Global analysis of gene expression by SV40 T antigen in the mouse small intestine epithelium

SV40 large T antigen (TAg) contributes to cell transformation, in part, by targeting two well characterized tumor suppressors, pRb and p53. TAg expression affects the transcriptional circuits controlled by Rb and by p53. We have performed a microarray analysis to examine the global change in gene expression induced by wild-type TAg and TAg-mutants, in an effort to link changes in gene expression to specific transforming functions. For this analysis we have used enterocytes from the mouse small intestine expressing TAg. Expression of TAg in the mouse intestine results in hyperplasia and dysplasia. Our analysis indicates that practically all gene expression regulated by TAg in enterocytes is dependent upon its binding and inactivation of the Rb-family proteins. Laser capture microdissection (LCM) was used to isolate villus enterocytes from three independent non-transgenic mice, four wild-type TAg transgenic mice, five N136 transgenic mice, three D44N transgenic mice and three 3213 transgenic mice. Total RNA was extracted from the enterocytes with the Pico Pure kit and amplified twice with the Ribo Amp kit (Arcturus). Amplified RNA was processed by the Genomics and Proteomics Core Laboratories at the University of Pittsburgh and hybridized to the Affymetrix GeneChip Mouse Genome 430 2.0 array. MAS 5.0 was used to obtain the present/absent calls and CEL files were normalized by RMA to obtain log2 expression values.