Vol. 3, 2025-2032, November 1997 Clinical Cancer Research 2025 Altered Methylation of the Human MDR1 Promoter Is Associated with Acquired Multidrug Resistance1 Phillip Kantharidis, Assam El-Osta, Michelle deSilva, Dominic M. P. Wall, Xiu F. Hu, Alison Slater, Gabriella Nadalin, John D. Parkin, and John R. Zalcberg2 Departments of Medical Oncology and Haematology, Austin Repatriation Medical Centre, West Heidelberg 308 1 . Victoria, Australia [D. M. P. W., J. D. P]: and Trescowthick Research Labs [P. K.. A. E-O., M. d., X. F. H., A. S., G. N.] and Division of Haematology and Medical Oncology [J. R. Z.], Peter MacCallum Institute, East Melbourne 3002, Victoria, Australia ABSTRACT One of the most important forms of drug resistance in acute myeboid leukemia is the multidrug resistance (MDR) phenotype, which is characterized by the expression of the MDRJ gene product, P-glycoprotein. Although a number of factors affect MDR1 gene expression, the genetic events that “switch on” the human MDRJ gene in tumor cells that were previously P-glycoprotein negative have remained elusive. Here, we report evidence that the methybation status of the human MDR1 promoter may serve as a basis for this “switch.” Based on Southern analysis using methylation- sensitive and methylation-insensitive restriction enzymes, a tight correlation was found between MDR phenotype and demethylation of the 5’ region of the MDRJ gene in a human T cell leukemia cell line. Similar results were obtained from the analysis of P-glycoprotein-positive and P-glycoprotein- negative samples of chronic lymphocytic leukemia. Treat- ment of the cell lines with the demethybating agent 5’- azadeoxycytidine altered the methybation pattern of the MDR1 promoter in P-glycoprotein.negative cells to resem- ble that of P-gbycoprotein-positive cells and activated the promoter such that MDR1 mRNA was now detectable. Treatment also resulted in an increased resistance to epiru- bicin and decreased daunomycin accumulation, both of which were reversible by verapamil, a characteristic of the classical MDR phenotype in cells expressing P-gbycoprotein. These results suggest that the MDR phenotype may be Received I 1/13/96; revised 4/25/97; accepted 7/3/97. 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 I8 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Anti-Cancer Council of Victoria and Department of Veterans’ Affairs (Canberra, ACT. Australia) and the Sir Edward Dunlop Medical Research Foundation (Melbourne, Victoria, Australia). 2 To whom requests for reprints should be addressed, at Division of Haematology and Medical Oncology. Peter MacCallum Cancer Insti- tute, East Melbourne 3000, Victoria, Australia. Phone: (613) 9656 1190; Fax: (613) 9656 1408; E-mail: zalcberg@petermac.unimelb.edu.au. acquired as a result of changes in methylation of the MDR1 promoter. INTRODUCTION Drug resistance remains a major obstacle in the successful treatment of human tumors by cytotoxic agents. Of the several forms of drug resistance described, the most extensively studied is classical MDR,3 which is characterized by the overexpression of the MDRJ gene product, Pgp; partial reversal of resistance by several modulators, including verapamil and cyclosporin A; and cross-resistance to a variety of naturally occurring cytotoxic agents (1 ). This plasma membrane glycoprotein functions as an energy-dependent drug efflux transporter, resulting in bower intracellular levels of a wide variety of chemotherapeutic agents that are substrates for Pgp (1). Despite the difficulties in suc- cessfully modulating this phenotype clinically (2), the expres- sion of Pgp is of prognostic significance in various hematolog- ical malignancies, including acute myeboid leukemia. In a series of unrelated studies, Pgp expression represented an adverse prognostic factor that independently predicted for survival, re- sponse rate, and duration of response (reviewed in Ref. 2). In human cancers in which the corresponding normal tissue expresses Pgp, the resulting tumors also appear to express this protein. These include carcinomas of the colon, liver, pancreas, and kidney (1). However, in drug-sensitive tumors, the acqui- sition of MDR during the course of chemotherapy is thought to be due to the selection of resistant mutants in the tumor cell population by drugs known to be substrates for Pgp. This model fits with the mutation-selection hypothesis for drug resistance in cancer (3), in which genetic changes in a small number of drug-sensitive cells ultimately lead to the development of re- sistance via a process of selection. This scenario is thought to be particularly relevant in hematological malignancies. Although the regulation of Pgp expression has been studied in several cell types and much has been learned about factors regulating its expression (4-8), the genetic events that switch on Pgp expression in Pgp-negative cells remain poorly defined. No mutations in the sequence of the MDR 1 promoter have been identified to account for the activation of the MDR 1 promoter in cells that express Pgp. Although point mutations have been reported in a number of osteosarcomas at nucleotides 103 and 107 downstream of the MDR1 transcription initiation site, they were found in untreated tumors, and their significance remains to be determined (9). Attempts have been made to identify transcription factors that may be altered or present in higher or lower levels in Pgp-positive cells compared to Pgp-negative cells. One report 3 The abbreviations used are: MDR, multidrug resistance: Pgp. P-gly- coprotein: CLL. chronic lymphocytic leukemia: azadC, 5 ‘-azadeoxycy- tidine; RT-PCR, reverse transcription-PCR. Research. on December 7, 2021. © 1997 American Association for Cancer clincancerres.aacrjournals.org Downloaded from