[CANCER RESEARCH 61, 1469 –1476, February 15, 2001] The Pharmacological Phenotype of Combined Multidrug-Resistance mdr1a/1b- and mrp1-deficient Mice Dennis R. Johnson, Rick A. Finch, 1 Z. Ping Lin, Caroline J. Zeiss, and Alan C. Sartorelli 2 Department of Pharmacology and Developmental Therapeutics Program, Cancer Center and Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut 06520 ABSTRACT Two major classes of plasma membrane proteins that actively extrude a wide range of structurally diverse hydrophobic amphipathic antineo- plastic agents from cells, with different mechanisms of action, lead to multidrug resistance. To study the importance of these ATP-binding cassette transporters to the toxicity of cancer chemotherapy agents, we have used mice genetically deficient in both the mdr1a and mdr1b genes [mdr1a/1b(/) mice], the mrp1 gene [mrp1(/) mice], and the com- bined genes mdr1a/1b and mrp1 [mdr1a/1b(/), mrp1(/) mice] and embryonic fibroblasts derived from wild-type mice and from the three gene knockout animals. The consequences of export pump deficiencies were evaluated primarily using vincristine and etoposide. Mice deficient in the three genes, mdr1a/1b and mrp1, exhibited a 128-fold increase in toxicity to vincristine and a 3–5-fold increase in toxicity to etoposide; increased toxicity to embryonic fibroblast cells from triple knockout mice also occurred with vincristine and etoposide. Vincristine, which normally does not express toxicity to the bone marrow and to the gastrointestinal mucosa when used at therapeutic doses, caused extensive damage to these tissues in mdr1a/1b(/), mrp1(/) mice. The findings indicate that the P-glycoprotein and mrp1 are compensatory transporters for vincristine and etoposide in the bone marrow and the gastrointestinal mucosa and emphasize the potential for increased toxicities by the combined inhibition of these efflux pumps. INTRODUCTION Resistance of tumor cells to multiple chemotherapeutic agents (MDR) 3 is a major obstacle to the treatment of most human cancers. The phenomenon of MDR confers upon malignant cells the ability to withstand exposure to lethal doses of many structurally unrelated antineoplastic agents. Multidrug resistance has been characterized by the overexpression of membrane-associated glycoproteins; the two most studied of these ABC transporters that have a role in drug efflux are the P-gly, discovered in 1976 by Juliano and Ling (1), and the MRP1, first reported in 1992 by Cole et al. (2), which represents a family of ABC transporters. There are at least five additional members of this family, including MRP2 (cMOAT, ABCC 2 ), MRP3 (MOAT-D, ABCC 3 ), MRP4 (MOAT-B, ABCC 4 ), MRP5 (MOAT-C, ABCC 5 ), and MRP6 (MOAT-E, ABCC 6 ). Structural homology within the MRP family is greatest for MRP1, MRP2, MRP3, and MRP6, whereas the structures of MRP4 and MRP5 are similar (Refs. 3–5 and references therein). The P-gly, expressed by the MDR1 gene in humans and two closely related genes, mdr1a and mdr1b, in the mouse, and MRP1 play central roles in export pump-mediated resistance through the active extrusion of a wide range of structurally diverse antineoplastic agents including the Vinca alkaloids, the epipodophyllotoxins, and the anthracyclines (3– 6). Although the P-gly transports free drugs, MRP1 can be con- sidered to be an organic anion transporter capable of transporting a broad spectrum of organic anion conjugates of glutathione, glucuronic acid, and sulfate. In addition, we have shown with etoposide (7) and Loe et al. (8) with vincristine that mrp1 also functions as a cotrans- porter of xenobiotics and glutathione. In keeping with these findings, we have also demonstrated that levels of GSH in mrp1(-/-) mice were elevated by 25–90% in most tissues, especially in those tissues that are known to express high levels of mrp1 (9). That tissue increases in GSH in mrp1(-/-) mice were not attributable to the increased synthesis of GSH was supported by the finding that the levels of - glutamylcysteine synthase, the rate-limiting enzyme in the synthesis of GSH, was not significantly different in any of the tissues of mrp1(+/+) and mrp1(-/-) mice (9). Prior to these findings, a variety of studies had provided evidence that GSH was required for the transport of chemo- therapeutic agents (see Ref. 3 for appropriate references). Schinkel et al. (10, 11) have derived mice deficient in the mdr1a gene [mdr1a(-/-)] and, in addition, have derived mice deficient in the mdr1b gene [mdr1b(-/-)], and in both the mdr1a and mdr1b genes [mdr1a/1b(-/-)]. All three of these gene knockout animals are normal in all measured physiological parameters, displaying normal viability, fertility, and life span, as well as normal levels of a range of serum enzymes, proteins, electrolytes, and hematological parameters. In clinical trials, the P-gly has often been shown to be elevated in the hematological malignancies, particularly after the failure of mul- tiple drug therapy (12). Thus, the MDR phenotype as a cause of resistance in acute myelocytic leukemia and multiple myeloma and possibly in the late stages of non-Hodgkin’s lymphoma and acute lymphocytic leukemia has been documented. The role of MDR1 gene expression in the clinical resistance of solid tumors, however, is currently not firmly established (13). Nonetheless, in several malig- nancies, such as acute myelocytic leukemia, various childhood can- cers, and advanced breast cancer, overexpression of the MDR1 gene has been shown to correlate with a poor response in patients receiving cancer chemotherapeutic agents (reviewed in Ref. 14). In colon can- cer, renal cell carcinoma, primary breast cancer, and osteosarcoma, clinical studies have shown that P-gly positivity is associated with aggressive tumor behavior and is a strong predictor of treatment outcome. Whether in these instances the P-gly is a marker for drug resistance, tumor aggressiveness, or both is currently unknown. We (9) and Wijnholds et al. (15) have shown that disruption of mrp1 did not affect the viability or fertility of mice, nor were hema- tological parameters or levels of serum enzymes, proteins, and elec- trolytes different in mrp1(-/-) and mrp1(+/+) mice. However, mrp1(-/-) mice were hypersensitive to a relatively large number of anticancer drugs (9, 15, 16). The demonstration that the lack of mrp1 in mrp1(-/-) mice led to toxicity to the oropharyngeal mucosa and the seminiferous tubules of the testis in etoposide phosphate-treated animals indicates that mrp1 protects these tissues against damage from mrp1 substrates (17). The expression of MRP1 has been detected in a number of human cancers and shown to be associated with drug Received 8/9/00; accepted 12/13/00. 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 R. A. F. is a Special Fellow of the Leukemia and Lymphoma Society. 2 To whom requests for reprints should be addressed, at Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Phone: (203) 785-4533; Fax: (203) 737-2045; E-mail: alan.sartorelli@yale.edu. 3 The abbreviations used are: MDR, multidrug resistance; ABC, ATP binding cassette; P-gly, P-glycoprotein; MRP1, multidrug resistance (-associated) protein; GSH, glutathi- one; mrp1(-/-), mrp1 gene deficiency; mdr1a/1b(-/-), mdr1a/1b gene deficiency; VBL, vinblastine; mdr1a/1b(-/-), mrp1(-/-), combined mdr1a/1b, mrp1 gene defi- ciency; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophe- nyl)-2H-tetrazolium; MTD, maximum tolerated dose. 1469 Research. on November 27, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from