Growth factor induction by the adenovirus type 5 E1A 12S protein is required for immortalization of primary epithelial cells.

Abstract

The 12S protein encoded by the adenovirus EIA region induces cellular DNA synthesis in and proliferation and immortalization of primary rat epithelial cells in the presence or absence of serum. It also induces the production of a growth factor(s) that stimulates epithelial cell proliferation. We have undertaken a mutational analysis of the 12S gene to determine the sequences required for these functions. We found that a region near the C-terminus of the 12S protein was required for growth factor induction. No activities have been defined previously for this region. Furthermore, we show that growth factor production was necessary for epithelial cells to survive past their normal life span in culture and to become immortalized. The ability to induce growth factor production required prior expression of EIA activities encoded by the N-terminus of the 12S protein, including activation of quiescent cells into the cell cycle, and an unknown activity that required expression of the first 13 amino acids of the gene. In addition, examination of the subcellular localization of mutant 12S polypeptides suggested new regions that affect the nuclear localization of ElA proteins.

A mechanism for Rb/p130-mediated transcription repression involving recruitment of the CtBP corepressor

“…In contrast, other experiments have provided evidence for a role for these sequences in the immortalizing function of E1A as well as to collaborate with adenovirus E1B in transformation (31). Although the basis for the apparent discrepancy in these results is unclear, the latter findings, indicating a requirement for the C-terminal domain in E1A function in immortalization and transformation with E1B, are certainly consistent with the findings that this domain interacts with CtBP and thus would disrupt the formation of the E2F-p130-CtIP-CtBP repressor complex.…”

Rac regulates the stability of the adherens junction and its components, thus affecting epithelial cell differentiation and transformation

“…The cells (WTras+WTrac) derived from primary epithelial cells that express only WT12S and the endogenous, WTras and rac are immortalized and not transformed (they cannot grow in soft agar or form tumors in animals). They resemble the original primary cells in appearance and expression of epithelial characteristics (Quinlan et al, 1988;Quinlan and Douglas, 1992;Gopalakrishnan and Quinlan, 1995;Fischer and Quinlan, 1998a) and see below.…”

“…Primary BRK cells were prepared from 5 day-old rats (Fisher F344, Harlan, IN, U.S.A.) as described previously (Quinlan et al, 1988). The cell lines have been described previously (Fischer et al, 1998).…”

Physical and Functional Interactions between the Corepressor CtBP and the Epstein-Barr Virus Nuclear Antigen EBNA3C

“…The Drosophila melanogaster homologue dCtBP mediates transcriptional repression by at least six different transcription factors, including Knirps, Kruppel, Snail, Zfh-1, Polycomb, and Hairy (16,17,20,22,28). CtBP also interacts with Xenopus and human Polycomb proteins (28), and human hCtBP acts as a corepressor for the ZEB transcription factor that is involved in the regulation of lymphocyte and muscle differentiation (23). The mouse homologue mCtBP is a corepressor for the NET member of the Ets family of transcription factors and oncogenes (3).…”

“…Although the precise significance of this interaction remains unknown, it is essential for the immortalization of primary rodent cells by E1A and has also been reported to negatively modulate E1A-mediated transformation, tumorigenicity, and metastasis (2,23,24,27,29). More recently it has been shown that this E1A-binding protein is one of a highly conserved family of (co)repressors of transcription.…”