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.     

Retinoblastoma protein (pRb), but not p107 or p130, is required for maintenance of enterocyte quiescence and differentiation in small intestine

The function of retinoblastoma protein (pRb) in the regulation of small intestine epithelial cell homeostasis has been challenged by several groups using various promoter-based Cre transgenic mouse lines. Interestingly, different pRb deletion systems yield dramatically disparate small intestinal phenotypes. These findings confound the function of pRb in this dynamic tissue. In this study, Villin-Cre transgenic mice were crossed with Rb (flox/flox) mice to conditionally delete pRb protein in small intestine enterocytes. We discovered a novel hyperplasia phenotype as well as ectopic cell cycle reentry within villus enterocytes in the small intestine. This phenotype was not seen in other pRb family member (p107 or p130) null mice. Using a newly developed crypt/villus isolation method, we uncovered that expression of pRb was undetectable, whereas proliferating cell nuclear antigen, p107, cyclin E, cyclin D3, Cdk2, and Cdc2 were dramatically increased in pRb-deficient villus cells. Cyclin A, cyclin D1, cyclin D2, and Cdk4/6 expression was not affected by absent pRb expression. pRb-deficient villus cells appeared capable of progressing to mitosis but with higher rates of apoptosis. However, the cycling villus enterocytes were not completely differentiated as gauged by significant reduction of intestinal fatty acid-binding protein expression. In summary, pRb, but not p107 or p130, is required for maintaining the postmitotic villus cell in quiescence, governing the expression of cell cycle regulatory proteins, and completing of absorptive enterocyte differentiation in the small intestine.     

The retinoblastoma protein-binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice.

Regulation of the cell cycle is a critical aspect of cellular proliferation, differentiation, and transformation. In many cell types, the differentiation process is accompanied by a loss of proliferative capability, so that terminally differentiated cells become postmitotic and no longer progress through the cell cycle. In the experiments described here, the ocular lens has been used as a system to examine the role of the retinoblastoma protein (pRb) family in regulation of the cell cycle during differentiation. The ocular lens is an ideal system for such studies, since it is composed of just two cell types: epithelial cells, which are capable of proliferation, and fiber cells, which are postmitotic. In order to inactivate pRb in viable mice, genes encoding either a truncated version of simian virus 40 large T antigen or the E7 protein of human papillomavirus were expressed in a lens-specific fashion in transgenic mice. Lens fiber cells in the transgenic mice were found to incorporate bromodeoxyuridine, implying inappropriate entry into the cell cycle. Surprisingly, the lens fiber cells did not proliferate as tumor cells but instead underwent programmed cell death, resulting in lens ablation and microphthalmia. Analogous lens alterations did not occur in mice expressing a modified version of the truncated T antigen that was mutated in the binding domain for the pRb family. These experimental results indicate that the retinoblastoma protein family plays a crucial role in blocking cell cycle progression and maintaining terminal differentiation in lens fiber cells. Apoptotic cell death ensues when fiber cells are induced to remain in or reenter the cell cycle.     

A simian virus 40 large T-antigen segment containing amino acids 1 to 127 and expressed under the control of the rat elastase-1 promoter produces pancreatic acinar carcinomas in transgenic mice.

The simian virus 40 large T antigen induces tumors in a wide variety of tissues in transgenic mice, the precise tissues depending on the tissue specificity of the upstream region controlling T-antigen expression. Expression of mutant T antigens that contain a subset of the protein's activities restricts the spectrum of tumors induced. Others showed previously that expression of a mutant large T antigen containing the N-terminal 121 amino acids (T1-121) under control of the lymphotropic papovavirus promoter resulted in slow-growing choroid plexus tumors, whereas full-length T antigen under the same promoter induced rapidly growing CPR tumors, T-cell lymphomas, and B-cell lymphomas. In those instances, the alteration in tumor induction or progression correlated with inability of the mutant large T antigen to bind the tumor suppressor p53. In the study reported here, we investigated the capacity of an N-terminal T antigen segment (T1-127) expressed in conjunction with small t antigen under control of the rat elastase-1 (E1) promoter to induce pancreatic tumors. The results show that pancreases of transgenic mice expressing T1-127 and small t antigen display acinar cell dysplasia at birth that progresses to neoplasia. The average age to death in these mice is within the range reported for transgenic mice expressing full-length T antigen under control of the E1 promoter. These results indicate that sequestering p53 by binding is not required for the development of rapidly growing acinar cell carcinomas. In addition, we provide evidence that small t antigen is unlikely to be required. Finally, we show that the p53 protein in acinar cell carcinomas is wild type in conformation.     

Differential interaction of temperature-sensitive simian virus 40 T antigens with tumor suppressors pRb and p53.

Previous studies have shown that simian virus 40 large T antigen transforms cells by binding and inactivating suppressors of cell cycle progression and tumor formation. Here, we characterize the interactions of five temperature-sensitive T antigens with the tumor suppressor proteins pRb and p53. All five mutant T antigens bind pRb at the nonpermissive temperature with efficiencies similar to that of wild-type T antigen. A single transformation-competent mutant, with a substitution of amino acid 186, binds p53 at the nonpermissive temperature. Four transformation-defective mutants, with a substitution at T antigen position 357, 422, 427, or 438, are temperature sensitive for the binding and inactivation of p53. Our findings provide a basis for understanding the behavior of cells transformed by temperature-sensitive T antigens.     

Inhibition of lens fiber cell morphogenesis by expression of a mutant SV40 large T antigen that binds CREB-binding protein/p300 but not pRb

Simian virus (SV) 40 large T antigen can both induce tumors and inhibit cellular differentiation. It is not clear whether these cellular changes are synonymous, sequential, or distinct responses to the protein. T antigen is known to bind to p53, to the retinoblastoma (Rb) family of tumor suppressor proteins, and to other cellular proteins such as p300 family members. To test whether SV40 large T antigen inhibits cellular differentiation in vivo in the absence of cell cycle induction, we generated transgenic mice that express in the lens a mutant version of the early region of SV40. This mutant, which we term E107KDelta, has a deletion that eliminates synthesis of small t antigen and a point mutation (E107K) that results in loss of the ability to bind to Rb family members. At embryonic day 15.5 (E15.5), the transgenic lenses show dramatic defects in lens fiber cell differentiation. The fiber cells become post-mitotic, but do not elongate properly. The cells show a dramatic reduction in expression of their beta- and gamma-crystallins. Because CBP and p300 are co-activators for crystallin gene expression, we assayed for interactions between E107KDelta and CBP/p300. Our studies demonstrate that cellular differentiation can be inhibited by SV40 large T antigen in the absence of pRb inactivation, and that interaction of large T antigen with CBP/p300 may be enhanced by a mutation that eliminates the binding to pRb.     

Atm is dispensable for p53 apoptosis and tumor suppression triggered by cell cycle dysfunction

Both p53 and ATM are checkpoint regulators with roles in genetic stabilization and cancer susceptibility. ATM appears to function in the same DNA damage checkpoint pathway as p53. However, ATM's role in p53-dependent apoptosis and tumor suppression in response to cell cycle dysregulation is unknown. In this study, we tested the role of murine ataxia telangiectasia protein (Atm) in a transgenic mouse brain tumor model in which p53-mediated apoptosis results in tumor suppression. These p53-mediated activities are induced by tissue-specific inactivation of pRb family proteins by a truncated simian virus 40 large T antigen in brain epithelium. We show that p53-dependent apoptosis, transactivation, and tumor suppression are unaffected by Atm deficiency, suggesting that signaling in the DNA damage pathway is distinct from that in the oncogene-induced pathway. In addition, we show that Atm deficiency has no overall effect on tumor growth and progression in this model.     

Cross‐talk between T‐Ag presence and pRb family and p53/p73 signaling in mouse and human medulloblastoma

The formation and progression of mudulloblastoma (MB) is poorly understood. However, somatic inactivation of pRb/p105, in combination with a somatic or a germ‐line TP53 inactivation, leads to MB in a mouse model. Presently, there is no specific evidence of pathway/s alterations for the other two members of the retinoblastoma family, pRb2/p130 and/or p107 in MB. JC virus (JCV) is a human polyomavirus. Although there is no firm evidence that this virus plays a causal role in human neoplasia, it has been clearly proven that JCV is highly oncogenic when injected into the brain of experimental animals. The mechanism of JCV‐induced tumorigenesis is not entirely clear. However, several studies relate the oncogenic properties of JCV mainly to its early protein large T‐antigen (T‐Ag), which is able to bind and inactivate both TP53 and Rb family proteins. Here, we compared the protein expression profiles of p53, p73, pRb family proteins, and PCNA, as main regulators of cell proliferation and death, in different cell lines of mouse primitive neuroectodermal tumors (PNET), either T‐Ag‐positive or ‐negative, and in human MB cell lines. Our goal was to determine if changes in the relative expression of these regulators could trigger molecular perturbations underlying MB pathogenesis in mouse and human cells. Our results support that the presence of JCV T‐Ag may interfere with the expression of pRb family proteins, specific p73 isoforms, and p53. In turn, this “perturbation” may trigger a network of signals strictly connected with survival and apoptosis. J. Cell. Biochem. 110: 182–190, 2010. © 2010 Wiley‐Liss, Inc.     

The molecular chaperone activity of simian virus 40 large T antigen is required to disrupt Rb-E2F family complexes by an ATP-dependent mechanism

The simian virus 40 large T antigen (T antigen) inactivates tumor suppressor proteins and therefore has been used in numerous studies to probe the mechanisms that control cellular growth and to generate immortalized cell lines. Binding of T antigen to the Rb family of growth-regulatory proteins is necessary but not sufficient to cause transformation. The molecular mechanism underlying T-antigen inactivation of Rb function is poorly understood. In this study we show that T antigen associates with pRb and p130-E2F complexes in a stable manner. T antigen dissociates from a p130-E2F-4-DP-1 complex, coincident with the release of p130 from E2F-4-DP-1. The dissociation of this complex requires Hsc70, ATP, and a functional T-antigen J domain. We also report that the "released" E2F-DP-1 complex is competent to bind DNA containing an E2F consensus binding site. We propose that T antigen disrupts Rb-E2F family complexes through the action of its J domain and Hsc70. These findings indicate how Hsc70 supports T-antigen action and help to explain the cis requirement for a J domain and Rb binding motif in T-antigen-induced transformation. Furthermore, this is the first demonstration linking Hsc70 ATP hydrolysis to the release of E2F bound by Rb family members.     

Inactivation of the retinoblastoma susceptibility protein is not sufficient for the transforming function of the conserved region 2-like domain of simian virus 40 large T antigen.

Simian virus 40 large T antigen interacts with three cellular proteins, pRb, p107, and p130, through a common binding site on the T antigen protein called the E1A conserved region 2-like (CR2-like) domain. Mutations in this domain inactivate the transforming activity of large T antigen. Since these mutations have been demonstrated to abolish binding to pRb and p107, and presumably therefore affect binding to p130, assessment of the relative roles of these three proteins in transformation of rodent fibroblasts by T antigen has been difficult. We have examined the role of T antigen-pRb interactions in transformation. We have introduced a mutant T antigen, which is unable to bind any of these three proteins, into primary mouse fibroblasts derived from the embryos of mice in which the Rb gene encoding the retinoblastoma protein had been disrupted. This mutant is unable to transform the Rb-negative fibroblasts, indicating that inactivation of pRb is not the sole function of the CR2-like domain in the induction of transformation of mouse fibroblasts by simian virus 40.     

Induction and bypass of p53 during productive infection by polyomavirus

Lytic infection by polyomavirus leads to elevated levels of p53 and induction of p53 target genes p21Cip1/WAF1 (p21) and BAX. This is seen both in polyomavirus-infected primary mouse cell cultures and in kidney tissue of infected mice. Stabilization of p53 and induction of a p53 response are accompanied by phosphorylation of p53 on serine 18, mimicking a DNA damage response. Stabilization of p53 does not depend on p19Arf interaction with mdm2. Cells infected by a mutant virus defective in binding pRb and in inducing G(1)-to-S progression show a greatly diminished p53 response. However, cells infected by wild-type virus and blocked from entering S phase by addition of mimosine still show a p53 response. These results suggest a role of E2F target genes in inducing a p53 response. Polyomavirus large T antigen coprecipitates with p53 phosphorylated on serine 18 and also with p21Cip1/WAF1. Implications of these and other findings on possible mechanisms of induction and override of p53 functions during productive infection by polyomavirus are discussed.     

Coordinate expression of the endogenous p53 gene in beta cells of transgenic mice expressing hybrid insulin-SV40 T antigen genes.

The expression of p53 has been evaluated during oncogenesis of the pancreatic beta cells in transgenic mice harboring hybrid insulin-SV40 T antigen genes. Significant levels of p53 are detected in all cells expressing large T antigen. In contrast, the protein is undetectable in normal beta cells. There is a complete correspondence between the onset of expression of T antigen and the appearance of the endogenous p53 protein. In tumors, the two proteins are found in a complex. In addition, free uncomplexed T antigen is detected in every cell which expresses the transgene. These results are consistent with the participation of p53 in T antigen-induced tumorigenesis in vivo. The early appearance of p53 in all beta cells expressing large T cannot readily explain the progression of a small fraction of these cells into solid tumors.     

Binding of p300/CBP co-activators by polyoma large T antigen.

Small DNA tumor viruses such as simian virus 40 (SV40) and polyomavirus (Py) take advantage of host cell proteins to transcribe and replicate their DNA. Interactions between the viral T antigens and host proteins result in cell transformation and tumor induction. Large T antigen of SV40 interacts with p53, pRb/p107/p130 family members, and the cyclic AMP-responsive element-binding protein (CREB)-binding protein (CBP)/p300. Py large T antigen is known to interact only with pRb and p300 among these proteins. Here we report that Py large T binds to CBP in vivo and in vitro. In co-transfection assays, Py large T inhibits the co-activation functions of CBP/p300 in CREB-mediated transactivation but not in NF-kappa B-mediated transactivation. p53 appears not to be involved in the functions of CREB-mediated transactivation and is not essential for large T:CBP interaction. Mutations introduced into a region of Py large T with homology to adenovirus E1A and SV40 large T prevent binding to the co-activators. These mutant large T antigens fail to inhibit CREB-mediated transactivation. The CBP/p300-binding Py mutants are able to transform established rat embryo fibroblasts but are restricted in their ability to induce tumors in the newborn mouse, indicating that interaction of large T with the co-activators may be essential for virus replication and spread in the intact host.     

Transgenic mouse models that explore the multistep hypothesis of intestinal neoplasia

SV-40 T antigen (TAg), human K-rasVal12, and a dominant negative mutant of human p53 (p53Ala143) have been expressed singly and in all possible combinations in postmitotic enterocytes distributed throughout the duodenal-colonic axis of 1-12-mo-old FVB/N transgenic mice to assess the susceptibility of this lineage to gene products implicated in the pathogenesis of human gut neoplasia. SV-40 TAg produces re-entry into the cell cycle. Transgenic pedigrees that produce K-rasVal12 alone, p53Ala143 alone, or K-rasVal12 and p53Ala143 have no detectable phenotypic abnormalities. However, K-rasVal12 cooperates with SV-40 TAg to generate marked proliferative and dysplastic changes in the intestinal epithelium. These abnormalities do not progress to form adenomas or adenocarcinomas over a 9-12-mo period despite sustained expression of the transgenes. Addition of p53Ala143 to enterocytes that synthesize SV-40 TAg and K-rasVal12 does not produce any further changes in proliferation or differentiation. Mice that carry one, two, or three of these transgenes were crossed to animals that carry Min, a fully penetrant, dominant mutation of the Apc gene associated with the development of multiple small intestinal and colonic adenomas. A modest (2-5-fold) increase in tumor number was noted in animals which express SV-40 TAg alone, SV-40 TAg and K-rasVal12, or SV-40 TAg, K-rasVal12 and p53Ala143. However, the histopathologic features of the adenomas were not altered and the gut epithelium located between tumors appeared similar to the epithelium of their single transgenic, bi-transgenic, or tri-transgenic parents without Min. These results suggest that (a) the failure of the dysplastic gut epithelium of SV-40 TAg X K-rasVal12 mice to undergo further progression to adenomas or adenocarcinomas is due to the remarkable protective effect of a continuously and rapidly renewing epithelium, (b) initiation of tumorigenesis in Min mice typically occurs in crypts rather than in villus-associated epithelial cell populations, and (c) transgenic mouse models of neoplasia involving members of the enterocytic lineage may require that gene products implicated in tumorigenesis be directed to crypt stem cells or their immediate descendants. Nonetheless, directing K-rasVal12 production to proliferating and nonproliferating cells in the lower and upper half of small intestinal and colonic crypts does not result in any detectable abnormalities.     

Loss of p53 and acquisition of angiogenic microRNA profile are insufficient to facilitate progression of bladder urothelial carcinoma in situ to invasive carcinoma

Activation of oncogenes or inactivation of tumor suppressors in urothelium is considered critical for development of urothelial cancer. Here we report cloning of the urothelium-specific promoter uroplakin-II (UPK II) and generation of transgenic mice in which expression of SV40 large T antigen is driven by UPK II promoter. Inactivation of tumor suppressor p53 and pRb in urothelium by SV40 T antigen resulted in urothelial carcinoma, resembling human high-grade carcinoma in situ. Specific deletion of p53 in urothelial cells using the newly generated UPK II-Cre mice results in normal bladders without any evidence of cancer. The high-grade carcinoma in situ in the UPK II-SV40 mice is associated with significant activation of angiogenic signals consisting of hypoxia-inducible factor-1α (HIF-1α) and VEGF and a down-regulation of thrombospondin-1. Interestingly, such pro-angiogenic activity was not associated with progression to invasive cancer. Analysis of bladder-associated microRNAs in carcinoma in situ lesions reveals a pro-angiogenic profile, with specific overexpression of miR-18a and miR-19a and down-regulation of miR-107. A group of microRNAs (miRs) identified as associated with invasive human urothelial cancer remained unchanged in this mouse model. Collectively, our results support the notion that activation of angiogenesis and loss of p53 are not sufficient for progression to invasive cancer. Our studies identify a new mouse model for bladder cancer that can be used to study factors that determine progression to an invasive phenotype of bladder cancer.     

Examination of the pRb-dependent and pRb-independent functions of E7 in vivo

High-risk human papillomaviruses encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. Although E7 protein is best known for its ability to inactivate the retinoblastoma tumor suppressor protein, pRb, many other activities for E7 have been proposed in in vitro studies. Herein, we describe studies that allowed us to define unambiguously the pRb-dependent and -independent activities of E7 for the first time in vivo. In these studies, we crossed mice transgenic for human papillomavirus 16 E7 to knock-in mice genetically engineered to express a mutant form of pRb (pRb(DeltaLXCXE)) that is selectively defective for binding E7. pRb inactivation was necessary for E7 to induce DNA synthesis and to overcome differentiation-dependent cell cycle withdrawal and DNA damage-induced cell cycle arrest. While most of E7's effects on epidermal differentiation were found to require pRb inactivation, a modest delay in terminal differentiation with resulting hyperplasia was observed in E7 mice on the Rb(DeltaLXCXE) mutant background. E7-induced p21 upregulation was also pRb dependent, and genetic Rb inactivation was sufficient to reproduce this effect. While E7-mediated p21 induction was partially p53 dependent, neither p53 nor p21 induction by E7 required p19(ARF). These data show that E7 upregulates the expression of p53 and p21 via pRb-dependent mechanisms distinct from the proposed p19-Mdm2 pathway. These results extend our appreciation of the importance of pRb as a relevant target for high-risk E7 oncoproteins.