Small T Antigen Research Articles

Suppression of Cancer Cell Migration and Invasion by Protein Phosphatase 2A through Dephosphorylation of μ- and m-Calpains

The mu- and m-calpains are major members of the calpain family that play an essential role in regulating cell motility. We have recently discovered that nicotine-activated protein kinase C iota enhances calpain phosphorylation in association with enhanced calpain activity and accelerated migration and invasion of human lung cancer cells. Here we found that specific disruption of protein phosphatase 2A (PP2A) activity by expression of SV40 small tumor antigen up-regulates phosphorylation of mu- and m-calpains whereas C2-ceramide, a potent PP2A activator, reduces nicotine-induced calpain phosphorylation, suggesting that PP2A may function as a physiological calpain phosphatase. PP2A co-localizes and interacts with mu- and m-calpains. Purified, active PP2A directly dephosphorylates mu- and m-calpains in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppresses nicotine-stimulated phosphorylation of mu- and m-calpains, which is associated with inhibition of calpain activity, wound healing, cell migration, and invasion. By contrast, depletion of PP2A/C by RNA interference enhances calpain phosphorylation, calpain activity, cell migration, and invasion. Importantly, C2-ceramide-induced suppression of calpain phosphorylation results in decreased secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential clinic relevance in controlling metastasis of lung cancer. These findings reveal a novel role for PP2A as a physiological calpain phosphatase that not only directly dephosphorylates but also inactivates mu- and m-calpains, leading to suppression of migration and invasion of human lung cancer cells.

Regulation of cell adhesion by PP2A and SV40 small tumor antigen: an important link to cell transformation.

The serine/threonine protein phosphatase 2A (PP2A) represents a large family of highly conserved heterotrimeric enzymes. Their critical importance in cell homeostasis is underlined by the fact that they are targets of natural toxins like the tumor promoter okadaic acid, and of simian virus 40 small tumor antigen (SV40 small t), a viral protein known to promote cell transformation. Furthermore, mutated or lower expression levels of PP2A subunits have been found in certain cancers. One major known event in PP2A-dependent cell transformation is the alteration of key signaling pathways that control cell growth and survival. In this review, we focus on how PP2A enzymes also affect cell adhesion and cytoskeletal dynamics, the disruption of which is linked to loss of cell polarity, increased cell motility and invasiveness. We also examine how those various pathways participate in the transforming activity of SV40 small t.

Interaction of retinoblastoma protein family members with large T-antigen of primate polyomaviruses

The retinoblastoma gene product pRb and other members of the Rb family of pocket proteins have a central role in the regulation of cell cycle progression. Soon after its discovery, pRb was found to interact with the transforming oncoproteins of DNA tumor viruses and this led to rapid advances in our understanding of the mechanisms of viral transformation and cell cycle progression. DNA viruses of the polyomavirus family have small, circular, double-stranded DNA genomes contained within non-enveloped icosahedral capsids and are highly tumorigenic in experimental animals. At least three types of polyomavirus infect humans: JC virus (JCV), BK virus (BKV) and Simian Vacuolating virus-40. The early region of these viruses encodes the transforming proteins large T-antigen and small t-antigen, which are involved in viral replication and also promote transformation of cells in culture and oncogenesis in vivo. Binding of T-antigen to pRb promotes the activation of the E2F family of transcription factors, which induce the expression of cellular genes required for S phase. In the context of lytic infection, this cell cycle progression is necessary for viral replication because polyomaviruses rely on S phase-specific host factors for their DNA synthesis. In the context of cellular transformation and tumorigenesis, T-antigen/pRB interaction is an indispensable event.

Protein Phosphatase 2A Enhances the Proapoptotic Function of Bax through Dephosphorylation

Bax is a major proapoptotic member of the Bcl2 family that is required for apoptotic cell death. We have recently discovered that Bax phosphorylation at serine 184 induced by nicotine through activation of protein kinase AKT abolishes its proapoptotic function in human lung cancer cells. Here we found that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor okadaic acid or specific disruption of PP2A activity by expression of SV40 small tumor antigen enhanced Bax phosphorylation, whereas C(2)-ceramide, a potent PP2A activator, reduced nicotine-induced Bax phosphorylation, suggesting that PP2A may function as a physiological Bax phosphatase. PP2A co-localized and interacted with Bax. Purified, active PP2A directly dephosphorylated Bax in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppressed nicotine-stimulated Bax phosphorylation in association with increased apoptotic cell death. By contrast, depletion of PP2A/C by RNA interference enhanced Bax phosphorylation and prolonged cell survival. Mechanistically C(2)-ceramide-induced Bax dephosphorylation caused a conformational change by exposure of the 6A7 epitope (amino acids 13-19) that is normally hidden at its N terminus that promoted the insertion of Bax into mitochondrial membranes and formation of Bax oligomers leading to cytochrome c release and apoptosis. In addition, PP2A directly disrupted the Bcl2/Bax association to liberate Bax from the heterodimer complex. Thus, PP2A may function as a physiological Bax regulatory phosphatase that not only dephosphorylates Bax but also activates its proapoptotic function.

Inhibition of PP2A, but not PP5, mediates p53 activation by low levels of okadaic acid in rat liver epithelial cells

The microbial toxin okadaic acid (OA) specifically inhibits PPP-type ser/thr protein phosphatases. OA is an established tumor promoter with numerous cellular effects that include p53-mediated cell cycle arrest. In T51B rat liver epithelial cells, a model useful for tumor promotion studies, p53 activation is induced by tumor-promoting (low nanomolar) concentrations of OA. Two phosphatases sensitive to these concentrations of OA, PP2A and protein phosphatase 5 (PP5), have been implicated as negative regulators of p53. In this study we examined the respective roles of these phosphatases in p53 activation in non-neoplastic T51B cells. Increases in p53 activity were deduced from levels of p21 (cip1) and/or the rat orthologue of mdm2, two p53-regulated gene products whose induction was blocked by siRNA-mediated knockdown of p53. As observed with 10 nM OA, both phospho-ser15-p53 levels and p53 activity were increased by 10 microM fostriecin or SV40 small t-antigen. Both of these treatments selectively inhibit PP2A but not PP5. siRNA-mediated knockdown of PP2A, but not PP5, also increased p53 activity. Finally, adenoviral-mediated over-expression of an OA-resistant form of PP5 did not prevent increased phospho-ser15-p53, p53 protein, or p53 activity caused by 10 nM OA. Together these results indicate that PP5 blockade is not responsible for OA-induced p53 activation and G1 arrest in T51B cells. In contrast, specific blockade of PP2A mimics p53-related responses to OA in T51B cells, suggesting that PP2A is the target for this response to OA.

Cross-platform expression profiling demonstrates that SV40 small tumor antigen activates Notch, Hedgehog, and Wnt signaling in human cells

BackgroundWe previously analyzed human embryonic kidney (HEK) cell lines for the effects that simian virus 40 (SV40) small tumor antigen (ST) has on gene expression using Affymetrix U133 GeneChips. To cross-validate and extend our initial findings, we sought to compare the expression profiles of these cell lines using an alternative microarray platform. METHODS: We have analyzed matched cell lines with and without expression of SV40 ST using an Applied Biosystems (AB) microarray platform that uses single 60-mer oligonucleotides and single-color quantitative chemiluminescence for detection. RESULTS: While we were able to previously identify only 456 genes affected by ST with the Affymetrix platform, we identified 1927 individual genes with the AB platform. Additional technical replicates increased the number of identified genes to 3478 genes and confirmed the changes in 278 (61%) of our original set of 456 genes. Among the 3200 genes newly identified as affected by SV40 ST, we confirmed 20 by QRTPCR including several components of the Wnt, Notch, and Hedgehog signaling pathways, consistent with SV40 ST activation of these developmental pathways. While inhibitors of Notch activation had no effect on cell survival, cyclopamine had a potent killing effect on cells expressing SV40 ST. CONCLUSIONS: These data show that SV40 ST expression alters cell survival pathways to sensitize cells to the killing effect of Hedgehog pathway inhibitors.

Recombinant Human Mullerian Inhibiting Substance Inhibits Long-term Growth of MIS Type II Receptor–Directed Transgenic Mouse Ovarian Cancers In vivo

Mullerian inhibiting substance (MIS) is a glycoprotein hormone that causes Mullerian duct regression in male embryos. In short-term experiments, recombinant human MIS (rhMIS) inhibits xenotransplanted human ovarian cancer cell lines that are thought to be of Mullerian origin. Because this highly lethal cancer has a high recurrence rate after conventional chemotherapy, new treatments are warranted. We examined whether rhMIS as a novel, nontoxic, naturally occurring growth inhibitor can be an effective anticancer drug in long-term studies in vivo against allograft tumors that recapitulate human ovarian carcinoma. Mouse ovarian carcinoma (MOVCAR) cell lines expressing the early region of the SV40 virus, including the large and small T-antigen genes under transcriptional control of a portion of the murine MIS receptor type II (MISRII) gene promoter, were derived from TgMISIIR-TAg transgenic mice. rhMIS was tested against MOVCAR cells in growth inhibition assays in vitro, and in vivo in 6-week-old female nude mice. Tumor growth in animals was measured at weekly intervals for up to 20 weeks. MOVCAR cells and tumors express MISRII by Western blot, immunohistochemical, and Northern blot analyses. rhMIS significantly inhibited MOVCAR cell growth in vitro and in vivo in three separate long-term allotransplantation experiments. Because rhMIS is an effective anticancer agent in in vitro and in long-term in vivo preclinical experiments against MISRII-positive tumors, we predict that rhMIS can be used safely and effectively to treat human ovarian malignancies.

Expression of JC virus regulatory proteins in human cancer: Potential mechanisms for tumourigenesis

JC virus (JCV) is a human polyomavirus that is the etiologic agent of the fatal demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML). JCV is also linked to some tumours of the brain and other organs as evidenced by the presence of JCV DNA sequences and the expression of viral proteins in clinical samples. Since JCV is highly oncogenic in experimental animals and transforms cells in culture, it is possible that JCV contributes to the malignant phenotype of human tumours with which it is associated. JCV encodes three non-capsid regulatory proteins: large T-antigen, small t-antigen and agnoprotein that interact with a number of cellular target proteins and interfere with certain normal cellular functions. In this review, we discuss how JCV proteins deregulate signalling pathways especially ones pertaining to transcriptional regulation and cell cycle control. These effects may be involved in the progression of JCV-associated tumours and may represent potential therapeutic targets.

MTOR-dependent Suppression of Protein Phosphatase 2A Is Critical for Phospholipase D Survival Signals in Human Breast Cancer Cells

A critical aspect of tumor progression is the generation of survival signals that overcome default apoptotic programs. Recent studies have revealed that elevated phospholipase D activity generates survival signals in breast and perhaps other human cancers. We report here that the elevated phospholipase D activity in the human breast cancer cell line MDA-MB-231 suppresses the activity of the putative tumor suppressor protein phosphatase 2A in a mammalian target of rapamycin (mTOR)-dependent manner. Increasing the phospholipase D activity in MCF7 cells also suppressed protein phosphatase 2A activity. Elevated phospholipase D activity suppressed association of protein phosphatase 2A with both ribosomal subunit S6-kinase and eukaryotic initiation factor 4E-binding protein 1. Suppression of protein phosphatase 2A by SV40 small t-antigen has been reported to be critical for the transformation of human cells with SV40 early region genes. Consistent with a critical role for protein phosphatase 2A in phospholipase D survival signals, either SV40 small t-antigen or pharmacological suppression of protein phosphatase 2A restored survival signals lost by the suppression of either phospholipase D or mTOR. Blocking phospholipase D signals also led to reduced phosphorylation of the pro-apoptotic protein BAD at the protein phosphatase 2A dephosphorylation site at Ser-112. The ability of phospholipase D to suppress protein phosphatase 2A identifies a critical target of an emerging phospholipase D/mTOR survival pathway in the transformation of human cells.

Activation of the Canonical Wnt Pathway during Genital Keratinocyte Transformation: A Model for Cervical Cancer Progression

Cervical carcinoma, the second leading cause of cancer deaths in women worldwide, is associated with human papillomavirus (HPV). HPV-infected individuals are at high risk for developing cervical carcinoma; however, the molecular mechanisms that lead to the progression of cervical cancer have not been established. We hypothesized that in a multistep carcinogenesis model, HPV provides the initial hit and activation of canonical Wnt pathway may serve as the second hit. To test this hypothesis, we evaluated the canonical Wnt pathway as a promoting factor of HPV-induced human keratinocyte transformation. In this in vitro experimental cervical carcinoma model, primary human keratinocytes immortalized by HPV were transformed by SV40 small-t (smt) antigen. We show that smt-transformed cells have high cytoplasmic beta-catenin levels, a hallmark of activated canonical Wnt pathway, and that activation of this pathway by smt is mediated through its interaction with protein phosphatase-2A. Furthermore, inhibition of downstream signaling from beta-catenin inhibited the smt-induced transformed phenotype. Wnt pathway activation transformed HPV-immortalized primary human keratinocytes even in the absence of smt. However, activation of the Wnt pathway in the absence of HPV was not sufficient to induce transformation. We also detected increased cytoplasmic and nuclear staining of beta-catenin in invasive cervical carcinoma samples from 48 patients. We detected weak cytoplasmic and no nuclear staining of beta-catenin in 18 cases of cervical dysplasia. Our results suggest that the transformation of HPV expressing human keratinocytes requires activation of the Wnt pathway and that this activation may serve as a screening tool in HPV-positive populations to detect malignant progression.

Effects of Simian Virus 40 Large and Small Tumor Antigens on Mammalian Target of Rapamycin Signaling: Small Tumor Antigen Mediates Hypophosphorylation of eIF4E-Binding Protein 1 Late in Infection

We report that late in a simian virus 40 (SV40) infection in CV-1 cells, there are significant decreases in phosphorylations of two mammalian target of rapamycin (mTOR) signaling effectors, the eIF4E-binding protein (4E-BP1) and p70 S6 kinase (p70S6K). The hypophosphorylation of 4E-BP1 results in 4E-BP1 binding to eIF4E, leading to the inhibition of cap-dependent translation. The dephosphorylation of 4E-BP1 is specifically mediated by SV40 small t antigen and requires the protein phosphatase 2A binding domain but not an active DnaJ domain. Serum-starved primary African green monkey kidney (AGMK) cells also showed decreased phosphorylations of mTOR, 4E-BP1, and p70S6K at late times in infection (48 h postinfection [hpi]). However, at earlier times (12 and 24 hpi), in AGMK cells, phosphorylated p70S6K was moderately increased, correlating with a significant increase in phosphorylation of the p70S6K substrate, ribosomal protein S6. Hyperphosphorylation of 4E-BP1 at early times could not be determined, since hyperphosphorylated 4E-BP1 was present in mock-infected AGMK cells. Elevated levels of phosphorylated eIF4G, a third mTOR effector, were detected in both CV-1 and AGMK cells at all times after infection, indicating that eIF4G phosphorylation was induced throughout the infection and unaffected by small t antigen. The data suggest that during SV40 lytic infection in monkey cells, the phosphorylations of p70S6K, S6, and eIF4G are increased early in the infection (12 and 24 hpi), but late in the infection (48 hpi), the phosphorylations of mTOR, p70S6K, and 4E-BP1 are dramatically decreased by a mechanism mediated, at least in part, by small t antigen.

Simian virus 40 small t antigen mediates conformation-dependent transfer of protein phosphatase 2A onto the androgen receptor.

The tumor antigens simian virus 40 small t antigen (ST) and polyomavirus small and medium T antigens mediate cell transformation in part by binding to the structural A subunit of protein phosphatase 2A (PP2A). The replacement of B subunits by tumor antigens inhibits PP2A activity and prolongs phosphorylation-dependent signaling. Here we show that ST mediates PP2A A/C heterodimer transfer onto the ligand-activated androgen receptor (AR). Transfer by ST is strictly dependent on the agonist-activated conformation of AR, occurs within minutes of the addition of androgen to cells, and can occur in either the cytoplasm or the nucleus. The binding of ST changes the conformation of the A subunit, and ST rapidly dissociates from the complex upon PP2A A/C heterodimer binding to AR. PP2A is transferred onto the carboxyl-terminal half of AR, and the phosphatase activity is directed to five phosphoserines in the amino-terminal activation function region 1, with a corresponding reduction in transactivation. Thus, ST functions as a transfer factor to specify PP2A targeting in the cell and modulates the transcriptional activity of AR.

PP2A-dependent transactivation of the cyclin A promoter by SV40 ST is mediated by a cell cycle-regulated E2F site

The Simian Virus 40 (SV40) small-t antigen (ST) plays an important role in driving cell proliferation, enhancing transformation by the large-T (LT) antigen. Potential targets of ST are the cyclin kinase inhibitor p27 and the cyclin A gene itself. Transactivation of the cyclin A promoter by ST depends on the interaction of ST with protein phosphatase 2A (PP2A) and occurs through a cell cycle-regulated E2F site near the transcription start site of the promoter. A third SV40 early protein, 17KT, also transactivates the cyclin A promoter but, in this case, transactivation depends on the dnaJ domain of the protein.

The agnoprotein of polyomaviruses: A multifunctional auxiliary protein

The late region of the three primate polyomaviruses (JCV, BKV, and SV40) encodes a small, highly basic protein known as agnoprotein. While much attention during the last two decades has focused on the transforming proteins encoded by the early region (small and large T-antigens), it has become increasingly evident that agnoprotein has a critical role in the regulation of viral gene expression and replication, and in the modulation of certain important host cell functions including cell cycle progression and DNA repair. The importance of agnoprotein is underscored by its expression during lytic infection of glial cells by JCV that occurs in progressive multifocal leukoencephalopathy (PML), and also in some JCV-associated human neural tumors particularly medulloblastoma. In this review, we will discuss the structure and function of agnoprotein in the viral life cycle during the course of lytic infection and the consequences of agnoprotein expression for the host cell.

Signaling and transcriptional changes critical for transformation of human cells by simian virus 40 small tumor antigen or protein phosphatase 2A B56gamma knockdown.

One set of genes sufficient for transformation of primary human cells uses the combination of Ha-Ras-V12, the telomerase catalytic subunit hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, the contribution of ST is poorly understood. The essential helper function of ST requires a functional interaction with protein phosphatase 2A (PP2A). Here we have identified changes in gene expression induced by ST and show that ST mediates these changes through both PP2A-dependent and PP2A-independent mechanisms. Knockdown of PP2A B56gamma subunit can substitute for ST expression to fully transform cells expressing LT, hTERT, and Ras-V12. We also identify those genes affected similarly in two cell lines that have been fully transformed from a common parental line by two alternative mechanisms, namely ST expression or PP2A B56gamma subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and transcriptional regulation. ST reduced surface expression of MHC class I molecules, consistent with a need for SV40 to evade immune detection. ST expression enabled cell cycle progression in reduced serum and src phosphorylation in anchorage-independent media, whereas B56gamma knockdown required normal serum levels for these phenotypes. Inhibitors of integrin and src signaling prevented anchorage-independent growth of transformed cells, suggesting that integrin and src activation are key ST-mediated events in transformation. Our data support a model in which ST promotes survival through constitutive integrin signaling, src phosphorylation, and nuclear factor kappaB activation, while inhibiting cell-cell adhesion pathways.

Role for PP2A in ARF signaling to p53.

Activation of the ARF-p53 tumor suppressor pathway is one of the cell's major defense mechanisms against cancer induced by oncogenes. The ARF-p53 pathway is dysfunctional in a high proportion of human cancers. The regulation of the ARF-p53 signaling pathway has not yet been well characterized. In this study polyoma virus (Py) is used as a tool to better define the ARF-p53 signaling pathway. Py middle T-antigen (PyMT) induces ARF, which consequently up-regulates p53. We show that Py small T-antigen (PyST) blocks ARF-mediated activation of p53. This inhibition requires the small T-antigen PP2A-interacting domain. Our results reveal a previously unrecognized role of PP2A in the modulation of the ARF-p53 tumor suppressor pathway.

Polyomavirus tumorantigens have a profound effect on gene expression in mouse fibroblasts.

Polyomavirus (Py) large and small tumorantigens together are competent to induce S phase in growth-arrested mouse fibroblasts. The capacity of the large tumorantigen to bind the pocket proteins, pRB, p130 and p107, is important for the transactivation of DNA synthesis enzymes and the cyclins E and A, while the interference of small tumorantigen with protein phosphatase PP2A causes a destabilization of the cdk2 inhibitor p27, and thus leads to strong cyclin E- and cyclin A-dependent cdk2 activity. Py small tumorantigen, in addition, is able to transactivate cyclin A. Hence, this protein might have a much wider effect on gene expression in arrested mouse fibroblasts than hitherto suspected. This may have a profound part in the known capacity of Py to form tumors in mice. Therefore, it was interesting to gain an insight into the spectrum of transcriptional deregulation by Py tumorantigens. Accordingly, we performed microarray analysis of quiescent mouse fibroblasts in the absence and presence of small or large tumorantigen. We found that the viral proteins can induce or repress a great variety of genes beyond those involved in the S phase induction and DNA synthesis. The results of the microarray analysis were confirmed for selected genes by several methods, including real-time PCR. Interestingly, a mutation of the binding site for pocket proteins in case of LT and for PP2A in case of ST has a variable effect on the deregulation of genes by the viral proteins depending on the gene in question. In fact, some genes are transactivated by LT as well as ST completely independent of an interaction with their major cellular targets, pocket proteins and PP2A, respectively.

LIVER-SPECIFIC PHYSIOLOGY OF IMMORTAL, FUNCTIONALLY DIFFERENTIATED HEPATOCYTES AND OF DEFICIENT HEPATOCYTE-LIKE VARIANTS

Five different immortalized transgenic hepatocyte cell lines derived from mice were investigated with respect to their potential to maintain the physiological properties of primary hepatocytes using chemically defined medium. This research completes a previous study by Klocke and coworkers in 2002, using gene expression analysis of the same cell lines by the respective physiological analysis for investigating the hepatocyte-like function. Three transgenic cell lines harboring a fusion gene derivative (construct 202) consisting of the complete SV40 early region, including the coding sequences for the transforming large and small tumor antigens, placed under the control of the murine metallothioneine 1-promotor/enhancer element, showed a hepatocyte-like function and physiology. They grew as a monolayer with a polygonal cell shape, consumed lactate, and secreted albumin at a cell-specific rate of 1.5 pg/h, which is in the range of primary hepatocytes. In addition, the potential of detoxifying ammonium could be maintained. Ammonium was metabolized and urea was produced and released into the medium. A complete urea cycle could be determined. A cell line established from neonatal transgenic mice and expressing a secretory variant of the human epidermal growth factor (IgEGF) under the control of the albumin promoter was characterized by an incomplete urea cycle. Another cell line isolated from the liver of homozygote neonatal p53-knockout mice showed no hepatocyte-specific functions but only properties of continuous cell lines. Specific nucleoside triphosphate (NTP) and uridine (U) ratios were used to characterize the differentiation status of the particular cell lines. A low NTP-U value was found for the three cell lines containing construct 202, which was identical to that observed for primary hepatocytes. In contrast, the cell line harvested from the liver of homozygote neonatal p53-knockout mice presented a NTP-U ratio characteristic for continuous cell lines. This study demonstrates that the four transgenic and the p53-knockout hepatocyte-derived cell lines can be used as models for investigating the conservation of tissue-specific functions in immortalized cells.

JC virus small tumor antigen promotes S phase entry

A number of studies revealed the detection of human polyomavirus, JC virus (JCV), in a variety of human tumors. Viral large tumor antigen (T-Ag) is known to play a major role in cell proliferation and transformation but the role of JCV small t-Ag in such processes remains completely unknown. Several studies from monkey polyomavirus, SV40, small tumor antigen (tAg) suggest that it significantly contributes to the large T-Ag-mediated cell transformation. Here, we investigated the effects of JCV small t-Ag on cell cycle progression and cell proliferation. Examination of subcellular distribution of small t-Ag by means of immunocytochemistry demonstrated that it mostly localizes to the cytoplasmic compartment of the cell. Cell cycle progression analysis of a human glioblastoma cell line, U-87MG, demonstrated that small t-Ag expressing cells enter S phase of the cell cycle at a significantly higher rate than controls when released from G0/G1 arrest. In addition, the growth rate of these cells is consistent with the rate of cell cycle progression and is significantly enhanced compared to controls. Moreover, analysis of the mechanism through which small t-Ag accelerates the cell cycle progression showed that JCV small t-Ag targets a major serine/threonine phosphatase, PP2A, and its interaction domain with this enzyme is mapped toward the middle portion of the protein, residues 82 to 124. Furthermore, examination of the cell cycle stage specific expression profiles of selected cyclins and cyclin-dependent kinases, particularly those active in G1/S and G2/M transition states demonstrated that the rate of the appearance of cyclin E, cyclin B, and cyclin E-dependent kinase 2 (Cdk2) substantially increased in small t-Ag positive cells as compared to controls when the cells were released from G0/G1 arrest. In parallel, in vitro analysis of cyclin E-associated H1 kinase activity of clones demonstrated that small t-Ag positive clones exhibited significantly higher activity than controls. Additionally, results from reporter gene assays showed that small tAg has the ability to activate the cyclin E promoter which is consistent

SV40 and Notch-I: multi-functionality meets pleiotropy.

We have discovered that Simian virus 40 (SV40) infection of human mesothelial cells induces Notch-1. Upregulation of Notch-1 is achieved at the transcriptional level and requires the activity of both the large (90-100 kDa) and the small (20 kDa) SV40 tumor antigens. Notch-1 upregulation is maintained in SV40-transformed mesothelial cell lines in tissue culture, and Notch-1 is overexpressed only in SV40-positive mesotheliomas. Chemical inactivation of Notch-1 causes cell growth arrest of SV40-transformed human mesothelial cells. Our findings indicate that Notch-1 activation plays an important role in SV40-mediated carcinogenesis. Notch-1 is a pleiotropic gene that influences cell differentiation, proliferation and apoptosis. The effects of Notch-1 activation are species and cell type specific, in that, in different species or cell type within the same species, Notch-1 signaling can cause opposite effects. In this chapter, we review some general aspects of Notch signaling, and how Notch signaling regulates differentiation, the cell cycle and apoptosis. Recent data involving Notch and human cancer will be discussed. Then, we will deal with the current information regarding a specific form of lung cancer (malignant mesothelioma) and the involvement of SV40 in the pathogenesis of this form of human cancer. We will enter into the details of SV40-mediated cell transformation, and the specific interaction between SV40 and primary human mesothelial cells when infected with SV40 in vitro.