DNA Methylation From The Cancer Perspective DNA methylation is an epigenetic modification, which can affect gene silencing, genetic imprinting, X-chromosome inactivation, and chromosome stability (1, 2, 3). While epigenetic factors are not inherited through normal Mendelian genetics, these alterations are reversible, research supports these components consistent across generations (4). The link between DNA methylation and cancer is controversial with disputable variables, such as location, mechanism, progression, detection, and genomic individualities per each patient (1-8). The dispute between hypomethylation and hypermethylation continues among the scientific community (7,8). While some believe one mechanism is responsible, current research suggests a combination of hypermethylation and hypomethylation induces cancer growth (7,8). The independence and integration of the two methylation mechanisms for cancer formation have been stressed equally by research (7,8). The methylation of certain genes also varies with the cancer type, p16 and RASSF1A are two known methylated genes in many forms, while GSTP1 gene is only found to be significantly hypermethylated in prostate and breast cancer (5,7). The mechanism of a certain gene serves as a variable; GSTP1 is found hypomethylated in acute myeloid leukemia patients (5). With increasing number of cancer forms, the layers of complex keep building, in the relationship between the DNA methylation and tumors formation and progression.
Mechanism Two sets of DNA methylase enzymes play a role in DNA methylation (2,3,5,6). The de novo DNMTs are responsible for initial methylation in a DNA sequence and maintenance DNMTs which copy DNA sequence methylations during DNA replication process (3,4). The de novo DNMTs prevent transcription factors from binding to the CpG dinulceotide, including AP-2, c-Myc/Myn, the cyclic AMP-dependent activator CREB, E2F, and NFkB (3). CP1 and MeCP2 MBD1, MBD2, MBD4, and Kaiso are the protein complex are affected by methylation (3).While MBD1, MBD2, MeCP2, and Kaiso are proteins which repress transcription by preventing histone deacetylase complexes on nucleosomes in cell cultures and in vivo (3).
Hypermethylation Hypermethylation is the abundance of methyl groups on a strand of DNA, which does not allow for gene transcription (1). This form of regulatory gene expression prevents the transcription of chromosomes that contain tumor suppressor genes (2,4-6). When a tumor suppressor gene is silenced in the human genome the corresponding protein is not formed (2). The result is the proliferation of damaged cells within the human body, which under normal circumstances would be inactive by the tumor suppressor proteins (2). The persistent formation of these damages cells without the protein necessary leads to cancer tumors. Many chromosomal region of DNA have be noted for hypermethylation, such as 3P, 11P, and 17p, across several cancer forms (2). Since specific chromosomes were not methylation in vivo samples and not observed through in vitro studies, the methylation only seen in cancerous samples implies the hypermethylation is not a required genetic modification, but does result in cancer (2,7,8). Research demonstrates hypermethylation of tumor suppressor genes can lead to gene inactivation and a selective growth model advantageous to the progression of cancerous cells (2,4-6). This was observed in retinoblastoma by transient transfection experimentation.
Hypomethylation The promotion of gene expression is observed with hypomethylation (2,4-6). The hypomethylation mechanism has been related to the transformation of normal cell to cancerous cells, but also cancer progression to advanced stages (5). The acceleration was observed through an increase in the methylation levels of malignant tumors were half the benign tumor concentrations of the 5-methyl cytosine complex (2). Thus, the malignant tumors showed a greater degree of hypomethylation. Hypomethylation is the lack of methyl groups in on DNA, which promotes gene transcription (1). When hypomethylation occurs on a chromosome which codes for a proto-ocogene cancer formation can occur (2,4-6). The underrepresentation of the normal amount of methyl groups allows gene transcription, which affects in a protein expression (2). Hypomethylation causes the production of uncontrolled damaged cell growth (5). This mechanism of methylation favors the oncogenisis process found in cancer patients, it has been shown the manipulation of hypomethylation can result in anticancer affects as a short term solution (5). However, this practice could also results in the advanced progression of already damaged cells undergoing chemotherapy treatment (5). The study of proto-oncogenes has been extensive in liver tumors for the -fos, c-myc, and Ha-ras, Ki-ras genes, which are hypomethylation in cancer patients (2). Leaukemia has research that shows both the hypermethylation and hypomethylation in various cases (2). This example demonstrates the significant variability between even the same cancer type, but different patients.
Location
DNA methylation occurs sporadically and unevenly throughout the genome (1-8). Special attention has been placed on the DNA methylation patterns in CpG islands and promoter regions of a gene (2,7,8). A CpG dinucleotide is the nucleotide sequence of a cytosine followed by a guanine base in DNA strand (2,5,6). These specific bases are advantageous and promote the mechanism of methylation by their chemical structure. The promoter region for the cell serves the purpose of the binding site for most transcription factors and various reactions can control gene transcription from the promoter region (2).
There appears to no specific methylation target location with a distinctive role for gene expression in eukaryotic organisms. DNA methylation can occur at the fifth carbon position of the cyostine nucleotide in a DNA sequence(2,4-6). While the frequency for the cytosine followed by a guanine show occur in equal frequency of 6%, the dinulceotide is only observed 6-10% of that portion, making the combination rare within the chromosome sequence, 1% of all nucleotides (5). The CpG nucleotide location further supports hypermethylation interference with eukaryotic gene expression because the of specificity of base pairing. When a cytosine base which is methylated is followed by a guanine which is hydrogen bonded to a complementary cytosine base pair. This formations creates two methyl groups sitting diagonally from one another in the middle of two DNA strands (5).
The patterns for DNA methylation can be compared in units of DMR (3). The DMR can be measured by various microarrays and gel electrophoresis or absorbance titrations (2,3,5). From these techniques a genomic maps for the specific type of cancer can begin to be developed through the similarities observed across of a large number of cancer patients (7,8).
Link to Diet Proper DNA replication depends on the presence of reactants necessary for methylation. Methyl groups are derived from the human diet through folic acid, B12 vitamin, methionine, betaine and choline (2). Hypomethylation has been observed in humans, which have minimal intake of the listed sources of methyl sources. Thus, the patients diagnosed with cancer are increasing the rate of progression with the lack of methyl groups(2). Humans with overall hypomethylation in the genome could have a greater susceptibility to cancer, since the conditions for the advancement are already presence (2). Diet can affect the progression of cancer and accelerate the advancement through hypomethylation.
Methodology Since the modifications made by DNA are being further developed the patterns which are tumor-specific, can begin to be used for human therapeutics, detection, diagnosis, and eventually prevention (2,5). Detection techniques for DMT are sodium bisulfite conversion, cDNA microarray, restriction genomic protein mapping, and CpG island microarrays (2,5,7,8).The sodium bisulfite assay transforms the cytosine to uracil bases in the unmethylated form, while ignoring methylated cytosine (2). The assay yields a clear map, which highlights hyper and hypomethylation DMTs (2). Another application to understand the role of DNA methylation is methylation-specific polymerase chain reaction (2,7,8). This allows the cell culture to reproduce the aberrant DNA methylation patterns and observed the whether the patterns are inherited down to the daughter generations (2).
Therapeutic Drugs Since epigenetic factor can be reversed to original state human therapeutics can also lead to advances in cancer treatment. Studies have demonstrated cell cultures from demethylating drugs can revert the silencing of tumor suppressor gene from hypermethylation (2). DNMT inhibitor drugs are cytosine analogues, such as Vidaza™, Dacogen™, DHAC, kymarabine, and zebularine (2). All of these inhibitor drugs prevent hypermethylation mechanism (2). The five are variations which contain a certain modification at the fifth position carbon in the pyrimidine ring, the location of normal methylation (2). Histone deacetylase (HDAC) inhibitors could also repress DNA methylation by histone deacetlyarion (2). This histone alteration triggers the activation of expression from hypermethylated regions, such as tumor suppressor genes, found in cancer patients (2,5). Further research is necessary to determine the interaction which occur when HDAC are taken in conjunction with DMNT. These drugs are still in the clinical trial phase.