Expression Of SV40 Large T-antigen Research Articles

RABL6A Promotes Pancreatic Neuroendocrine Tumor Angiogenesis and Progression In Vivo.

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet β cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.

Aberrant granulosa cell-fate related to inactivated p53/Rb signaling contributes to granulosa cell tumors and to FOXL2 downregulation in the mouse ovary.

Ovarian granulosa cell tumors (GCTs) are indolent tumors of the ovary affecting women at all ages and potentially displaying late recurrence. Even if there is still little information regarding the mechanisms involved in GCT development and progression, FOXL2 would be a major tumor suppressor gene in granulosa cells. We analyzed the mechanisms underlying GCT initiation and progression by using mice with targeted expression of SV40 large T-antigen in granulosa cells (AT mouse), which develop GCTs. Consistent with patients, AT mice with developing GCTs displayed increased levels in circulating anti-Müllerian hormone (AMH), estradiol and androgens, as well as decreased FOXL2 protein abundance. Very few mice developed metastases (1 out of 30). In situ analyses revealed that GCT initiation resulted from both increased granulosa cell survival and proliferation in large antral follicles. Tumorigenesis was associated with the combined inactivation of p53 and Rb pathways, as shown by the impaired expression of respective downstream targets regulating cell apoptosis and proliferation, i.e., Bax, Bak, Gadd45a, Ccna2, Ccne1, E2f1, and Orc1. Importantly, the expression of FOXL2 was still present in newly developed GCTs and its downregulation only started during GCT growth. Collectively, our experiments provide evidence that disrupted p53/Rb signaling can drive tumor initiation and growth. This model challenges the current paradigm that impaired FOXL2 signaling is a major switch of granulosa cell tumorigenesis, albeit possibly contributing to tumor growth.

Establishment of keratinocyte cell lines from human hair follicles

The advent of organotypic skin models advanced the understanding of complex mechanisms of keratinocyte differentiation. However, these models are limited by both availability of primary keratinocytes and donor variability. Keratinocytes derived from cultured hair follicles and interfollicular epidermis were immortalized by ectopic expression of SV40 and hTERT. The generated keratinocyte cell lines differentiated into stratified epidermis with well-defined stratum granulosum and stratum corneum in organotypic human skin models. They behaved comparable to primary keratinocytes regarding the expression of differentiation-associated proteins, cell junction components and proteins associated with cornification and formed a barrier against biotin diffusion. Mechanistically, we found that SV40 large T-antigen expression, accompanied by a strong p53 accumulation, was only detectable in the basal layer of the in vitro reconstructed epidermis. Inhibition of DNA-methylation resulted in expression of SV40 large T-antigen also in the suprabasal epidermal layers and led to incomplete differentiation of keratinocyte cell lines. Our study demonstrates the generation of keratinocyte cell lines which are able to fully differentiate in an organotypic skin model. Since hair follicles, as source for keratinocytes, can be obtained by minimally invasive procedures, our approach enables the generation of cell lines also from individuals not available for skin biopsies.

Naked Mole Rat Cells Have a Stable Epigenome that Resists iPSCReprogramming.

SummaryNaked mole rat (NMR) is a valuable model for aging and cancer research due to its exceptional longevity and cancer resistance. We observed that the reprogramming efficiency of NMR fibroblasts in response to OSKM was drastically lower than that of mouse fibroblasts. Expression of SV40 LargeT antigen (LT) dramatically improved reprogramming of NMR fibroblasts. Inactivation of Rb alone, but not p53, was sufficient to improve reprogramming efficiency, suggesting that NMR chromatin may be refractory to reprogramming. Analysis of the global histone landscape revealed that NMR had higher levels of repressive H3K27 methylation marks and lower levels of activating H3K27 acetylation marks than mouse. ATAC-seq revealed that in NMR, promoters of reprogramming genes were more closed than mouse promoters, while expression of LT led to massive opening of the NMR promoters. These results suggest that NMR displays a more stable epigenome that resists de-differentiation, contributing to the cancer resistance and longevity of this species.

P53/Rb inhibition induces metastatic adrenocortical carcinomas in a preclinical transgenic model.

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Pan-genomic analyses identified p53/Rb and WNT/β-catenin signaling pathways as main contributors to the disease. However, isolated β-catenin constitutive activation failed to induce malignant progression in mouse adrenocortical tumors. Therefore, there still was a need for a relevant animal model to study ACC pathogenesis and to test new therapeutic approaches. Here, we have developed a transgenic mice model with adrenocortical specific expression of SV40 large T-antigen (AdTAg mice), to test the oncogenic potential of p53/Rb inhibition in the adrenal gland. All AdTAg mice develop large adrenal carcinomas that eventually metastasize to the liver and lungs, resulting in decreased overall survival. Consistent with ACC in patients, adrenal tumors in AdTAg mice autonomously produce large amounts of glucocorticoids and spontaneously activate WNT/β-catenin signaling pathway during malignant progression. We show that this activation is associated with downregulation of secreted frizzled related proteins (Sfrp) and Znrf3 that act as inhibitors of the WNT signaling. We also show that mTORC1 pathway activation is an early event during neoplasia expansion and further demonstrate that mTORC1 pathway is activated in ACC patients. Preclinical inhibition of mTORC1 activity induces a marked reduction in tumor size, associated with induction of apoptosis and inhibition of proliferation that results in normalization of corticosterone plasma levels in AdTAg mice. Altogether, these data establish AdTAg mice as the first preclinical model for metastatic ACC.

Embryonic Retinal Tumors in SV40 T-Ag Transgenic Mice Contain CD133+ Tumor-Initiating Cells

Human retinoblastomas form during the proliferative phase of retina development and are caused by mutations that result in absent or functionally defective Rb protein. Similar tumors occur in mice only when multiple Rb gene family members are absent. We asked if retinal tumors can arise from an undifferentiated retinal cell. The tumor-initiating cells isolated from these tumors that formed in early embryonic murine retinas were characterized. Transgenic mice were created using a Pax6 promoter to target expression of SV40 large T-antigen (T-Ag) in the undifferentiated murine embryonic retina. T-Ag, which sequesters all Rb family proteins and p53, is expressed in the retina and lens by murine embryonic day 10 (E10) and tumors are observed by E12.5. A cell line that is adherent in serum-containing media and forms neurospheres in supplemented serum-free media was developed from retinal tumors isolated on postnatal day 7. In all, 1.5% of attached cells form neurospheres when transferred to serum-free medium. All cultured cells express T-Ag, confirming that they derive from the original tumors; 0.5% of adherent cells express detectable levels of CD133. CD133+ FACS-sorted cells cultured in serum-free medium form 3-fold more neurospheres than do CD133- cells. Six of seven mice injected with CD133+ cells and one of seven mice injected with CD133- cells formed tumors during a 6-month period. Unlike primary adherent cells, primary and secondary tumors heterogeneously express markers of stem cells and differentiation similar to human retinoblastoma. CD133+ tumor-initiating cells can originate from proliferating undifferentiated precursor cells.

Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic β cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas that expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic β cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.

VentX trans-Activates p53 and p16ink4a to Regulate Cellular Senescence

Cell senescence is a process of irreversible arrest of cell proliferation and plays an important role in tumor suppression. Recent studies showed that Wnt inhibition is a trigger of cellular senescence. Using methods of reverse genetics, we recently identified VentX, a human homolog of the vertebrate Xenopus Vent family of homeobox genes, as a novel Wnt repressor and a putative tumor suppressor in lymphocytic leukemia. Here, we show that VentX is a direct transcriptional activator of p53-p21 and p16ink4a-Rb tumor suppression pathways. Ectopic expression of VentX in cancer cells caused an irreversible cell cycle arrest with a typical senescence-like phenotype. Conversely, inhibition of VentX expression by RNA interference ameliorated chemotherapeutic agent-induced senescence in lymphocytic leukemia cells. The results of our study further reveal the mechanisms underlying tumor suppression function of VentX and suggest a role of VentX as a potential target in cancer prevention and treatment.

Mutant p53 initiates a feedback loop that involves Egr-1/EGF receptor/ERK in prostate cancer cells

Early Growth Response-1 (Egr-1) is overexpressed in human prostate tumors and contributes to cancer progression. On the other hand, mutation of p53 is associated with advanced prostate cancer, as well as with metastasis and hormone independence.This study shows that in prostate cell lines in culture, Egr-1 overexpression correlated with an alteration of p53 activity due to the expression of SV40-TAg or to a mutation in the TP53 gene. In cells containing altered p53 activity, Egr-1 expression was abolished upon pharmacological inhibition or RNAi silencing of p53. Although forced expression of wild-type p53 was not sufficient to trigger Egr-1 transcription, four different mutants of p53 were shown to induce Egr-1. Direct binding of p53 to the EGR1 promoter could not be detected. Instead, Egr-1 transcription was driven by the ERK1/2 pathway, since it was abrogated by specific inhibitors of MEK. Egr-1 increased the transcription of HB-EGF, amphiregulin and epiregulin, resulting in autocrine activation of the EGFR (epidermal growth factor receptor) and downstream MEK/ERK cascade. Thus, mutant p53 initiates a feedback loop that involves ERK1/2-mediated transactivation of Egr-1, which in turn increases the secretion of EGFR ligands and stimulates the EGFR signaling pathway. Finally, p53 may further regulate this feedback loop by altering the level of EGFR expression.

Precise conditional immortalization of mouse cells using tetracycline‐regulated SV40 large T‐antigen

Cellular immortalization provides a way for expansion and subsequent molecular characterization of rare cell types. Ideally, immortalization can be achieved by the reversible expression of immortalizing proteins. Here, we describe the use of conditional immortalization based on a modified tetracycline-regulated system for the expression of SV40 large T-antigen in embryonic stem (ES) cells and mice. The modified system relies on a codon improved reverse tetracycline transactivator (irtTA) fused to the ligand-binding domain (LBD) of the androgen receptor (irtTA-ABD) or of a mutated glucocorticoid receptor (irtTA-GBD*). Induction of T-antigen is conferred only after addition of two ligands, one to activate the LBD (mibolerone for irtTA-ABD or dexamethasone for irtTA-GBD*) and one to activate the tetracycline transactivator (doxycycline). In ES cells, changes in gene expression upon large T induction were limited and reversible upon deinduction. Similarly, expression of T-antigen was very tightly regulated in mice. We have isolated and expanded bone marrow mesenchymal stem cells that could be genetically manipulated and maintained their differentiation properties after several passages of expansion under conditions that induce the expression of large T-antigen.

Interferon-γ induces regression of epithelial cell carcinoma: critical roles of IRF-1 and ICSBP transcription factors

We have developed an epithelial cell carcinoma model for studying efficacy of IFNgamma gene therapy and have identified components of IFNgamma-signaling pathway responsible for its direct anti-tumor actions. The tumor results from ectopic expression of SV40 Large T-Antigen (SV40 T-Ag) oncogene in lens of transgenic mouse (alphaT3) and complete regression of the tumor is induced by targeting expression of IFNgamma into malignant lens cells. Inflammatory cells are absent in lens of alphaT3 or DT (co-expressing IFNgamma and SV40-T-Antigen) mice and the transformed lens cells are non-immunogenic, suggesting non-involvement of immunologic cells. We show that IFNgamma has direct growth-inhibitory effects on tumor cells, induces death of tumor cells by apoptosis and that these effects are mediated by two transcription factors, IRF-1 (interferon-regulatory factor-1) and ICSBP (interferon-consensus sequence-binding protein) induced by IFNgamma. Furthermore, stable transfection with ICSBP or IRF-1 construct inhibits lens carcinoma cell growth by upregulating Caspase-1, p21(WAF1) and p27 expression. In contrast, tumor progression in alphaT3 lens correlates with inhibition of IRF-1 and ICSBP expression. Our results suggest that IFNgamma gene therapy maybe effective in malignant diseases for which DNA tumor viruses are etiologic agents and that antitumor actions of IRF-1/ICSBP can be exploited therapeutically to circumvent adverse clinical effects associated with IFN therapy.

Hrs, a Mammalian Master Molecule in Vesicular Transport and Protein Sorting, Suppresses the Degradation of ESCRT Proteins Signal Transducing Adaptor Molecule 1 and 2

The degradation and sorting of cytoplasmic and cell-surface proteins are crucial steps in the control of cellular functions. We previously identified three mammalian Vps (vacuolar protein sorting) proteins, Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and signal transducing adaptor molecule (STAM) 1 and -2, which are tyrosine-phosphorylated upon cytokine/growth factor stimulation. Hrs and the STAMs each contain a ubiquitin-interacting motif and through formation of a complex are involved in the vesicle transport of early endosomes. To explore the mechanism and cellular function of this complex in mammalian cells, we established an Hrs-defective fibroblastoid cell line (hrs(-/-)); embryos with this genotype died in utero. In the hrs(-/-) cells only trace amounts of STAM1 and STAM2 were detected. Introduction of wild-type Hrs or an Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) fully restored STAM1 and STAM2 expression, whereas mutants with no STAM binding ability (Hrs-dC2, Hrs-dM) failed to express the STAMs. This regulated control of STAM expression by Hrs was independent of transcription. Interestingly, STAM1 degradation was mediated by proteasomes and was partially dependent on the ubiquitin-interacting motif of STAM1. Revertant Hrs expression in hrs(-/-) cells not only led to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also restored STAM1 levels in early endosomes and eliminated the enlarged endosome phenotype caused by the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins.

Apoptosis in RAW 264.7 cells exposed to 4-hydroxy-2-nonenal: dependence on cytochrome C release but not p53 accumulation

The toxic reactive aldehyde lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) is thought to be a major contributor to oxidant stress-mediated cell injury. HNE induced apoptosis in RAW 264.7 murine macrophage cells in a dose-dependent manner within 6–8 h after exposure. Expression of the antiapoptotic protein Bcl-2 in stably transfected RAW 264.7 cells prevented HNE-induced internucleosomal DNA fragmentation and apoptosis, and these cells resume growth after a temporary (24–48 h) growth delay. While parental RAW 264.7 cells released mitochondrial cytochrome c within 3 h after HNE exposure, expression of Bcl-2 prevented cytochrome c release. In control cells, p53 protein levels peaked at 6–9 h after HNE exposure and then declined, while in Bcl-2 expressing cells, p53 levels were maximal at 6–9 h and remained elevated up to 96 h. Expression of SV40 large T-antigen, which forms a stable complex with p53 protein, via stable transfection-blocked transactivation of the p53-regulated gene p21 WAF1/CIP1, but did not affect induction of apoptosis by HNE, suggesting that p53 function is not important in HNE-induced apoptosis. These results suggest that cytochrome c release, but not p53 accumulation, plays an essential role in HNE-induced apoptosis in RAW 264.7 cells.

Targeted Expression of SV40 Large T-antigen to Visceral Smooth Muscle Induces Proliferation of Contractile Smooth Muscle Cells and Results in Megacolon

Many pathological conditions result from the proliferation and de-differentiation of smooth muscle cells leading to impaired contractility of the muscle. Here we show that targeted expression of SV40 large T-antigen to visceral smooth muscle cells in vivo results in increased smooth muscle cell proliferation without de-differentiation or decreased contractility. These data suggest that the de-differentiation and proliferation of smooth muscle cells, seen in many pathological states, may be independently regulated. In the T-antigen transgenic mice the increased smooth muscle cell proliferation results in thickening of the distal colon. Consequently the distal colon becomes hyper-contractile and impedes the flow of digesta through the colon resulting in enlargement of the colon oral to the obstruction. These transgenic mice thus represent a novel model of megacolon that results from increased smooth muscle cell proliferation rather than altered neuronal innervation.

A human pituitary adenoma cell line proliferates and maintains some differentiated functions following expression of SV40 large T-antigen

Human pituitary cells proliferate very slowly in vitro. Only a few cell lines have been established, and these have been used mainly for short-term studies. To obtain immortalized cell lines of human pituitary adenomas for in vitro studies, we infected adenoma cells with a replication-defective recombinant human adenovirus, which contains an SV40 early large T-antigen. One of the cell lines (HP75), which has been studied in culture during 60 passages, has been extensively characterized. It expressed the large T-antigen protein and its mRNA, as well as the genes for FSH-β, LH-β and α-subunit (α-SU) of gonadotropin hormone.

Novel Smooth Muscle Cell Lines from Transgenic Mice Harboring Temperature-sensitive SV40 Large T-antigen Gene. Temperature-dependent Expression of Smooth Muscle Myosin Heavy Chain-1 and Calponin Genes

We have established novel vascular smooth muscle cell lines (SVS30 and SVS24 cells) which retain the expression of specific markers for smooth muscle cells, such as α-actin, smooth muscle myosin heavy chain-1, and calponin, from transgenic mice harboring the temperature-sensitive SV40 large T-antigen gene. SVS cell lines showed temperature-dependent growth and the expression of SV40 large T-antigen. Interestingly, protein and mRNA levels of smooth muscle myosin heavy chain-1 and calponin seen in culture at the non-permissive temperature (39°C) were higher than those at the permissive temperature (33°C). These results suggest that SV40 large T-antigen affects the expression of smooth muscle-specific markers in SVS cell lines, and that some of the characters in SVS cell lines can be controlled by culture temperature. SVS cell lines should be quite valuable tools with which to study the regulation of phenotypic modulation of smooth muscle cells, and to identify smooth muscle specific transcription factors which involve the expression of smooth muscle myosin heavy chain-1 and calponin genes.

Intranuclear Redistribution of SV40T, p53, and PML in a Conditionally SV40T-Immortalized Cell Line

We have previously reported that EBNA-5, one of the Epstein–Barr virus-encoded proteins, accumulates in the nuclear bodies containing PML, the promyelocytic leukemia associated protein. In this study, we examine the intranuclear distribution of SV40 large T-antigen (SV40T), the p53 tumor suppressor protein (p53), and PML in a conditionally immortalized cell line, IDH4. In IDH4 cells, the expression of SV40T is regulated by a dexamethasone (Dex)-driven promoter. Withdrawal of Dex results in down-regulation of SV40T and growth arrest, whereas addition of Dex to the growth-arrested cells results in up-regulation of SV40T and proliferation. In proliferating IDH4 cells, SV40T is concentrated in nuclear dots that are also positive for p53. Many of these dots are juxtaposed to PML positive structures but do not colocalize with them. After removal of Dex, SV40T–p53 dots gradually disappear, while the PML structures remain. Induction of SV40T in nonproliferating IDH4 cells causes a coordinated redistribution of SV40T and p53. The immunostaining for SV40T and p53 is first weak, then strong with a homogeneous distribution, and 3–4 days later becomes dot-like again. This reappearance of SV40T–p53 dots coincides with the recovery of proliferation in restimulated IDH4 cells. Also, the p53 pattern correlates with the SV40T pattern with regard to both morphology and intensity during both suppression and induction of SV40T. Taken together, our data suggest that (i) the level of p53 is coregulated with the level of SV40T in a dose-dependent fashion; (ii) the formation of SV40T–p53 nuclear dots correlates with the transformed phenotype; (iii) the SV40T–p53 dots localize preferentially to the neighborhood of PML bodies which are already present in normal cells.

Telokin expression is mediated by a smooth muscle cell-specific promoter

The carboxy terminus of the smooth muscle myosin light chain kinase (smMLCK) is expressed as an independent protein, telokin. Western and Northern blotting analyses demonstrated that telokin protein and mRNA are expressed at high levels only in adult and embryonic smooth muscle tissues and cells. In vitro transfection assays in A10 smooth muscle cells identified a functional promoter located in an intron in the 3' region of the smMLCK gene that directs the smooth muscle cell-specific transcription of telokin. To test the cell specificity of the telokin promoter in vivo, transgenic mice were generated in which the telokin promoter was used to drive expression of SV40 large T-antigen. Expression of T-antigen in the transgenic mice paralleled that of the endogenous telokin gene. High levels of T-antigen expression were observed in smooth muscle tissues of the digestive, urinary, and reproductive tracts, with lower levels of expression in airway and vascular smooth muscle. Expression was restricted to smooth muscle cells, with no expression detected in any other cell type.

Non-random jumping translocations as a result of SV40 large T-antigen expression in benign human tumor cells.

In an attempt to analyse the cytogenetic effects caused by SV40 large T-antigen expression in cells of human benign tumors we transfected cells of an uterine leiomyoma characterized by a primary reciprocal translocation t(12;14)(q15;q24) and a pleomorphic adenoma of the salivary gland with an inversion inv(12) (q15q24.1) using a construct coding for SV40 large and small T-antigen. The most interesting finding was not a generally destabilized karyotype, but the strictly non-random involvement of two chromosomal breakpoints, i.e. 5p13 and 10q11 in jumping translocations, never described before as a result of SV40 transformation. In addition we were able to show by non-radioactive in situ hybridization that there was no direct relationship between the integration site of the construct and the pre-disposition of 5p13 and 10q11 to somatic recombination. The jumping translocations with consistent breakpoints observed closely resemble the cytogenetic situation seen in a variety of human tumors with specific translocations. Based on the findings described here it is tempting to assume that the expression of SV40 large T-antigen can induce specific karyotypic alterations following an unknown trans-acting mechanism.

Distinct hypomyelinated phenotypes in MBP‐SV40 large T transgenic mice

To study the effect of SV40 large T-antigen expression in myelin-forming cells of both the central and peripheral nervous system, a series of transgenic mice were generated expressing the SV40 large T-antigen under control of the myelin basic protein (MBP) promoter. Two neurologic phenotypes, designated A and B, appeared among individual transgenic founders and their progeny. The A mice developed a severe action tremor at about 10 days of age that progressed into periods of convulsions and early death by three to four weeks of age. In contrast, the B mice exhibited a progressive hindlimb ataxia and had a more normal lifespan. The A mice displayed hypomyelinating lesions in the central nervous system (CNS), whereas the B mice had lesions in either the peripheral nervous system (PNS) alone or in both the PNS and CNS. Immunohistochemical staining of spinal cord sections of a type A mouse showed a substantial depletion in MBP. Moreover, T-antigen-positive cells appeared predominantly in white matter tracts as randomly distributed single cells. Double labeling immunocytochemistry demonstrated that some of these T-antigen-positive cells were positive for oligodendrocyte differentiation markers MBP and O4. Thus, T-antigen expression appeared to coincide with a terminal stage of oligodendrocyte differentiation.