Aberrant promoter hypermethylation of tumor-associated genes leading to their inactivation is a common event in many cancer types. Using a sensitive restriction-multiplex PCR method, we studied the promoter hypermethylation profile of the p16, p15, hMLH1, MGMT and E-cad genes in oral squamous cell carcinoma (OSCC) of Indians. We analyzed a total of 51 samples for the p15 tumor-suppressor gene and 99 samples for each of the remaining genes. Our studies indicate an incidence of promoter hypermethylation of 23% each for p16 and p15, 8% for hMLH1, 41% for MGMT and 35% for E-cad. We observed aberrant hypermethylation of the promoter region of at least 1 of these genes in 74.5% of cases (n ‫؍‬ 51) for which all the 5 genes were studied. Abnormal methylation was detected in tumors irrespective of stage and location in the oral cavity, whereas no abnormal methylation was detectable in normal oral squamous tissues obtained from 25 OSCC patients. Oral squamous cell carcinoma (OSCC) is one of the most widely prevalent cancer types in developing countries, including India, and is associated with tobacco and alcohol abuse. 1 Although recent research has given deeper insight into the etiology of the disease, its occurrence as a potentially fatal disease continues unabated. It is therefore essential to identify and develop newer risk markers for diagnosis and therapy of OSCC.Methylation profiling of promoter regions is essential for understanding the regulation of imprinted genes, X-chromosome inactivation and the role of various tumor-suppressor genes in the genesis of different types of cancer. 2,3 Methylation profiling of tumor-suppressor genes is a potentially powerful diagnostic tool for early detection of various types of cancer. 3-5 Assessment of methylation inactivation is important in assigning functional roles of genes and may have implications in the function of other interacting proteins. 3,6 Inactivation of several tumor-associated genes, especially tumor-suppressor genes, has been attributed to aberrant hypermethylation of their promoter regions. 2,3,6 We included in our study 5 tumor-suppressor genes: p16, a CDK inhibitor involved in regulation of the cell cycle by the cyclin D-Rb pathway, control of which is lost in virtually all tumor types; 7 p15, another CDK inhibitor involved in cell-cycle regulation; 8 hMLH1, the human homolog of bacterial MutL, involved in mismatch repair; 9 MGMT, the gene involved in repair of methylated guanosine residues formed due to alkylated carcinogens; and E-cadherin, involved in homotypic epithelial cell-cell adhesion. 10 All of these genes are known to be inactivated by methylation in various cancers. 3,11 Methylation profiling of CpG sites originally involved digestion of sample DNA using a methylation-sensitive restriction enzyme followed by Southern hybridization 7,12 or PCR using primers flanking the restriction site. [13][14][15] Using these assays, methylation patterns could not be determined from very small amounts of DNA and the occurrence of false-negatives due to degra...

The ADH1C*2/*2/MTHFR 677TT genotype combination appears to be more susceptible for OSCC, since it showed a 20-fold increase in risk in heavy drinkers and a 5.9- and 2.8-fold increase in risk respectively in moderate drinkers and light drinkers. This study suggests the association of ADH1C*2/*2/MTHFR 677TT genotype combination as a risk factor for OSCC in alcoholics.

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The DNA binding proteins in a nuclear membrane fraction that can synthesize DNA in vitro (referred to as "nuclear membrane complex") and in the cytoplasm of adenovirus infected and uninfected cells were isolated and characterized. Suspension cultures of human KB cells infected with human adenovirus 2 were treated with 25 mu-g/ml of arabinosylcytosine starting at 2 hr to block the synthesis of viral structural proteins, and then labeled with (3H)leucine from 6 to 24 hr after infection. Uninfected cells were treated similarly and labeled with (14C)leucine. The 3H-labeled proteins (infected cells) and 14C-labeled proteins (uninfected cells) isolated from the cytoplasm were mixed, as were the corresponding proteins isolated from the membrane complex, and each mixture was fractionated by stepwise elution from single-stranded DNA-cellulose columns. From 50 to 60% of the labeled protein in the membrane complex from infected cells and 40 to 50% of that from uninfected cells bound to DNA-cellulose in 0.05 M NaCl. Much less protein from the cytoplasm was bound to DNA cellulose, 20% from infected cells and 11% from uninfected cells. Gel electrophoresis of the mixture of 3H- and 14C-labeled proteins eluted from DNA-cellulose by different concentrations of NaCl revealed the following. (1) The 0.15 and 0.40 M NaCl eluates from the membrane complex of infected and uninfected cells contained a heterogenous mixture of similar polypeptides. (2) The 0.6 M NaCl eluate from the membrane complex derived from infected cells contained two major DNA binding proteins with molecular weights of 75,000 and 45,000 that were absent from uninfected cells. Large quantities of these two proteins were present in highly purified form in the 0.6 M NaCl eluate from the cytoplasm of infected cells. The DNA binding proteins of molecular weight 75,000 and 45,000 that are present in the cytoplasm are identical with those present in the membrane complex, as established by coelectrophoresis. (3) Two major cell-specific proteins of molecular weight 40,000 and 15,000-17,000 were present in the 2 M NaCl eluate of the membrane complex from uninfected and infected cells. A major cell-specific protein of molecular weight 33,000 was present in the 0.15 and 0.4 M NaCl eluates of the uninfected and infected cell cytoplasmic fractions. Aanalysis of cells labeled at 2-6 hr after infection in the absence of arabinosyl cytosine indicated that the synthesis of the DNA binding proteins of molecular weight 75,000 and 45,000 begins early after infection prior to the onset of viral DNA replication.

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