Overexpression of the ATP-binding Cassette Half-Transporter, ABCG2 (MXR/BCRP/ABCP1), in Flavopiridol-resistant Human Breast Cancer Cells 1 Robert W. Robey, Wilma Y. Medina-Pe ´rez, Kenryu Nishiyama, Tyler Lahusen, Keisuke Miyake, Thomas Litman, Adrian M. Senderowicz, Douglas D. Ross, and Susan E. Bates 2 Developmental Therapeutics Department, Medicine Branch, National Cancer Institute [R. W. R., W. Y. M-P., K. N., K. M., T. L., S. E. B.], and Molecular Therapeutics Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland 20892 [T. L., A. M. S.], and University of Maryland Greenebaum Cancer Center and Department of Medicine, University of Maryland School of Medicine, and Baltimore Veterans Medical Center, Department of Veterans Affairs, Baltimore, Maryland 21201 [D. D. R.] ABSTRACT We sought to characterize the interactions of flavopiri- dol with members of the ATP-binding cassette (ABC) trans- porter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 M was able to prevent MRP-mediated calcein efflux, whereas Pgp- mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 M. To determine putative mechanisms of resistance to flavopiridol, we ex- posed the human breast cancer cell line MCF-7 to incremen- tally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1000 nM flavopiridol and was found to be 24-fold resistant to fla- vopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overex- pressing cells, cytotoxicity studies were repeated in the pres- ence of 5 M of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiri- dol, mitoxantrone, SN-38, and topotecan was restored. Mi- toxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol. INTRODUCTION Flavopiridol (HMR 1275, L86-8275), an N-methylpiperidi- nyl, chlorophenyl flavone, is the first cdk 3 inhibitor used in human clinical trials (1). Flavopiridol can cause both a G 1 and G 2 -M cell cycle arrest, which is thought to be attributable to three biochemical effects: (a) it inhibits cdk1, cdk2, cdk4, and cdk7 in a competitive manner with respect to ATP; (b) it inhibits the activity of the cdk7/cyclin H complex, thus preventing necessary activating phosphorylation of cdks; and (c) it down- regulates cyclin D1 and cyclin D3, which are necessary for cdk4 and cdk6 activation (2, 3). Flavopiridol has also been shown to induce apoptosis in a number of cell lines (4 –9). As the clinical development of flavopiridol is pursued, it becomes important to evaluate mechanisms of cellular resist- ance. Although a number of resistance mechanisms are possible, we focused on the ABC family of transporter proteins that may mediate efflux of anticancer agents, thereby reducing intracel- lular drug concentrations. Pgp and MRP are two of the most extensively studied ABC transporters and are known to confer resistance to a wide variety of structurally unrelated cytotoxic agents (10, 11). The newly described mitoxantrone resistance protein, MXR or ABCG2, is an ABC half-transporter that is thought to dimerize to function and has been shown to confer resistance to mitoxantrone, anthracyclines, and to the campto- thecins topotecan and SN-38 (12–16). Except for minor se- quence differences, MXR is identical to BCRP reported by Doyle et al. (13) and to the placental ABC protein (ABCP1), which was reported by Allikmets et al. (17) and is expressed in high levels in the placenta. The Human Gene Nomenclature Committee has suggested that MXR/BCRP/ABCP1 be renamed ABCG2, 4 and such terminology will be used hereafter. Recent studies have shown that flavopiridol is able to inhibit MRP-mediated transport and increase MRP-related Received 5/18/00; revised 9/13/00; accepted 9/15/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Funded in part by Grant RO1-CA77545 from the National Cancer Institute, NIH and a Merit Review Grant from the Department of Veterans Affairs (to D. D. R.). 2 To whom requests for reprints should be addressed, at Medicine Branch, National Cancer Institute, NIH, Building 10, Room 12N226, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-4916; Fax: (301) 402-0172; E-mail: sebates@helix.nih.gov. 3 The abbreviations used are: cdk, cyclin-dependent kinase; ABC, ATP- binding cassette; Pgp, P-glycoprotein; MRP, multidrug resistance-asso- ciated protein; BCRP, breast cancer resistance protein; ABCP1, placen- tal ABC protein 1; FTC, fumitremorgin C; 5-FU, 5-fluorouracil. 4 Internet address: http://www.gene.ucl.ac.uk/users/hester/abc.html. 145 Vol. 7, 145–152, January 2001 Clinical Cancer Research Research. on September 14, 2021. © 2001 American Association for Cancer clincancerres.aacrjournals.org Downloaded from