One of the most striking system used for the analysis of the role of DNA methylation in carcinogenesis was

an APCMIN mouse model analysed by Laird et al. [71]. APCMIN mouse carries a germline mutation in APC gene and develop hundreds of intestinal polyps. The inhibition of methyltransferase with 5-aza-2'-deoxycytidine in these mouse resulted in the reduction of polyp number from 113 to only 2. Although the mechanism by which demethylation reduces the polyp formation remains unclear, these results provide an important evidence for the involvement of methylation in carcinogenesis About 60% of human genes are associated with unique CpG islands [2], and recently it has been estimated that the human genome contains about 29,000 CpG islands. These normally unmethylated CpG islands may become methylated in cancer cells, and the event is associated with loss of expression of flanking genes. Thus, abnormal de novo methylation of CpG islands in human cancer cells represents one of the most prevalent molecular markers yet identified. Important question to ask is CpG islands which has C in methylated state except when the gene is transcribed. But in cancer cells CpG islans are kept in unmethylated state but why cancer cells fails to methylate ? Loss of cell cycle control resulting in unrestrained cell proliferation is a hallmark trait of cancer cells, and increased cell proliferation might be required for epigenetic changes in cancer cells [28]. Failure of CpG islands to become remethylated in non-dividing cells [28] suggests that de novo methylation of CpG islands occurs only in cells that are dividing. DNA methyl-CpG binding domain proteins (MBDs) and DNMT1 may recruit HDACs to methylated promoters, which, in turn, deacetylate histones, to maintain chromatin in the repressed state. It means MBD and DNMT recruits HDAC for the purpose of methylation of promoter but since DNMT1 has been recruited it will deactylate the chromatin and keep it in repressed state. So conclusion is MBD+DNMT1 causes promoter methylation and gene inactivation and for this HDAC is involved which ultimately leads to chromatin repression. Together with the observations that MBD-containing co-repressor complexes associate with methylated CpG islands, it has been suggested that histone modifications are secondary to DNA methylation. However, mutations in a putative methyltransferase specific for histone H3 lysine 9 result in loss of cytosine methylation in fungus [64], providing the first evidence that histone methylation can initiate DNA methylation. How histone H3 methylation recruits a DNMT has not been resolved. in plants, it has recently been shown that CpG island methylation depends on histone H3 methyltransferase [65], suggesting that histone methylation can actually direct DNA methylation. Finally, a specific histone code has been proposed to target methylation to DNA. Modifications of histones, such as by deacetylation, methylation or phosphorylation, may be somehow required to initiate or maintain DNA methylation, perhaps by recruiting proteins, such as MBDs, transcriptional activators or repressors, and these interactions then determine which genes are expressed or silenced. Above line means that modification of histones by methylation, deactylation or phosphorylation is required for maintaining or initiating DNA methylation and the mechanism for this has been explained by recruitment of proteins such as MBDs. transcriptional activators or repressors and interaction of these proteins determine genes to be activated or silenced.

Based on this observation, it has been suggested that histone modifications are the primary event in initiating gene silencing whereas CpG island hypermethylation, a subsequent event, establishes a permanent state of gene inactivation. On the basis of above explained paragraph, it becomes clear that histone modifications are the primary for initiating gene silencing whereas CpG island hypermethylation is next step which insures a stable state of gene inactivation. Future research will reveal how chromatin remodeling contributes to epigenetic regulation of gene expression and perhaps control of the CpG island methylation machinery

CpG hypermethylation and subsequent inactivation of important genes, such as tumor suppressor genes, can provide a selective growth advantage to cancer cells. In colorectal cancer hypermethylation of a number of genes has been shown to start in normal mucosa and early- and pre-neoplasia, and CpG island methylation increases as a function of age. Nevertheless, there is substantial evidence that CpG island hypermethylation occurs early in the neoplastic process. For example, hypermethylation of the p16 INK4a gene is frequently seen during metaplastic progression in Barretts esophagus [72,73], lung [74], cervical [74] and gastric carcinoma. Hypermethylation of 14-3-3 sigma is an early event in breast cancer [77]. The association of promoter hypermethylation of VHL [78], MLH1 [79,80], and BRCA1 [81,82] with familial cancers are additional examples of epigenetics being causative, but the possibility that hypermethylation of these genes might be late events in the non-familial cancers cannot be overlooked. Evidences suggests that hypermethylationof CpG islands and genes occur early during the carcinogenesis process but there is no evidence to prove that hyprmethylation cannot occur during the late stages of carcinogenesis. De novo methylation usually takes place at the outskirts of a promoter CpG island and progressively spreads into the core of the island [83]. It is not clear why protection of CpG islands and the neighboring sequences from abnormal DNA methylation is lost in cancer cells. From the above fact that de novo methylation progresses from the outskirtsof CpG islands to core indicates a kind of protective mechanism guarding the CpG island but how this protective mechanism is lost in cancerous cells is unknown. Studies suggest that some sequences in the promoter region may serve as docking sites to attract repression complexes, including HDACs, MBDs, DNMTs and others, from which the exonic sequences become hypermethylated and their histones become hypoacetylated and methylated. Promoter region has some docking site for repression complex consisting of MDB, DNMT, HDAC and others which cause hypermethylation of exonic sequences and causes hypoacetylation and methylation of histone proteins (more condensed chromatin means suppressed gene expression). Recognition sequences within and/or around CpG dinucleotides may also play a role in the association of proteins to methylated cytosines. Cytosine methylation by proteins is guided by recognition sequences around CpG dinucleotides. The interference of these proteins in the binding of transcription factors that prefer unmethylated sequences was recently reported [88], suggesting that transcription factor abundance may compete with promoter methylation machinery and this may also be relevant to the initiation of CpG island hypermethylation in cancer.

The proteins which are responsible for cytosine methylation, interfere with transcription factors so there exists competition between transcription factors and proteins of methylation machinery. Non-promoter CpG islands also appear to be more susceptible to aberrant methylation than their nearby respective promoter sequences, and methylation can begin in exonic regions and then spread to CpG islands in other locations, including promoter regions. The density or number of methylated CpG dinucleotides can exert long distance transcriptional repression. Because of their heritable nature, hypermethylated CpG islands leave molecular footprints from which the event of epigenetic progression can be reconstructed during tumorigenesis and therefore, are useful markers for molecular classification of different tumor types. A high-throughput screening approach for profiling methylation alterations of CpG islands in ovarian tumors and identifying candidate markers for diagnosis and prognosis of the disease. This novel microarray approach, called differential methylation hybridization (DMH), allows us to do a global analysis of DNA methylation in ovarian and other carcinomas. By using DMH, we have shown that aberrant DNA methylation is a frequent epigenetic event in ovarian cancer, and we have also revealed tumor groups with distinctly different methylation profiles. Furthermore, patients defined by these groups differed markedly in the time until disease recurrence following chemotherapy; in addition, a higher degree of CpG island methylation was associated with early disease recurrence after chemotherapy. A select group of CpG island loci was also revealed in the study which may be useful as epigenetic markers. for predicting chemotherapy outcome in patients with ovarian cancer. Further, comparing epigenetic changes in early- versus late-stage ovarian cancer may provide a rationale on which to base the need for aggressive therapy. In an initial study, treatment of ovarian cancer cells with DNMT inhibitor 5-aza-20-deoxycytidine (DAC, a cytidine analogue that sequesters DNMT after its incorporation into genomic DNA) resulted in reactivation of some hypermethylated genes, but no change in expression levels of methylated genes was observed in cells treated with the HDAC inhibitor trichostatin A (TSA). However, treatment with DAC plus TSA resulted in the synergistic upregulation of many methylated silenced genes, and several methylation-unrelated genes were also upregulated by the drug combination. We are currently using this approach for screening gene promoter function and activity of promoters in ovarian cancer. The potential to reverse DNA methylation and reexpress tumor suppressor genes and key control pathways in cancer cells presents attractive and exciting clinical possibilities. Experimental results have shown that reexpression of epigenetically silenced hMLH1 by treatment with the DAC sensitized cisplatin-resistant ovarian cancer cells to chemotherapeutic agents both in vitro [98] and in vivo. Reversal of the methylated estrogen receptor (ERa) gene in human ER-negative breast cancer cell lines resulted in re-expression of functional ER and breast cancer cells were then able to respond to the antiestrogen therapies. Methylation profiling of tumors could provide a more focused test for reactivation of methylation-silenced genes as therapeutic targets and form a rational basis for future treatment strategies designed to alter this fundamental process in cancer.