14-3-3 EXPRESSION CHANGES IN HUMAN ADENOCARCINOMAS OF THE COLON

Gavin Young , Vijayababu MR, Jesse D. Martinez

14-3-3 EXPRESSION CHANGES IN HUMAN ADENOCARCINOMAS OF THE COLON

The 14-3-3 family is a group of intracellular proteins found in nearly every eukaryotic organism. In humans there are 7 isoforms denoted by the Greek letters beta, gamma, zeta, eta, epsilon, sigma and theta. These proteins form dimers in the cell and serve as scaffolds to promote interactions of various other regulatory proteins. There are thousands of known interactions. Previous studies have shown disturbances in native 14-3-3 levels among many cancers. Our study aims to establish the normal and cancerous expression levels of all 7 isoforms in human colon adenocarcinomas and correlate 14-3-3 expression changes with epigenetic silencing. Finding a correlation between regulation of 14-3-3 and the establishment of colon tumors might suggest future drug targets or diagnostic procedures.

14-3-3 protein expression levels were established by RT-PCR analysis. Purified whole-RNA extracts were run against each of the seven human isoforms and normalized to the housekeeping gene GAPDH. The sample consisted of 71 colon adenocarcinoma and 52 normal frozen tissue samples taken from patients of varying ages (18-93) and mostly Caucasian background (85%) through surgical resection. Here we present statistically significant (p < 0.05) data showing decreased levels of 14-3-3 sigma, eta, and zeta were observed among colon adenocarcinomas compared to normal tissue.  These data suggest a link between 14-3-3 protein expression levels and the development of colon cancers.

To further explore the mechanisms behind 14-3-3 expression changes, we examined the methylation status of sigma, eta, and zeta isoforms in twelve selected samples. Genomic DNA was extracted from frozen tissue and subjected to bisulfite treatment. Small segments (~600 bp) surrounding each gene’s promoter region were amplified by PCR and sequenced. Our data identifies novel CpG methylation sites in zeta and eta promoters and suggests an epigenetic mechanism for 14-3-3 silencing during carcinogenesis.

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