grants CA06927 and RR05539, NIH Grant CA53893 (to K. D. T.), and by an appropriation from the Commonwealth of Pennsylvania.

² Present address: Viro-Pharma, Malvern, PA 19355.

³ To whom requests for reprints should be addressed, at Department of Pharmacology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. Phone: (215) 728-3137; Fax: (215) 728-4333.

Received 12/22/97. Accepted 2/16/98.

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