Induction of interferon-stimulated genes by Simian virus 40 T antigens.

Simian virus 40 (SV40) large T antigen (TAg) is a multifunctional oncoprotein essential for productive viral infection and for cellular transformation. We have used microarray analysis to examine the global changes in cellular gene expression induced by wild-type T antigen (TAg(wt)) and TAg-mutants in mouse embryo fibroblasts (MEFs). The expression profile of approximately 800 cellular genes was altered by TAg(wt) and a truncated TAg (TAg(N136)), including many genes that influence cell cycle, DNA-replication, transcription, chromatin structure and DNA repair. Unexpectedly, we found a significant number of immune response genes upregulated by TAg(wt) including many interferon-stimulated genes (ISGs) such as ISG56, OAS, Rsad2, Ifi27 and Mx1. Additionally, we also observed activation of STAT1 by TAg(wt). Our genetic studies using several TAg-mutants reveal an unexplored function of TAg and indicate that the LXCXE motif and p53 binding are required for the upregulation of ISGs.

Simian virus 40 T-antigen-mediated gene regulation in enterocytes is controlled primarily by the Rb-E2F pathway.

Simian virus 40 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 (TAg(wt)) 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(wt) 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. To further dissect the role of the Rb family in the induction of intestinal hyperplasia, we have screened several lines of transgenic mice expressing a truncated TAg (TAg(N136)), which is able to interfere with the Rb pathway but lacks the functions associated with the carboxy terminus of the protein. This analysis confirmed the pivotal association between the Rb pathway and the induction of intestinal hyperplasia and revealed that upregulation of p53 target genes is not associated with the tumorigenic phenotype. Furthermore, we found that TAg(N136) was sufficient to induce intestinal hyperplasia, although the appearance of dysplasia was significantly delayed.

A structure-guided mutational analysis of simian virus 40 large T antigen: identification of surface residues required for viral replication and transformation.

Simian virus 40 large T antigen (TAg) transforms cells in culture and induces tumors in rodents. Genetic studies suggest that TAg interaction with the chaperone hsp70 and tumor suppressors pRb and p53 may not be sufficient to elicit complete transformation of cells. In order to identify additional cellular factors important for transformation, we designed mutations on the solvent-exposed surface of TAg. We hypothesized that surface residues would interact directly with cellular targets and that the mutation of these residues might disrupt this interaction without perturbing TAg's global structure. Using structural data, we identified 61 amino acids on the surface of TAg. Each surface amino acid was changed to an alanine. Furthermore, five patches containing clusters of charged amino acids on the surface of TAg were identified. Within these patches, we selectively mutated three to four charged amino acids and thus generated five mutants (patch mutants 1 to 5). We observed that while patch mutants 3 and 4 induced foci in REF52 cells, patch mutants 1 and 2 were deficient in focus formation. We determined that the patch 1 mutant is defective in p53 binding, thus explaining its defect in transformation. The patch 2 mutant can interact with the Rb family members and p53 like wild-type TAg but is unable to transform cells, suggesting that it is defective for action on an unknown cellular target essential for transformation. Our results suggest that the histone acetyltransferase CBP/p300 is one of the potential targets affected by the mutations in patch 2.

Cell-type specific regulation of gene expression by simian virus 40 T antigens.

SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examined global gene expression in SV40-transformed mouse embryo fibroblasts, and in enterocytes obtained from transgenic mice. SV40 transformation alters the expression of approximately 800 cellular genes in both systems. Much of this regulation is observed in both MEFs and enterocytes and is consistent with T antigen action on the Rb-E2F pathway. However, the regulation of many genes is cell-type specific, suggesting that unique signaling pathways are activated in different cell types upon transformation, and that the consequences of SV40 transformation depends on the type of cell targeted.

Enterocyte proliferation and intestinal hyperplasia induced by simian virus 40 T antigen require a functional J domain.

Transgenic mice expressing the simian virus 40 large T antigen (TAg) in enterocytes develop intestinal hyperplasia that progresses to dysplasia with age. This induction requires TAg action on the retinoblastoma (Rb) family of tumor suppressors and is independent of the p53 pathway. In cell culture systems, the inactivation of Rb proteins requires both a J domain in TAg that interacts with hsc70 and an LXCXE motif that directs association with Rb proteins. Together these elements are sufficient to release E2Fs from their association with Rb family members. We have generated transgenic mice that express a J domain mutant (D44N) in villus enterocytes. In contrast to wild-type TAg, the D44N mutant is unable to induce enterocyte proliferation. Histological and morphological examination revealed that mice expressing the J domain mutant have normal intestines without loss of growth control. Unlike mice expressing wild-type TAg, mice expressing D44N do not reduce the protein levels of p130 and are also unable to dissociate p130-E2F DNA binding complexes. Furthermore, mice expressing D44N in a null p130 background are still unable to develop hyperplasia. These studies demonstrate that the ectopic proliferation of enterocytes by TAg requires a functional J domain and suggest that the J domain is necessary to inactivate all three pRb family members.

The mismatch repair system is required for S-phase checkpoint activation.

Defective S-phase checkpoint activation results in an inability to downregulate DNA replication following genotoxic insult such as exposure to ionizing radiation. This 'radioresistant DNA synthesis' (RDS) is a phenotypic hallmark of ataxia-telangiectasia, a cancer-prone disorder caused by mutations in ATM. The mismatch repair system principally corrects nucleotide mismatches that arise during replication. Here we show that the mismatch repair system is required for activation of the S-phase checkpoint in response to ionizing radiation. Cells deficient in mismatch repair proteins showed RDS, and restoration of mismatch repair function restored normal S-phase checkpoint function. Catalytic activation of ATM and ATM-mediated phosphorylation of the protein NBS1 (also called nibrin) occurred independently of mismatch repair. However, ATM-dependent phosphorylation and activation of the checkpoint kinase CHK2 and subsequent degradation of its downstream target, CDC25A, was abrogated in cells lacking mismatch repair. In vitro and in vivo approaches both show that MSH2 binds to CHK2 and that MLH1 associates with ATM. These findings indicate that the mismatch repair complex formed at the sites of DNA damage facilitates the phosphorylation of CHK2 by ATM, and that defects in this mechanism form the molecular basis for the RDS observed in cells deficient in mismatch repair.

Caspase-3 mediated cleavage of BRCA1 during UV-induced apoptosis.

The breast cancer suppressor protein, BRCA1 plays an important role in mediating cell cycle arrest, apoptosis and DNA responses to DNA damage signals. In this study, we show that BRCA1 level is downregulated during UV-induced apoptosis by caspase-3 mediated cleavage. Cleavage of BRCA1 by caspase-3 produced a fragment that contained the C-terminal of the molecule. Accordingly, treatment of cells with caspase-3 inhibitor or mutation of a specific caspase-3 cleavage site (DLLD) at amino acid 1151-1154 of BRCA1 abolished cleavage and consequential accumulation of the BRCA1 C-terminal fragment. Whereas expression of the non-cleavable BRCA1 (D/A 1154) mutant conferred the resistance phenotype to UV-induced cell death, expression of the cleaved BRCA1 C-terminus induced cell death in the absence of UV. Examination of the mechanism of C-terminus-induced cell death revealed that the cleaved fragment triggers the apoptotic response through activation of BRCA1 downstream effectors, GADD45 and JNK. Altogether, results of our study demonstrate a functional role for caspase-3 mediated cleavage of BRCA1 during UV-induced apoptosis.