T Antigen Research Articles

WU Polyomavirus in Children, Canada

WU polyomavirus was detected in nasopharyngeal aspirates in 2 (2.5%) of 79 children with respiratory infections (both infected with respiratory syncytial virus) and in 5 (6.4%) of 78 asymptomatic children during the same winter season in Canada. The strains were closely related to Australian and American viruses based on analysis of large T antigen (TAg) and VP2 genes. The pathogenic role of WU virus is still uncertain.

Development of an AT2-deficient proximal tubule cell line for transport studies.

Angiotensin II is a major regulatory peptide for proximal tubule Na(+) reabsorption acting through two distinct receptor subtypes: AT(1) and AT(2). Physiological or pathological roles of AT(2) have been difficult to unravel because angiotensin II can affect Na(+) transport either directly via AT(2) on luminal or peritubular plasma membranes of proximal tubule cells or indirectly via the renal vasculature. Furthermore, separate systemic and intratubular renin-angiotensin systems impart considerable complexity to angiotensin's regulation. A transport-competent, proximal tubule cell model that lacks AT(2) is a potentially useful tool to assess cellular angiotensin II regulation. To this end, AT(2)-receptor-deficient mice were bred with an Immortomouse, which harbors the thermolabile immortalization gene SV40 large-T antigen (Tag), and AT(2)-receptor-deficient [AT(2) (-/-)], Tag heterozygous [Tag (+/-)] F(2) offspring were selected for cell line generation. S1 proximal tubule segments were microdissected, and epithelial cell outgrowth was expanded in culture. Cells that formed confluent, electrically resistive monolayers were selected for cryopreservation, and one isolate was extensively characterized for conductance (2 mS/cm(2)), short-circuit current (Isc; 0.2 microA/cm(2)), and proximal tubule-specific Na3(+) - succinate (DeltaIsc = 0.8 microA/cm(2) at 2 mM succinate) and Na3(+) - phosphate cotransport (DeltaIsc = 3 microA/cm(2) at 1 mM phosphate). Light microscopy showed a uniform, cobblestone-shaped monolayer with prominent cilia and brush borders. AT(2) receptor functionality, as demonstrated by angiotensin II inhibition of ANF-stimulated cGMP synthesis, was absent in AT(2)-deficient cells but prominent in wild-type cells. This transport competent cell line in conjunction with corresponding wild type and AT(1)-deficient lines should help explain angiotensin II signaling relevant to Na(+) transport.

Genetic networks in nonpermissive temperature‐induced cell differentiation of Sertoli TTE3 cells harboring temperature‐sensitive SV40 large T‐antigen

To identify the molecular basis by which nonpermissive temperature (NPT) induces cell differentiation in Sertoli TTE3 cells harboring temperature-sensitive SV40 large T-antigen, we performed global scale microarray and computational gene network analyses. In TTE3 cells, inactivation of the large T-antigen by a NPT at 39 degrees C led to cell differentiation accompanying elevation of transferrin, a marker for differentiation of Sertoli cells, and CDKN1A, a cyclin-dependent kinase inhibitor. Of the 22,690 probe sets analyzed, NPT down-regulated 498 probe sets and up-regulated 432 probe sets by >2.0-fold. Hierarchical clustering analysis showed six gene clusters. In the down-regulated cluster I, a significant genetic network including fibronectin 1 was associated with cellular growth and proliferation. In up-regulated cluster IV, a significant genetic network including CDKN1A was associated with cellular differentiation. The present results provide additional novel insights into the molecular basis of cell differentiation induced by NPT in cells.

Transgenic model of smooth muscle cell cycle reentry: expression pattern of the collageneous matrix

We have previously shown that genetically induced smooth muscle cell (SMC) cycle reentry in transgenic mouse models expressing the SV40 T antigen (TAg) resulted in adaptive arterial remodeling. The present investigation targeted the in vitro expression pattern of the collageneous matrix associated with TAg-induced SMC cycle modulation. SMC cultures were established from the transgenic model expressing temperature-sensitive TAg. This allowed inducible transgene expression at the permissive temperature of 33 degrees C compared with the restrictive temperature of 39.5 degrees C. To distinguish a transgene effect from a temperature effect, SMCs with constitutively expressed TAg were used as controls. Data were obtained using array technology, Northern blotting, reverse transcription polymerase chain reaction, and zymography. TAg-induced SMC cycle reentry resulted in significant down-regulation of matrix metalloproteinase (MMP)-3, whereas MMP-2, -9, and -11 were not influenced. In addition, SMC cycle reentry resulted in significantly increased RNA levels of procollagen alpha2(IV), procollagen alpha2(V), and procollagen alpha1(XI), whereas procollagen alpha1(III) and procollagen alpha1(VIII) were down-regulated. Studies of the RNA expression levels of granulocyte-macrophage colony-stimulating factor revealed an up-regulation of this proinflammatory and matrix-modulating cytokine. This transgenic model provides evidence that TAg-induced cell cycle reentry is associated with a complex modulation of the collageneous matrix. Factors identified in this in vitro study reveal a comprehensive expression pattern of candidates, which might allow the vessel to undergo adaptive arterial remodeling under in vivo conditions. Our results will give rise to further investigations to elaborate on this hypothesis and to improve understanding of the role of such factors in vascular diseases.

Molecular construction of bionanoparticles: chimaeric SIV p17–HIV I p6 nanoparticles with minimal viral protein content

VLPs (virus-like particles) are promising delivery vectors for molecular therapy, since they combine the major advantages of viral vectors with significantly fewer viral vector disadvantages. The present paper describes the molecular construction of chimaeric VLPs based on minimal SIV (simian immunodeficiency virus) and HIV1 components. A chimaeric protein was constructed by fusion of SIV matrix protein (p17) and HIV1 p6 protein, and we demonstrated that the chimaeric proteins assemble as 80 nm nanoparticles containing approximately 7700 chimaeric protein units. Chimaeric VLPs are released from HEK-293T cells (human embryonic kidney cells expressing the large T-antigen of simian virus 40) and are fully encapsulated with lipid membrane. Chimaeric VLPs are produced at 3.7-fold higher levels when compared with SIV p17 VLPs owing to duplication of a PTAP (Pro-Thr-Ala-Pro) domain previously shown as essential for virus particle release. The chimaeric VLPs constructed in the present paper were efficiently pseudotyped with vesicular-stomatitis-virus glycoprotein, as shown by immunoprecipitation assays.

Proapoptotic Function of the Nuclear Crk II Adaptor Protein

Crk II and Crk L have both cytosolic and nuclear functions. While Crk L is a bona fide nuclear signaling protein because of its ability to bind tyrosine-phosphorylated STAT5 and act as a transcriptional coactivator, the function of nuclear Crk II is less well understood. The present study was undertaken to investigate whether Crk II is in the nucleus, how Crk II translocates into the nucleus, whether it possesses a functional NES, and to determine if nuclear Crk II affects cell cycle checkpoints and promotes apoptosis. Toward this goal, we used several independent techniques to show that a significant percentage of the total endogenous Crk II partitions in the nucleus in mammalian cells, where it forms distinct complexes with DOCK180, Wee1, and Abl. We found no evidence that Crk II bound to Crm1 nor that the localization of GFP-Crk II was sensitive to LMB, an inhibitor of Crm1. To better define the significance of nuclear Crk II localization, we generated a GFP-Crk II protein (GFP-Crk-nuc) fused to three tandem nuclear localization signals derived from the SV40 large T-antigen. GFP-Crk-nuc exhibited exclusive nuclear localization, and in contrast to wild-type Crk, GFP-Crk-nuc expressing cells could not be propagated upon selection in G418-containing media, suggesting nuclear accumulation of Crk II caused either growth arrest or apoptosis. When transiently transfected cells were FACS sorted, GFP-expressing cells showed defective cell adhesion on tissue culture surfaces and showed an increased level of apoptosis assessed by pycnotic nuclei, annexin V staining, and PARP cleavage. Although we found that Crk II bound to the cell cycle protein Wee1, expression of GFP-Crk-nuc did not induce a G2/M cell cycle block or cause increased Cdc2 Tyr15 phosphorylation. Finally, upon UV stimulation, we found that endogenous Crk II translocated to the nucleus and potentiated the extent of UV-inducible apoptosis after 4 h. These data suggest that nuclear compartmentalization of Crk II antagonizes its cytoskeletal functions and assign a proapoptotic role to the nuclear pool of Crk II.

Functional Characterization of BK Virus-Specific CD4+T Cells with Cytotoxic Potential in Seropositive Adults

BK polyomavirus (BKV) reactivation is associated with a failure of T cell immunity in kidney transplant patients, and may lead to BKV-associated nephropathy (BKVN) and loss of the allograft. BKV reactivation in hematopoietic stem cell transplant recipients is associated with hemorrhagic cystitis. We have investigated T cell responses to overlapping peptide mixtures corresponding to the whole BKV major T antigen (TAg) and major capsid protein (VP1) in peripheral blood mononuclear cell samples from a cohort of healthy BKV-seropositive subjects. The majority of these individuals possessed populations of both CD8(+) and CD4(+) T cells specific for these BKV antigens. After expansion in culture, the majority of the BKV-specific CD4(+) T cells, in addition to expressing CD40L (CD154), secreted both interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, contained both granzyme A and granzyme B, and degranulated/mobilized CD107 in response to antigen-specific stimulation. These T cells thus represent potentially functional BKV-specific cytotoxic CD4(+) T lymphocytes. Secretion of both TNF-alpha and IFN-gamma by CD154(+)CD4(+) T cells on BKV-specific stimulation was associated with higher levels of granzyme B and a higher proportion of degranulating cells compared with CD154(+)CD4(+) T cells producing only IFN-gamma or neither cytokine. These healthy subjects also harbored populations of functional CD8(+) T cells specific for one or more of three newly defined HLA-A 02-restricted cytotoxic T lymphocyte epitopes within the BKV TAg as well as two HLA-A 02-restricted epitopes within the BKV VP1 we have previously described. The BKV-specific CD4(+) T cells characterized in this study may play a part in maintaining persistent memory T cell responses to the virus and thus contribute to the immune control of BKV in healthy individuals.

Fixed Bed Reactor for Solid-Phase Surface Derivatization of Superparamagnetic Nanoparticles

The functionalization of nanoparticles is conditio sine qua non in studies of specific interaction with a biological target. Often, their biological functionality is achieved by covalent binding of bioactive molecules on a preexisting single surface coating. The yield and quality of the resulting coated and functionalized superparamagnetic iron oxide nanoparticles (SPIONs) can be significantly improved and reaction times reduced by using solid-phase synthesis strategies. In this study, a fixed bed reactor with a quadrupole repulsive arrangement of permanent magnets was assayed for SPION surface derivatization. The magnet array around the fixed bed reactor creates very high magnetic field gradients that enables the immobilization of SPIONs with a diameter as low as 9 nm. The functionalization on the surface of immobilized 25 nm 3-(aminopropyl)trimethoxysilane-coated SPIONs (APS-SPIONs) was performed using fluorescein-isothiocyanate directly, and by the SV40 large T-antigen nuclear localization signal peptide (PKKKRKVGC) conjugated to acryloylpoly(ethylene glycol)-N-hydroxysuccinimide, where the PEG reagent is conjugated first to create a functionalized nanoparticle and the peptide is added to the acryloyl group. We show that the yield of reactant grafted on the surface of the APS-coated SPIONs was higher in solid-phase within the fixed bed reactor compared to conventional liquid-phase chemistry. In summary, the functionalization of SPIONs using a magnetically fixed bed reactor was superior to the liquid-phase reaction in terms of the yield, reaction times required for derivatization, size distribution, and scalability.

Regulation of Human Lung Adenocarcinoma Cell Migration and Invasion by Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is expressed and secreted in response to mitogens and integrin-dependent cell adhesion. Once released, autocrine MIF promotes the activation of RhoA GTPase leading to cell cycle progression in rodent fibroblasts. We now report that small interfering RNA-mediated knockdown of MIF and MIF small molecule antagonism results in a greater than 90% loss of both the migratory and invasive potential of human lung adenocarcinoma cells. Correlating with these phenotypes is a substantial reduction in steady state as well as serum-induced effector binding activity of the Rho GTPase family member, Rac1, in MIF-deficient cells. Conversely, MIF overexpression by adenovirus in human lung adenocarcinoma cells induces a dramatic enhancement of cell migration, and co-expression of a dominant interfering mutant of Rac1 (Rac1(N17)) completely abrogates this effect. Finally, our results indicate that MIF depletion results in defective partitioning of Rac1 to caveolin-containing membrane microdomains, raising the possibility that MIF promotes Rac1 activity and subsequent tumor cell motility through lipid raft stabilization.

PD2-1-8: Promotion of Squamous Differentiation and Tumorigenesis in Mouse Lung Expressing Human Keratin 14

Squamous cell carcinoma accounts for 20% of all human lung cancers, and is strongly linked to cigarette smoking. Historically it has been the first lung cancer associated with distinct premalignant intraepithelial changes that evolve through basal cell hyperplasia, squamous metaplasia and increasing dysplasia to carcinoma in situ (CIS) and invasive carcinoma. However, it has been difficult to mimic this sequence in genetically engineered mice (GEM). Because lung squamous cell carcinoma, its progenitor cells (basal cells) and precursor lesions express high levels of human keratin 14 (hK14), we hypothesized that K14 is critical for squamous differentiation and carcinogenesis in the pulmonary epithelium that normally lacks squamous differentiation. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous features and K14 immunoreactivity, hK14 gene was constitutively expressed in mouse airways using Clara cell specific 10kDa protein (CC10) promoter. In order to investigate the effect of hK14 on chemically-induced lung tumorigenesis, mice were given tobacco-specific nitrosamine NNK (4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol), a major systemic carcinogen in cigarettes by intraperitoneal injections (100mg/kg body weight, 3 times). To further explore the role of hK14 in carcinogenesis, CC10-hK14 mice were crossed with the previously established CC10-SV40 TAg mice, a model for aggressive lung carcinomas. CC10-hK14 transgenic (TG) mice developed normally except at the age of 1 year they were 20% lighter (p= 0.014) than their wild type (WT) littermates. Light and electron microscopic examination of TG mice failed to show any evidence for overt squamous features in the airways such as keratinization, intercellular bridges or tonofilaments. However, by RT-PCR, quantitative-RT-PCR and immunohistochemistry (IHC), TG mouse lungs were positive for hK14, while wild type (WT) lungs did not show any expression. Notably, we also detected variable levels of the squamous differentiation markers involucrin, loricrin, small proline rich proteins and transglutaminase 1, an enzyme that mediates cornified cell envelope formation, a hallmark of squamous differentiation in TG lungs, while WT lungs were consistently negative. By 48 weeks the lungs of NNK-exposed TG mice almost tripled in weight (p=0.011), contained larger tumors and revealed a three-fold increase (p=0.002) in tumor multiplicity compared to the lungs of their WT littermates. No tumors were found in saline-exposed control mice. The bi-transgenic CC10-hK14-TAg mice became moribund or died of massive lung tumors on average at 18 weeks of age. Their lungs typically increased five-fold in weight and contained hK14 by RT-PCR, while the lungs of CC10-SV40 Tag mice had smaller tumors that were negative for hK14. While the tumors in TG mice were adenomas or adenocarcinomas, they expressed involucrin by IHC. Constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, which normally lacks K14 containing cells. Furthermore, it markedly enhances the progression of both chemically- and SV40 TAg- induced tumors. Our study is significant because it provides a novel in vivo model for a common premalignant pulmonary lesion in humans, characterized by hK14 expression and squamous differentiation. It will also be a potentially useful pre-clinical tool for chemoprevention research.

A novel role of Rac1 GTPase in JCV T-antigen-mediated β-catenin stabilization

Wnt signaling follows canonical and non-canonical pathways to regulate a variety of processes during cellular homeostasis and development. The large T-antigen (T-Ag) of the human neurotropic JC virus, has been shown to modulate the Wnt-signaling pathway via interaction with beta-catenin, one of the most important components of the canonical Wnt pathway. Here, we have identified an alternative non-canonical pathway that allows T-Ag to recruit Rac1 for stabilizing beta-catenin by inhibiting its ubiquitin-dependent proteasomal degradation. We demonstrate that inhibition of Rac1 by its dominant negative mutant, RacN17, abrogates T-Ag-mediated stabilization of beta-catenin yet exhibits no impact on the transcriptional activity of beta-catenin. Results from immunocytochemistry revealed that together with T-Ag, a pool of beta-catenin appears at the cell surface, particularly at the membrane ruffles where active Rac1 is positioned. Interestingly, cooperativity between T-Ag and beta-catenin leads to activation of Rac1, which in turn, stimulates its association with beta-catenin. These observations unravel the interplay between beta-catenin and Rac1 that is initiated by T-Ag and results in stabilization of beta-catenin and its presence in cell membrane ruffles.

The sweet nature of cardioprotection

glucose, glutamine, and glucosamine are metabolites that can alter cardioprotection ([1][1], [3][2], [8][3], [9][4], [13][5]–[15][6]). Numerous studies suggest that one molecular mechanism by which these metabolites protect cells is though their conversion to uridine diphosphate- N -

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.

On the mechanism of cerebral accumulation of cholestanol in patients with cerebrotendinous xanthomatosis

The most serious consequence of sterol 27-hydroxylase deficiency in humans [cerebrotendinous xanthomatosis (CTX)] is the development of cholestanol-containing brain xanthomas. The cholestanol in the brain may be derived from the circulation or from 7alpha-hydroxylated intermediates in bile acid synthesis, present at 50- to 250-fold increased levels in plasma. Here, we demonstrate a transfer of 7alpha-hydroxy-4-cholesten-3-one across cultured porcine brain endothelial cells (a model for the blood-brain barrier) that is approximately 100-fold more efficient than the transfer of cholestanol. Furthermore, there was an efficient conversion of 7alpha-hydroxy-4-cholesten-3-one to cholestanol in cultured neuronal and glial cells as well as in monocyte-derived macrophages of human origin. It is concluded that the continuous intracellular production of cholestanol from a bile acid precursor capable of rapidly passing biomembranes, including the blood-brain barrier, is likely to be of major importance for the accumulation of cholestanol in patients with CTX. Such a mechanism also fits well with the observation that treatment with chenodeoxycholic acid, which normalizes the level of the bile acid precursor, results in a reduction of cholestanol-containing xanthomas even in the brain.

Suppressive effects of antiandrogens, finasteride and flutamide on development of prostatic lesions in a transgenic rat model

Transgenic (TG) rats bearing a probasin promoter/simian virus 40 T antigen (SV40 Tag) construct were treated with antiandrogens to examine their ability to suppress prostate carcinogenesis. Finasteride and flutamide were administered to 10-week-old TG rats five times a week for 2, 5 and 7 weeks. Antiandrogen-treated prostates exhibited atrophic glandular structures with almost no expression of SV40 Tag and only weak signals for androgen receptors. Furthermore, quantitative data for ventral prostate adenocarcinomas showed significant decrease with antiandrogen treatment. Both finasteride and flutamide had the ability to suppress SV40 Tag-driven carcinogenesis through their different antiandrogenic mechanisms, suggesting that this TG model is suitable for exploring the potential of agents to inhibit prostate cancer development.

Interleukin-4 impairs granzyme-mediated cytotoxicity of Simian virus 40 large tumor antigen-specific CTL in BALB/c mice.

In this report we analyzed the impact of interleukin-4 (IL-4) on tumor-associated simian virus 40 (SV40) large T-antigen (TAg)-specific CD8+ cytotoxic T cells during rejection of syngeneic SV40 transformed mKSA tumor cells in BALB/c mice. Strikingly, challenge of naïve mice with low doses of mKSA tumor cells revealed a CD8+ T cell-dependent prolonged survival time of naïve IL-4-/- mice. In mice immunized with SV40 TAg we observed in IL-4-/- mice, or in wild type mice treated with neutralizing anti-IL-4 monoclonal antibody, a strongly enhanced TAg-specific cytotoxicity of tumor associated CD8+ T cells. The enhanced cytotoxicity in IL-4-/- mice was accompanied by a significant increase in the fraction of CD8+ tumor associated T-cells expressing the cytotoxic effector molecules granzyme A and B and in granzyme B-specific enzymatic activity. The data suggest that endogenous IL-4 can suppress the generation of CD8+ CTL expressing cytotoxic effector molecules especially when the antigen induces only a very weak CTL response.

Diversity of escape variant mutations in Simian virus 40 large tumor antigen (SV40 Tag) epitopes selected by cytotoxic T lymphocyte (CTL) clones

To better understand the relationship between epitope variation and tumor escape from immune surveillance, SV40 T antigen-transformed B6/K-0 cells were subjected to selection with individual CTL clones specific for the SV40 T antigen H-2D b-restricted epitopes I or V. CTL-resistant populations were isolated from a majority of the selection cultures and substituted epitope sequences were identified within most of the resistant populations. Tag sequences deleted of all or portions of the selection-targeted epitope were identified, but in lower numbers compared to epitope sequences bearing single residue substitutions. Relatively few flanking residue substitutions were identified, and only in epitope I-targeted selections. The diversity (numbers and epitope residue locations) of substituted epitope residue positions varied between selections. These findings suggest that the scope of spontaneously occurring mutations that could allow for escape from individual CD8+ T cell clones is large.

Reversible transfection of human melanocytes mediated by Cre/loxP site‐specific recombination system and SV40 large T antigen

To study the reversible transfection of human melanocytes mediated by simian virus 40 large T antigen (SV40LTAg) and Cre/loxP site-specific recombination system. The reconstructed SV40LTAg-EGFP-neo-loxP vector was transfected into primary cultured human melanocytes with Sofast(TM) transfection reagent and the positive cells were selected using G418. After expanding culture of these positive cell clones, the expression of SV40LTAg was detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent method. After that, these positive cells were infected by virus supernatant of Cre-ER(T2) retrovirus vector and Cre recombinase was induced to act by tamoxifen. On the 6th and 10th day after Cre recombinase acting, the expression of SV40LTAg was detected using the same methods as above, and cell tumorigenicity was studied using soft agar assay, athymic mouse study and karyotype analysis. On 10th day after tamoxifen treatment, cell biological characters were identified with immunofluorescent staining and transmission electron microscopy. Then these cells were transplanted into vitiligo animal model to observe their melanogenesis ability in vivo. The genome DNA and total RNA were isolated from the positive cells transfected by SV40LTAg (designated as MCT) and specific 288 bp fragment was amplificated using PCR and RT-PCR methods. The results of immunofluorescence confirmed the expression of SV40LTAg in cell nucleus. On the 6th day after tamoxifen treatment in infected cells by Cre-ER(T2) retrovirus vector (designated as MCT-Cre), there could be detected SV40LTAg expression, but on 10th day, there could not be detected SV40LTAg expression in cells. These results showed that the excised efficiency of Cre recombinase increased along with time prolongation, and would obtain complete recombination efficiency. The identification of MCT-Cre cell biological characters showed that these cells had normal parent-cell-like cell phenotype and no tumorigenicity in vitro. The pigmentation started in 4 weeks and formed black macula in 3 months after grafting. The pathological results showed that there had been significant melanocytes and melanin accumulation in epidermis and some hair follicle in transplanted area, which confirmed that MCT-Cre had melanogenesis function in vivo. Human melanocytes could be mediated by reversible transfection by SV40LTAg and Cre/loxP site-specific recombination system, which had stable parent-cell-like phenotypic characters and no tumorigenicity in vitro; moreover, these cells still had melanogenesis function in vivo.

Conditional Neuronal Simian Virus 40 T Antigen Expression Induces Alzheimer-Like Tau and Amyloid Pathology in Mice

A large body of evidence has shown the activation of a cohort of cell cycle regulators and the duplication of DNA in degenerating neurons of Alzheimer's disease (AD) brain. Activation of these regulators and duplication of chromosomes precede neurodegeneration and formation of neurofibrillary tangles (NFTs), one of the diagnostic lesions of AD. These findings, in combination with evidence for cell cycle regulation of amyloid precursor protein and tau, has led to the hypothesis that reentry into the cell cycle underlies AD pathogenesis. To test this hypothesis directly, we have created transgenic mice with forced cell cycle activation in postmitotic neurons via conditional expression of the simian virus 40 large T antigen (TAg) oncogene. We show that TAg mice recapitulate the cell cycle changes seen in AD and display a neurodegenerative phenotype accompanied by tau pathology and NFT-like profiles. Moreover, plaque-like amyloid deposits, similar to those seen in AD, are also observed in the brains of TAg mice. These data provide support for an essential role of ectopic cell cycle activation in the generation of the characteristic pathological hallmarks of AD. Furthermore, our TAg mice are the first model to develop NFTs and amyloid pathology simultaneously and in the absence of any human transgenes. These mice will be useful for further defining the nongenetic mechanisms in AD pathogenesis and for the development of cell cycle-based therapies for AD.

Repression of intestinal drug metabolizing enzymes by the SV40 large T antigen

Toxic compounds such as carcinogens are removed from the body by the action of a series of detoxifying enzymes and transporters expressed in the liver and the small intestine. We have found that intestinal epithelial cells expressing the SV40 large T antigen (TAg) contain significantly lower levels of mRNAs, encoding several drug metabolizing/detoxifying enzymes and transporters compared to their non-transgenic littermates. In addition, TAg blocks the induction of these mRNAs by xenobiotics. The repression depends on an intact LXCXE motif in TAg, suggesting that inactivation of the retinoblastoma (Rb) family of tumor suppressors plays a role in the process. These results imply that a functional Rb pathway in the intestine is necessary for the expression of the detoxification system used to clear carcinogens, and suggest that loss of this tumor suppressor might alter susceptibility to chemical injury. In addition, the effect of TAg on the detoxification pathway appears to be tissue-specific, as its ectopic expression in the liver failed to suppress the P450 enzymes. The TAg-mediated suppression of drug metabolizing/detoxifying enzymes may have broad implications in the metabolism and mechanism of action of both carcinogens and prescription drugs.