Use of transgenic mice reveals cell-specific transformation by a simian virus 40 T-antigen amino-terminal mutant.

Abstract

We have used the multifunctional transforming protein, simian virus 40 T antigen, as a probe to study the mechanisms of cell growth regulation in the intact organism. T antigen appears to perturb cell growth, at least in part, by stably interacting with specific cellular proteins that function to maintain normal cell growth properties. Experiments in cultured cells indicate that at least three distinct regions of simian virus 40 T antigen have roles in transformation. Two regions correlate with the binding of known cellular proteins, p53, pRB, and p107. A third activity, located near the amino terminus, has been defined genetically but not biochemically. By targeting expression of wild-type and mutant forms of T antigen to distinct cell types in transgenic mice, we have begun to systematically determine which activities play a role in tumorigenesis of each cell type. In this study, we sought to determine the role of the amino-terminal transformation function with such an analysis of the T-antigen mutant d11135. This protein, which lacks amino acids 17 to 27, retains the p53-, pRB-, and p107-binding activities yet fails to transform cells in culture. To direct expression in transgenic mice, we used the lymphotropic papovavirus transcriptional signals that are specific for B and T lymphocytes and the choroid plexus epithelium of the brain. We show here that although defective in cell culture, d11135 specifically induced the development of thymic lymphomas in the mouse. Expression of the protein was routinely observed in B-and T-lymphoid cells, although B-cell abnormalities were not observed. Choroid plexus tumors were observed only infrequently; however, d11135 was not consistently expressed in this tissue. Within a given transgenic line, the penetrance of T-cell tumorigenesis was 100%X but appeared to require secondary events, as judged from the clonal nature of the tumors. These experiments suggest that the amino-terminal region of T antigen has a role in the transformation of certain cell types (such as fibroblasts in culture and B lymphocytes) but is dispensable for the transformation of T lymphocytes.Simian virus 40 (SV40) large T antigen efficiently disrupts normal cell growth control in a variety of cell types in culture and in test animals (reviewed in references 17 and 29). The diversity of cells susceptible to the effects of T antigen has been demonstrated most dramatically in transgenic mice, in which the growth properties of over 20 cell types have been altered by this protein (reviewed in references 1 and 22). Current hypotheses suggest that T antigen induces neoplastic transformation by direct interaction with cellular proteins that normally function to regulate cell proliferation (18,20,27). Genetic analyses of T antigen in cultured cells indicate that the protein possesses at least three transforming functions (10,35,42,52). One of these functions corresponds to the ability of T antigen to bind both pRB (9)

Different functions are required for initiation and maintenance of immortalization of rat embryo fibroblasts by SV40 large T antigen

“…Dl1135 was previously found to be inactive in all transformation assays Michalovitz et al, 1987;Yaciuk et al, 1991;Symonds et al, 1993;Srinivasan et al, 1997). Our results showing that it was unable to immortalize REFs directly but could maintain the growth of tsa14 cells, suggests that some of the functions initially required to immortalize REFs may not be required once the immortal state has been established.…”

“…The ®rst of these was indicated by mutant 5080 which contains a substitution of amino acid 584 that lies within one of two hydrophobic stretches (amino acids 571 ± 589 and 619 ± 627) required for stability of the tertiary structure (Tevethia et al, 1988;Peden et al, 1989;Tack et al, 1989;Zhu et al, 1991Zhu et al, , 1992. The second region is colinear with the p53 binding domain.…”

Transforming functions of Simian Virus 40

“…The role of the J domain has been less studied in transgenic models, and the reports so far indicate an important -although tissue-speci®c-dependency on the J domain to induce tumors (Chen et al, 1992;Ratineau et al, 2000;Symonds et al, 1993). In each of these cases, the amino-terminal fragment of LT is su cient to drive Fox et al, 1989;Zennaro et al, 1998;Le Menuet et al, 2000Bone Behringer et al, 1988Knowles et al, 1990;Jensen et al, 1993;Wilkie et al, 1994 Brain andnervous system Brinster et al, 1984;Reynolds et al, 1988;Chen et al, 1989;Theuring et al, 1990;Chen and Van Dyke, 1991;Perraud et al, 1992;Smith et al, 1992;Jensen et al, 1993;Chiu et al, 2000Cartilage Cheah et al, 1995Digestive system Li et al, 1995Kim et al, 1994;Hauft et al, 1992;Asa et al, 1996Eye Theuring et al, 1990Penna et al, 1998;Beermann et al, 1999Heart Behringer et al, 1988Kidney Theuring et al, 1990Liver Dyer and Messing, 1989Sandgren et al, 1989;Sepulveda et al, 1989;Butel et al, 1990;Manickan et al, 2001Lung Sandmoller et al, 1994Wikenheiser et al, 1992Lymphoid tissue Reynolds et al, 1988Chen et al, 1989Mammar...…”

Tissue-specific inactivation of p53 tumor suppression in the mouse.

“…The protein extract containing 5 mg of total protein from spleen, 2 mg from brain, 0.1 mg from liver, and 0.5 mg from thymus was precleared with Staphylococcus aureus-protein A followed by incubation with the appropriate monoclonal antibody (300 ~1 of hybridoma supernatant) for 1 hr at 4~ The antibody-protein complexes were immunoprecipitated with S. aureus, the immunoprecipitates washed three times with SNNTE [5% sucrose, 0.5 mM NaC1, 0.1% NP-40, 50 mM Tris (pH 7.4), and 5 mM EDTA] followed by one wash with NTE [50 mM NaC1, 10 mM Tris (pH 7.4), and 1 mM EDTA], resuspended in Laemmli protein loading buffer, and electrophoresed in 15% polyacrylamide-SDS gels. Proteins were transferred to nitrocellulose (BioBlot-NC, Costar), and immunoblotting was performed as described previously using L2SI-labeled protein A detection (Symonds et al 1993). To detect p53/p53DD complexes, liver lysate containing 5 mg of total protein was used for each immunoprecipitation.…”

“…some cases (see below), TgTTRp53DD-4 mice were examined as well. Tumor tissue (control p53) from a transgenic mouse expressing a mutant T antigen (d11135) that stabilizes p53 was used to detect wild-type p53 (Symonds et al 1993). Both wild-type and p53DD were immunoprecipitated with mouse monoclonal antibody PAb421, which recognizes an epitope in the carboxyl terminus (see A), followed by Western blot analysis using PAb421 as the primary antibody.…”