Journal: PLoS ONE

Article Title: Overexpression of EGFR in Head and Neck Squamous Cell Carcinoma Is Associated with Inactivation of SH3GL2 and CDC25A Genes

doi: 10.1371/journal.pone.0063440

Figure Lengend Snippet: a) Representative autoradiographs showing deletion and MA in HNSCC samples at D9S157 marker loci. (i) LOH, loss of heterozygosity, (ii) MA-I, microsatellite size alteration of one allele, (iii) LMA microsatellite size alteration of of one allele and LOH in other allele. b) Genetic alterations of CDC25A analyzed by microsatellite based deletion mapping showing deletion and MA in HNSCC at D3S3560 marker loci (i) LOH loss of heterozygosity, (ii) MA-I microsatellite size alteration of one allele. (iii) MA2 microsatellite size alteration of both alleles. Arrow heads indicate the lost allele and star indicates the allele with MA. Representative gel electrophorogram showing the methylation status of c) SH3GL2 and d) CDC25A in tumor samples and in corresponding normal sample by MSRA. SH3GL2 showed methylation in tumor sample and absence of methylation in adjacent normal samples. Promoter methylation of CDC25A was absent in both tumor and normal samples. h, amplicon obtained with primer for HpaII digested DNA; m, amplicon obtained with primer for MspI digested DNA; u, amplicon obtained with primer for undigested DNA. e) The methylation analysis by MSRA was validated by MSP after bisulphate modification of DNA. The sample #2123T showed the methylalaton specific PCR band, but #2123N and #2935TN showed unmethylation specific PCR band. U; amplicon obtained with primer for modified unmethylated DNA, M amplicon obtained with primer for modified methylated DNA, T tumor DNA, N corresponding normal tissue DNA. f) Frequency of overall alterations of the genes SH3GL2 and CDC25A observed in dysplasia and different stages of HNSCC samples. Significant increase in alteration with tumor progression is shown by asterisk. Q-RT PCR showing reduced expression of g) SH3GL2 and h) CDC25A in HNSCC tumor. Bars represented the gene expression normalized to β2-microglobulin and relative to adjacent normal tissues using the 2? -ddCt method. The line illustrates the mean decreased level of the genes. X-axis indicates samples.

Article Snippet: The promoter methylation of the candidate genes SH3GL2 and CDC25A was analyzed by PCR-based methylation sensitive restriction analysis (MSRA) method using MspI/HpaII restriction enzymes (Sibenzyme, Russia) using standard procedure as describe previously .

Techniques: Marker, Methylation, Amplification, Modification, Reverse Transcription Polymerase Chain Reaction, Expressing