A Medline search from January, 1966 to July, 2005 was done with the keywords “methylation” and “oral cancer”. Studies identified were screened for those that focused on tissues with primary oral cancer and that looked directly at DNA methylation by use of methylation-specific PCR, PCR-based restriction enzyme assay, and sequencing after sodium bisulphite treatment. We included only a few studies of oral squamous-cell carcinoma cell lines, since the focus of the review is primary tissue.
ReviewPromoter methylation and inactivation of tumour-suppressor genes in oral squamous-cell carcinoma
Introduction
Squamous-cell carcinoma of the oral cavity has long been known to be the endpoint of many genetic changes (figure). A visible change in the histology of the oral mucosa is accompanied by mutations or loss of DNA. Various genetic events have been investigated in the context of mutations or disruptions in the DNA sequence, which lead to loss of function of tumour-suppressor genes or activate oncogenes. These cumulative changes can lead to cellular atypia and eventually cancer.1
Although many important genes and gene products have been identified through DNA changes, no single unifying pathway has been identified that accounts for all oral squamous-cell carcinomas. Rather, accumulation of many varied genetic modifications results in changes of crucial pathways that maintain cellular homoeostasis. Promoter methylation is an alternative form of gene silencing, which relies on epigenetic factors rather than direct DNA mutations.
Many regions rich in cytosine–guanine, also known as CpG islands, have been noted within the human genome. These islands are located upstream from the promoter region at the 5′ end of a gene, and up to half of all human genes seem to have CpG islands.2 Three known DNA methyltransferases are known to have the ability to add methyl groups to the 5′ cytosine of a C–G dinucleotide. Addition of these methyl groups to the CpG islands silences genes by facilitating the association of methylated DNA with a methyl-binding complex. This methylation ultimately leads to histone deacetylation, which then prevents transcription of the subsequent gene.2 Thus, gene products are not produced, even though the DNA coding sequence is maintained.
For many decades, methylation-induced gene silencing has been recognised as being needed for developmental regulation.3 Methylation is involved in carcinogenesis, and many tumour-suppressor genes have been investigated in various tumours.4, 5, 6 These studies suggest that methylation-induced gene silencing is as important as gene mutation or loss of heterozygosity in the progression to cancer.
Carcinogenesis might not be the result of just one mechanism of gene suppression. Rather than focus only on methylation of tumour-suppressor genes, many studies7, 8, 9, 10 have investigated the role of the combination of loss of heterozygosity and of methylation to establish the importance of each with the gene of interest. The incidence of promoter methylation is enhanced in those tumour suppressor genes that do not have loss of heterozygosity as a prominent mechanism. Because genes can be silenced through at least two mechanisms, promoter methylation might explain why studies have shown widely different results of methylation status, since loss of heterozygosity could vary within their study samples.
In this review, we discuss genes that have been implicated in oral squamous-cell carcinoma and the clinical implications and future possibilities.
Section snippets
Molecular techniques
The molecular techniques used to detect methylation have evolved from the Southern blot to more sensitive quantitative-PCR techniques. In the past, investigators used methylation-specific restriction enzymes followed by Southern-blot analysis to probe for predicted fragments, which are produced by methylation-specific cutting of DNA fragments, to show the presence or absence of methylation.11 This technique needed a large quantity of DNA and was not very sensitive. Similarly, PCR-based
Methylated genes in oral cancer
Many genes in oral-cancer tissue have been tested for methylation. These tumour-suppressor genes all have a mechanistic basis for their role in carcinogenesis and are generally implicated in other tumour types. However, matched or paired healthy tissue should be tested in conjunction with affected tissue, since seemingly healthy tissue can have low rates of methylation, especially in patients who are smokers.22 Tissue adjacent to tumours that is histologically normal23, 24, 25 and premalignant
Clinical importance
Despite few data for gene-promoter methylation in the oral cavity, measurement of such patterns has shown promise in cancer detection schemes. Because of the sensitivity and specificity of PCR-based assays (especially quantitative methylation-specific PCR) and the potential for use in high-throughput assays, determination of methylation status in oral cancers has great potential for early detection, monitoring, and treatment.
The creation of methylation gene panels could be useful for cancer
Conclusion
Promoter methylation is a powerful and ubiquitous mechanism of gene silencing. Initially discovered to be a mechanism for developmental control, it plays an important part in the development of many tumour types. Various genes have been implicated in squamous-cell carcinoma of the oral cavity with differing results. However, methylation is important in the development of oral squamous-cell carcinoma, and other tumour-suppressor genes targeted by promoter methylation will no doubt be described
Search strategy and selection criteria
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