DNA Methylation and Cancer

Alterations in DNA methylation are regarded as epigenetic and not genetic changes, because although epigenetic changes affect the structure of DNA, they do not materially affect the genetic code. In recent years, numerous studies have demonstrated that a close correlation exists between methylation and transcriptional inactivation, supporting the notion that not only genetic changes, but also epigenetic changes can contribute to the carcinogenic process (Strathdee et al., 2002; Yan et al., 2001). The pattern of methylation observed in cancer generally shows a dramatic shift compared with that of normal tissue. The methylation pattern in tumors consists of a global hypomethylation, in conjunction with localized hypermethylation at CpG islands (Goelz et al., 1985). This regional hypermethylation at CpG islands is associated with the transcriptional inactivation of cancer related genes (Momparler and Bovenzi, 2000).

Recent studies have demonstrated that hypermethylation of CpG islands may be implicated in tumorigenesis, acting as a mechanism to inactivate specific gene expression of a diverse array of genes (Baylin et al., 2001). Genes that have been reported to be regulated by CpG hypermethylation, include tumor suppressor genes, cell cycle related genes, DNA mismatch repair genes, hormone receptors and tissue or cell adhesion molecules (Yan et al., 2001). For example, tumor-specific deficiency of expression of the DNA repair genes MLH1 and MGMT (Herman, 1998; Simpkins, 1999) and the tumor suppressors, p16, CDKN2 and MTS1, has been directly correlated to hypermethylation (Jones, 1999; Merlo et al., 1995). Increased CpG island methylation can result in the inactivation of these genes resulting in increased levels of genetic damage, predisposing cells to later genetic instability which then contributes to tumor progression (Strathdee and Brown, 2002).

Hypermethylation is now the most well characterized epigenetic change to occur in tumors, and it is found in virtually every type of human neoplasm. Promoter hypermethylation is as common as the disruption of classic tumor-suppressor genes in human cancer by mutation and possibly more so (Baylin and Herman, 2000). Approximately 50% of the genes that cause familial forms of cancer when mutated in the germ line are also known to undergo methylation-associated silencing in various sporadic forms of cancer (Jones and Baylin, 2002).

In cancer, the dynamics of genetic and epigenetic gene silencing are very different. Somatic genetic mutation leads to a block in the production of functional protein from the mutant allele. If a selective advantage is conferred to the cell, the cells expand clonally to give rise to a tumor in which all cells lack the capacity to produce protein. In contrast, epigenetically mediated gene silencing occurs gradually. It begins with a subtle decrease in transcription, fostering a decrease in protection of the CpG island from the spread of flanking heterochromatin and methylation into the island. This loss results in gradual increases of individual CpG sites, which vary between copies of the same gene in different cells (Jones and Baylin, 2002).

Techniques to Study Epigenetics

TECHNIQUES TO STUDY EPIGENETICS

Links to Protocols, Methods and Techniques in order to study Epigenetic changes:

Epigenetic Cell Growth and Nucleic Acid Protocols:

Cell Lines and Cell Culture

Seeding, Passaging and Freezing Cells

Cell Stimulation Protocol

Preparation of Cytosolic Extracts

Total Cellular RNA Extraction

 

Polymerase Chain Reactions in Epigenetics:

Reverse Transcriptase Polymerase Chain Reaction RT-PCR

Methylation Specific PCR

Methods and Protocols to Analyze DNA Methylation and Epigenetic Changes:

Treatment of Cells with 5-Aza-2′-Deoxycytidine

Sodium Bisulfite Treatment of DNA

PCR Methods to study Epigenetics

Methylation Specific PCR

Methylation Sensitive Restriction Endonuclease Enzymes to study DNA Methylation and Epigenetic Changes

Methylation Sensitive Restriction Enzymes

Southern Blotting Method for DNA Methylation Detection

Combined Bisulfite Restriction Analysis COBRA

Bisulfite PCR Single Strand Conformation Polymorphism SSCP

PCR Fluorescence Melting Curve Analysis

Methylation Sensitive Single Nucleotide Primer Extension Ms-SNuPE

Identification of CpG Islands Exhibiting Altered Methylation Patterns (ICEAMP)

Hairpin-Bisulfite Polymerase Chain Reaction PCR

 

Histone Modification Protocols:

Chromatin Immunoprecipitation

 

Genome-Wide Approaches to studying Epigenetics:

Epigenomic Approaches

Treatment of Cells with 5-Aza-2′ – Deoxycytidine Protocol and Method

Treatment of Cells with 5-Aza-2′-Deoxycytidine

5-Aza-2′-Deoxycytidine: Demethylating Agents and Reactivation of Silenced Genes

Genes inappropriately silenced by structural chromatin changes that involve DNA methylation can be reactivated by demethylating agents, that can reverse these changes and, therefore, restore principal cellular pathways. This results in gene re-expression and reversion of some aspects of the transformed state. The demethylating agent 5-azacytidine and its deoxy derivative 5-aza-2’deoxycytidine were first synthesized in Czechoslavakia as potential chemotherapeutic agents for cancer (Cihak, 1974). These agents are incorporated into the nucleic acids of dividing cells, where they act as mechanism-based inhibitors of DNA methytransferases. They inactivate DNA cytosine C5- methyltransferases through the formation of stable complexes between the 5-aza-2′-deoxycytidine residues in DNA and the enzyme, thereby mimicking a stable transition state intermediate when bound to the methyltransferase enzyme (Sheikhnejad et al., 1999).

These powerful inhibitors of DNA methylation, can restore gene function to treated cells in culture, which has indicated that they may have potential in treating patients with malignant disease (Lubbert, 2000; Jones and Baylin, 2002).

METHODS and MATERIALS: A Protocol for the Treatment of Cells with 5-Aza-2′-Deoxycytidine

Cells are seeded at a density of 5×10 5 /100-mm dishes, cultured for 48 hours, and treated with 0, 50, or 100 m M 5-aza-dC (Sigma Chemical Co., St. Louis, MD) (Li et al., 2001).

Forty-eight hours after treatment, cells are washed with PBS and fresh medium was added. Cells are further incubated for another 48 h before isolated total cellular RNA.

For protein studies, cells are seeded at a density of 5×10 5 /100-mm dishes, cultured for 24 h, and treated with 0, 1, 2, 3, 5, and 10 m M 5-aza-dC. After 5 days, cell supernatants are harvested and centrifuged at 1,800 rpm to pellet and remove any cell debris. Supernatants are subsequently transferred to a new tube and analyzed for protein concentrations or stored at -20 0 C .

Analysis of DNA Methylation by Sequencing of Sodium Bisulfite-treated DNA

Sequencing of Sodium Bisulfite-treated DNA | DNA Methylation Analysis

 

SODIUM BISULFITE INDUCED OXIDATIVE DEAMINATION OF GENOMIC DNA

In single-stranded DNA, sodium bisulfite preferentially deaminates cytosine residues to uracil, compared with a very slow rate of deamination of 5-methylcytosine to thymine (Shapiro et al., 1973). Frommer et al. (1992) utilized this difference in bisulfite reactivity for genomic sequencing of 5-methylcytosine residues, by fully denaturing total genomic DNA and treating total genomic DNA with sodium bisulfite under conditions such that cytosine is converted stoichiometrically to uracil, but 5-methylcytosine remains nonreactive. The DNA is initially denatured by alkali treatment prior to treatment with bisulfite. The second part of the procedure involves PCR amplification of the region of interest in the bisulfite-reacted DNA to yield a fragment in which all uracil, formerly cytosine, and thymine residues have been amplified as thymine and only 5-methylcytosines have been amplified as cytosine. The bisulfite reaction yields DNA strand products, which are no longer complementary. PCR primers can therefore be designed, such that a specific pair can only bind to one of the bisulfite-reacted DNA strands. Primers for each strand will differ in every position where there is a C or G in the original sequence (Frommer et al., 1992).

If the PCR products from the bisulfite-treated DNA are cloned and individual clones are sequenced, the sequences will provide methylation maps of single DNA strands from individual DNA molecules in the original genomic DNA sample. The procedure yields a sequence and methylation pattern specific for each strand of the original genomic DNA. The position of each 5-methylcytosine will be given by a positive band on a sequencing gel (Frommer et al., 1992).

 

METHODS and MATERIALS: A Protocol for the Analysis of DNA Methylation by Sequencing of Sodium Bisulfite-treated DNA

Genomic DNA is isolated using the Qiagen DNA KitTM and subjected to sodium bisulfite treatment to modify unmethylated cytosine to uracil, using the CpGenome TM DNA Modification Kit (Intergen Company, Oxford , UK ) and following the manufacturer’s instructions. The conditions for PCR were as follows: 1 cycle at 95 0 C for 15 min; 40 cycles of 94 0 C for 1 min, 60 0 C for 1 min and 72 0 C for 1 min; and 1 cycle of 72 0 C for 10 min. QIAGEN has recently developed an excellent kit for methylation analsys that should be sought if you are able to.

PCR products are then separated on a 1 % agarose gel, stained with ethidium bromide, and visualized under ultraviolet (UV) light. The PCR bands are subsequently cut from the gel with a sharp razor, pooled together and purified using the QIAGEN gel extraction kit (QIAGEN Inc., Valencia, CA) according to the vendor’s instructions. The purified DNA product is subsequently ligated to a TA cloning vector, TOPO pCR2.1 Sequencing Vector (Invitrogen, Grand Island , NY ). 3 m l of the dilution is then added to DH5 a -T1 competent cells and incubated in ice for 30 minutes. Cells are then heat-shocked for 45 seconds in a 42 o C heat block, and then placed on ice for a further 2 minutes. 250 m l SOC medium is added to each vial of cells. The vials are subsequently shaken at 225 RPM, at 37 o C for one hour, after which the content of each vial is spread on agar plates containing 100 m g/ml of ampicillin. Plates are then incubated overnight at 37 o C for the production of positive colonies.

Five-ten ampicillin resistant colonies grown on agar plates are selected and cultured overnight in Luria-Bertani (LB) medium composed of 1.0% NaCl, 1.0% tryptone and 0.5% yeast extract (DIFCO, MD, USA), pH 7.0 supplemented with 100 m g/ml of ampicillin (Sigma-Aldrich, St. Louis, MO). The ampicillin provides a selective pressure for the exclusive growth of positive colonies. Plasmid DNA is isolated using the QIAGEN Miniprep Kit (QIAGEN Inc., Valencia , CA ), following the manufacturer’s instructions. Screening for positive plasmids is done by a restriction digest. 1 m l of purified plasmid, with 1 m l of EcoRI (Fermentas Inc., MD, USA ), 1 m l of Buffer EcoRI and 17 m l of dH20, are incubated for 2 hours at 37 o C. The reaction mixture is subsequently seperated on a 2.5% agarose gel, stained with ethidium bromide and visualized under UV light. Positive clones are then sequenced using M13 primers (Invitrogen), and analyzed for percentage methylation of specific CpG dinucleotides. Data analsis consists of plotting CpG island dinucleotide positions and percentage of cytosines methylated.

Methylation-Specific PCR

Methylation-Specific PCR Protocol and Method

MSP can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of cloning or methylation-sensitive restriction enzymes. The assay consists of initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA.

MSP requires very small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed in DNA extracted paraffin-embedded samples (Herman et al., 1996).

METHODS and MATERIALS: A Protocol for Methylation-Specific PCR, MSP

One micro g of sodium bisulfite-treated genomic DNA is used for PCR amplification using MSP primers (Li et al., 2001) (MSP-Methylated and MSP-Unmethylated). The methylation-specific primers included in which nucleotides corresponding to potentially methylated cytosines where retained.

The primer combination to amplify unmethylated DNA included in which the nucleotides corresponding to cytosine nucleotides were changed to thymine (sense primer) or adenine (antisense primer) (Herman et al., 1996). The PCR conditions are as follows: 1 cycle of 95oC for 15 minutes; 40 cycles of 95 o C for 30 seconds, 50 o C for 30 seconds, and 72 o C for 45 seconds; and 1 cycle of 72 o C for 5 minutes.

The methylation-specific and nonmethylated DNA-specific primers yield different PCR products, respectively, when constructed. Depending on the primers created, differences in PCR temperatures and cycle optimizations are necessary.