Methylation of the hMLH1, p16, and MDR1 genes in colorectal carcinoma: associations with clinicopathological features
Introduction
Methylation of promoter-associated CpG islands has been implicated as an important alternative to mutation in the inactivation of tumor suppressor genes during carcinogenesis. Silencing of expression by methylation has been reported for a wide range of cancer-related genes including those involved in cell cycle control, DNA repair and chemoresistance [1]. Recent evidence indicates that some sporadic cases of colorectal cancers (CRCs) exhibit concordant methylation of several genes including hMLH1 and p16. It is proposed that these tumors may account for the majority of sporadic microsatellite instability (MSI+) cases through methylation of the DNA repair gene hMLH1 and that these progress along a pathway distinct from that of tumors exhibiting p53 mutation [2], [3]. Two types of target loci have been proposed by Toyota and colleagues [2]: the methylation of type A genes occurs in an age-dependent manner and is also found in the colonic mucosa, whereas the methylation of type C genes is confined to tumor tissue. Further work is required however to clearly establish the existence of these two distinct types of target genes for methylation.
The aims of the present study were firstly to further characterize the clinicopathological features of CRC tumors showing evidence of CpG island methylation and secondly to identify other genes that may be methylated in these tumors. We investigated the methylation status of seven cancer-related genes in a series of 58 primary CRCs previously characterized for MSI status [4]. The hMLH1 and p16 genes were chosen because they are known to be frequently methylated in MSI+ tumors [2]. The multidrug resistance (MDR1) and thymidylate synthase (TS) genes were chosen because of their proposed roles in the resistance of tumor cells to chemotherapeutic drugs [5], [6]. Both the ataxia-telangiectasia mutated (ATM) and poly (ADP-ribose) polymerase (PARP) genes were chosen because of their involvement in the detection of DNA damage [7], [8]. The p21/waf1/cip1 gene was investigated because of its crucial role in cell cycle regulation [9]. The methylation status of MDR1, ATM and p21 has not previously been reported for CRC, while that of the TS and PARP genes has not been reported for any cancer type.
Section snippets
Tumor DNA
Tissue samples were obtained from patients undergoing surgery for CRC at Sir Charles Gairdner Hospital, Western Australia, and stored at −80°C prior to DNA extraction by standard methods. Of 58 tumor samples analyzed for methylation, 18 were selected because of their previous characterization as MSI+ [4]. All MSI+ tumors were located in the proximal colon. The remaining 40 MSI− tumors originated in the proximal (20 cases) or distal (20 cases) colon. These three subgroups were evenly matched for
Detection of methylation in the hMLH1, p16 and MDR1 genes using Ms-SNuPE
Ms-SNuPE primers were designed to analyze the methylation status of either two or three specific CpG sites within the promoter-associated CpG island of each gene. Of the seven genes investigated we detected methylation only in the hMLH1, p16 and MDR1 genes (Fig. 1A–C, respectively). In all tumors showing methylation of these genes, a signal was always detected at each of the CpG sites examined. Methylation of hMLH1, p16 or MDR1 was not detected in any of the normal colon mucosa samples. None of
Discussion
We have used both Ms-SNuPE and SSCP techniques to investigate the methylation status of seven cancer-related genes in a series of 58 primary human CRCs. As reported by others, we found methylation of CpG islands within the promoter regions of the hMLH1 and p16 genes [2], [17]. Although methylation of the MDR1 gene has been described in hematopoietic malignancies [18], [19], this is the first report of its methylation in CRC. We found no evidence for methylation of the TS, ATM, PARP or p21 genes
Acknowledgements
The authors are grateful to Professor House for the provision of frozen colorectal tumor specimens, and G Gnanasampanthan for assistance with SSCP and Ms-SNuPE gels. This work was supported by The Raine Medical Research Foundation.
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