Elsevier

Experimental Cell Research

Volume 290, Issue 2, 1 November 2003, Pages 177-194
Experimental Cell Research

Brief review
The rise of DNA methylation and the importance of chromatin on multidrug resistance in cancer

https://doi.org/10.1016/S0014-4827(03)00342-2Get rights and content

Abstract

In recent years, the different classes of drugs and regimens used clinically have provided an improvement in tumour management. However, treatment is often palliative for the majority of cancer patients. Transformed cells respond poorly to chemotherapy mainly due to the development of the multidrug resistance (MDR) phenotype. Response to treatment does not generally result in complete remission and disease cure is uncommon for patients presenting with advanced stage cancer. Successful treatment of cancer requires a clearer understanding of chemotherapeutic resistance. Here, we examine what is known of one of the most extensively studied mechanisms of cellular drug resistance. The human multidrug resistance gene 1 (MDR1) is associated with expression of p-glycoprotein (Pgp). A transmembrane protein, Pgp acts as an efflux pump and reduces intracellular drug levels and thus its effectiveness as an antitumor agent. The precise mechanism of transcriptional regulation has been unclear due to the complex regulatory nature of the gene. It has become increasingly apparent that trans-activation or genetic amplification is by no means the only mechanism of activation. Consequently, alternative pathways have received more attention in the area of epigenetics to help explain transcriptional competence at a higher level of organization. The goal of this article is to highlight important findings in the field of methylation and explain how they impinge on MDR1 gene regulation. In this review, we cover the current information and postulate that epigenetic modification of MDR1 chromatin influences gene transcription in leukaemia. Finally, we explore transcriptional regulation and highlight recent progress with engineered ZFP's (zinc finger proteins).

Section snippets

Transcriptional regulation of MDR1

Much of our current understanding of transcriptional regulation comes from reporter-based studies and in vitro reconstitution systems under various physiological conditions. The assortment of transcriptional cofactors assembling on the MDR1 promoter and sequence elements implicated in regulating gene expression undoubtedly reflect the complexity of regulation. Gene activation studies using expression vectors can drive transcription and it has been demonstrated that protein factors can

MDR1 initiator sequence

The human MDR1 promoter was isolated and sequenced using a series of overlapping cDNA clones representing the full length MDR1 transcript [18]. Transfer of the cDNA sequence into Pgp negative cells confered drug resistance [19]. The MDR1 gene initiates transcription at 136 and 140 bases upstream from the +1 ATG initiation methionine codon [18]. There is an additional downstream promoter region with a CAAT consensus box (−113 to −118) from the initiation start site and two GC box regions (−51 to

Y-box and YB-1

The Y-box consensus sequence positioned at −82 to −73 is shown to be important for basal expression [33]. In fact, YB-1 is associated with intrinsic MDR1 gene transcription in primary breast cancers [34]. YB-1 cDNA transfection mediates MDR1 gene over-expression in drug sensitive breast epithelial cells. In contrast to normal human breast tissue, YB-1 mRNA is over-expressed in breast cancers. Furthermore, in 27 untreated breast cancer patients, YB-1 is over-expressed in the cytoplasm of all

NF-R binding proteins and CAAT- and GC-motifs

The CAAT- and GC-box like motifs are essential cis-acting elements for MDR1 promoter activity [35]. The identification of three DNA binding proteins, NF-R1, NF-R2 and NF-R3 show binding to these sequences. NF-R1, a 110 kDa DNA binding protein recognizes both -CAAT and -GC like sequences and its presence was verified by Southwestern blotting in both drug sensitive and resistant cell lines. In contrast, NF-R2 and NF-R3 binding proteins bind to the −CAAT and −GC sequences, respectively.

Since the

Effects of Sp-1 and other binding proteins

Binding sites for Sp-1 have previously been described in the murine adenosine deaminase gene [39] and in the TATA-less promoter of the serotonin 1a receptor gene [40]. In addition, TATA-less/GC-rich promoters bind Sp-1 related cofactors (TATA-binding protein associated factors, TAFs) and facilitate binding to TFIID, aiding in the assembly of the transcription complex [41], [42].

The MDR1 promoter contains multiple GC-like boxes which are believed to be involved in transcriptional activity [18],

Unifying the separate elements with chromatin and transcriptional silencing

In order to understand transcriptional competence, we should first consider some of the key concepts in relation to epigenetic events and the importance of chromatin determinants. In one of the first studies to indirectly examine the role of methylation and nucleosome assembly on transcription, Graessmann et al., injected methylated and unmethylated variants of the Herpes simplex virus thymidine kinase (tk) gene into rodent cells in order to monitor tk activity [48], [49]. The results of these

Transfected MDR1 is active in drug sensitive cells

In one of the first reports examining gene regulation, Ueda and colleagues demonstrated transcriptional regulation of MDR1 in gene reporter experiments using drug sensitive and resistant cell lines [20]. It was anticipated that drug sensitive cells would have no promoter activity while resistant cells would have high levels of activity, reflecting the differences in the endogenous expression of activators in these cells. Surprisingly, promoter activity was inconsistent with endogenous MDR1

The rise of DNA methylation

Despite the efforts to understand the regulatory mechanisms controlling MDR1 gene expression, its epigenetic status is an important though largely unexplored parameter. We propose that the mechanism of transcriptional regulation is governed by an inverse correspondence. Hypermethylation of the MDR1 promoter is associated with dominant transcriptional repression and this is reinforced by chromatin immunoprecipitation (ChIP) experiments. In order to monitor the molecular determinants associated

Clinical relevance of MDR1 gene methylation

As a starting point, immortalised cell lines derived from cancer cells are ideal for DNA methylation analysis. Cultured cell lines provide homogenous cell populations that are convenient to use and provide a permanent window into the cell that can be dynamically and temporally examined. Cell treatments with methylation inhibitors have demonstrated a bona fide role for methylation in transcriptional silencing [115]. The role of methylation is well established in gene repression in cultured cell

Chromatin remodelling and “designer” transcription factors

The assembly of specialized chromatin structures on methylated DNA may explain why the MDR1 gene is transcriptionally silent. Understanding the mechanism of gene regulation is critical if future attempts to control MDR1 are to be successful. The evidence so far points to a model of transcriptional regulation closely associated with DNA methylation and chromatin modification. These studies are now challenging the way we view MDR1 silencing beyond our simple understanding of “textbook”

Acknowledgements

Assam El-Osta is a Senior Research Fellow with the Fragile X (FRAXA) Research Foundation. A. El-Osta dedicates this work to the memory of his good friend and mentor Alan P. Wolffe, who built on the foundation of gene regulation by the raising of questions and teaching of epigenetic mechanisms and their significance on chromatin and transcription.

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