Apobec-T is widely expressed in normal and cancerous gastrointestinal tissues and its inducible overexpression alters growth in NIH3T3 cells

Abstract

Background: Apobec-1 is an RNA-specific cytidine deaminase which mediates a single C to U change in the nuclear apolipoproteinB mRNA in mammalian small intestine. Human apobec-1 gene expression is regulated through differential altemative splicing, whereby an alternate splice acceptor site results in tissue and developmental stage specific exclusion of exon 2. This splice variant results in a frame shift in the processed mRNA which generates a novel 36 amino acid polypeptide, apobec-T, mRNA encoding apobec-T predominates during intestinal ontogenesis and was found to be overexpressed in human colorectal tumors when compared to matched normal tissue. Our aims in this study were to further examine the distribution of apobec-T expression in normal and cancerous gastrointestinal tissues and to evaluate the in vivo effects of apobec-T overexpression on cell proliferation by means of inducible expression. Results: Immunocytochemical staining of paraffin sections of normal stomach, small intestine and colon using an antipeptide antibody confirms the presence of apobec-T throughout the human gastrointestinal tract. Immunocytochemical analysis of 3 gastric adenocarcinomas, 5 small intestinal adenocarcinomas and 6 colonic adenocarcinomas revealed the presence of apobec-T in a diffuse pattern within epithelial cells throughout the tumors. 7 tubular adenomas also demonstrated the presence of apobec-T staining. Interestingly, 5 of 5 hepatic metastases from colon carcinomas demonstrated apobec-T staining. None of 5 primary hepatocellular carcinomas examined demonstrated apobec-T mRNA or protein, suggesting that the presence of apobec-T may be a useful surrogate marker of luminal GI tract malignancy. In order to determine the effects of apobec-T expression on cell proliferation, the splice variant mRNA was reverse transcribed and cloned downstream of a tetracycline-response element and transfected into NIH 3T3 cells expressing the tetR/VP16 fusion. One clone was identified in which expression of apobec-T could be induced 17-fold following withdrawal of tetracycline. Maximal induction of apobec-T in this cell line represents I l-fold higher levels of apobec-T mRNA than is expressed in normal adult jejunum. These cells were plated in 96 well dishes, +/tetracycline, and an XTT assay was performed daily on replicate plates to quantitate cell number. On days 4-6 after induction the cells expressing apobec-T were significantly fewer in number when compared to matched uninduced cells (paired t-test, p<0.01). Conclusions:Apobec-T peptide is detectable in normal and cancerous gastrointestinal tissues. High level overexpression of apobec-T in NIH3T3 cells utilizing a tetracycline-inducible system results in a significant decrease in cell number after 4 days of growth. Apobec-T may function as a modulator of cellular proliferation or apoptosis.