Intestinal Dysplasia Induced by Simian Virus 40 T Antigen Is Independent of p53

Abstract

Transgenic mice expressing simian virus 40 large T antigen in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. Hyperplasia is dependent on T antigen binding to the retinoblastoma (pRb) family of tumor suppressor proteins. Mice expressing a truncated T antigen that inactivates the pRb-family, but is defective for binding p53, exhibit hyperplasia but do not progress to dysplasia. We hypothesized that the inhibition of the pRb family leads to entry of enterocytes into the cell cycle, resulting in hyperplasia, while inactivation of p53 is required for progression to dysplasia. Therefore, we examined T antigen/p53 complexes from the intestines of transgenic mice. We found that T antigen did not induce p53 stabilization, and we could not detect T antigen/p53 complexes in villus enterocytes. In contrast, T antigen expression led to a large increase in the levels of the cyclin-dependent kinase inhibitor p21. Furthermore, mice in which pRb was inactivated by a truncated T antigen in a p53 null background exhibited intestinal hyperplasia but no progression to dysplasia. These data indicate that loss of p53 function does not play a role in T antigen-induced dysplasia in the intestine. Rather, some unknown function of T antigen is essential for progression beyond hyperplasia.Large T antigen (TAg), an oncogene encoded by the small DNA tumor virus simian virus 40 (SV40), is a powerful tool for elucidating the mechanisms of growth regulation and control of cell proliferation. Expression of this viral protein is sufficient to transform multiple primary cell types (reviewed in references 19 and 47), and when expressed ectopically in transgenic mice, it causes neoplasia in numerous tissues (reviewed in reference 47).Transformation induced by T antigen is accomplished by targeting cellular components. For example, the amino-terminal region of T antigen (Fig. 1) inactivates the retinoblastoma (pRb) family of tumor suppressors via an LXCXE motif (reviewed in reference 37) that mediates binding to pRb proteins and a J domain (56) that interacts with hsc70 and participates in pRb inactivation. T antigen alleviates the growth-suppressive functions of the Rb family by a J-domain-dependent mechanism (52,(54)(55)(56)69) and in the case of murine polyomavirus by both J-domain-dependent and -independent mechanisms (50). The carboxy-terminal region of T antigen binds, stabilizes, and inactivates the tumor suppressor p53 (12, 31, 42) (Fig. 1). Some reports argue that the elimination of pRb and p53 tumor suppressor functions is the only T antigen activity that contributes to transformation (23). However, other reports suggest that T antigen targets additional cellular proteins and that these interactions contribute to transformation as well (1,16,31,46,62).Studies with transgenic mice have revealed that T antigen is able to induce neoplasia in numerous cell types (reviewed in reference 47). A major complication to the interpretation of the neoplastic phenotype is that the changes induced depend on the cell type...

Simian Virus 40 T-Antigen-Mediated Gene Regulation in Enterocytes Is Controlled Primarily by the Rb-E2F Pathway

“…Goat anti-mouse antibody linked to peroxidase (Sigma A2554) was Immunohistochemistry. Small intestines were processed for immunohistochemistry as described elsewhere (20). Assessment of the proliferative status was obtained after mice were given an intraperitoneal injection of 5-bromo-2Ј-deoxyuridine (BrdU) (Sigma) (120 mg/kg of body weight) 2 hours prior to their sacrifice.…”

“…Material enriched in intestinal villi and crypts was prepared and analyzed by Western blotting as described previously (20). An appropriate dilution of TAg-mouse monoclonal PAb416 (11) was used as a primary antibody.…”

“…Furthermore, the ability of TAg wt to induce ectopic enterocyte proliferation depends on the presence of E2F2 (28). Surprisingly, p53 is not stabilized by TAg in enterocytes, nor do TAg-p53 interactions play any role in enterocyte transformation (20).…”

“…Intestinal villi and crypts are localized in different regions of the tissue and therefore can be readily isolated from the underlying submucosa and muscularis. This allows us to prepare proteins or nucleic acids from cell populations greatly enriched for nonproliferating, terminally differentiated cells located in the villi or from their proliferating, multipotent progenitors located in the crypts (20,33).…”

Two Independent Regions of Simian Virus 40 T Antigen Increase CBP/p300 Levels, Alter Patterns of Cellular Histone Acetylation, and Immortalize Primary Cells

“…Mouse embryo fibroblasts (MEFs) were harvested from 13.5-day-old FVB embryos as previously described (36) and grown at 37°C in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin. MEF lines expressing different SVT mutants were previously generated (37,38 , N136 dl89-97 , N136 F98A , N136 E9K , N136 K53R , N136 D2K , and N136 E107,108K ) were obtained by infection with retroviral (pBabePuro) or lentiviral (pL6.3; Invitrogen) constructs encoding the different mutants under the control of the CMV promoter.…”