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Overexpression of the Multidrug Resistance-associated Protein (MRP) Gene in Vincristine but not Doxorubicin-selected Multidrug-resistant Murine Erythroleukemia Cells¹

Center for Adaptation Genetics and Drug Resistance and the Departments of Molecular Biology and Microbiology and of Medicine, Tufts University School of Medicine and the New England Medical Center, Boston, Massachusetts 02111 [C. A. S., P. M. F., R. L. M., J-M. L., S. B. L], and the Department of Medicine, Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 [C. A. S., D. L. T.]

Multidrug-resistant sublines of the murine erythroleukemia cell line PC4 were sequentially selected in increasing vincristine concentrations (5–160 ng/ml). The low- and intermediate-level resistant cell lines, selected in 40 ng/ml of vincristine, demonstrated resistance to Vinca alkaloids and to an epipodophyllotoxin but little or none to an anthracycline. The expression of murine mdr genes, as analyzed by Northern blotting, revealed a baseline expression of murine mdr2 in parental cells that was unchanged in the drug-resistant cell lines. Overexpression of mdr3 was observed only in the highest-level resistant cell line, PC-V160, whereas mdr1 mRNA was not detected in any of the cell lines. The polymerase chain reaction, using mdr3-specific primers, excluded the possibility that low levels of P-glycoprotein expression contributed to the resistance phenotype in the low and intermediate-level resistant cell lines. Northern blot analysis using a human complementary DNA probe for the multidrug resistance-associated protein (MRP) demonstrated overexpression of murine mrp in each of the vincristine-selected sublines. Genomic amplification of the mrp gene was coincident with mrp overexpression. The expression of mrp was also examined in two series of previously characterized doxorubicin-selected cell lines derived from parental PC4 and C7D murine erythroleukemia cells. In contrast to the vincristine-selected cell lines, overexpression of mrp was not detected. These studies demonstrate that, in murine erythroleukemia cells selected for vincristine resistance, overexpression of murine mrp occurred prior to that for murine mdr. In contrast to human MRP, selection for vincristine, but not doxorubicin resistance, resulted in the overexpression of murine mrp.

¹ This investigation was supported in part by Clinical Investigator Award CA-01613 from the National Cancer Institute (to C. A. S.), USPHS Awards T32-C A09429 (to R. L. M.) and 1F32-CA08553 (to P. M. F.), the Arnold D. Imperatore Research Scholarship of the National Leukemia Association (to S. B. L.), and CA 59341 from the National Cancer Institute (to S. B. L.).

² Present address: Barnard Cancer Center, Washington University School of Medicine, St. Louis, MO 63110.

³ To whom requests for reprints should be addressed, at Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111.

Received 4/28/94. Accepted 8/29/94.

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