Mutant Of SV40 Research Articles

Functions and evolution of FAM111 serine proteases.

Proteolysis plays fundamental and regulatory roles in diverse cellular processes. The serine protease FAM111A (FAM111 trypsin-like peptidase A) emerged recently as a protease involved in two seemingly distinct processes: DNA replication and antiviral defense. FAM111A localizes to nascent DNA and plays a role at the DNA replication fork. At the fork, FAM111A is hypothesized to promote DNA replication at DNA-protein crosslinks (DPCs) and protein obstacles. On the other hand, FAM111A has also been identified as a host restriction factor for mutants of SV40 and orthopoxviruses. FAM111A also has a paralog, FAM111B, a serine protease with unknown cellular functions. Furthermore, heterozygous missense mutations in FAM111A and FAM111B cause distinct genetic disorders. In this review, we discuss possible models that could explain how FAM111A can function as a protease in both DNA replication and antiviral defense. We also review the consequences of FAM111A and FAM111B mutations and explore possible mechanisms underlying the diseases. Additionally, we propose a possible explanation for what drove the evolution of FAM111 proteins and discuss why some species have two FAM111 proteases. Altogether, studies of FAM111 proteases in DNA repair, antiviral defense, and genetic diseases will help us elucidate their functions and the regulatory mechanisms.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Chromosomal instability induced by increased BIRC5/Survivin levels affects tumorigenicity of glioma cells

BackgroundSurvivin, belonging to the inhibitor of apoptosis (IAP) gene family, is abundantly expressed in tumors. It has been hypothesized that Survivin facilitates carcinogenesis by inhibition of apoptosis resulting in improved survival of tumorigenic progeny. Additionally, Survivin plays an essential role during mitosis. Together with its molecular partners Aurora B, Borealin and inner centromere protein it secures bipolar chromosome segregation. However, whether increased Survivin levels contribute to progression of tumors by inducing chromosomal instability remains unclear.MethodsWe overexpressed Survivin in U251-MG, SVGp12, U87-MG, HCT116 and p53-deficient U87-MGshp53 and HCT116p53−/− cells. The resulting phenotype was investigated by FACS-assisted cell cycle analysis, Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and in a U251-MG xenograft model using immune-deficient mice.ResultsOverexpression of Survivin affected cells with knockdown of p53, cells harboring mutant p53 and SV40 large T antigen, respectively, resulting in the increase of cell fractions harboring 4n and >4n DNA contents. Increased γH2AX levels, indicative of DNA damage were monitored in all Survivin-transduced cell lines, but only in p53 wild type cells this was accompanied by an attenuated S-phase entry and activation of p21waf/cip. Overexpression of Survivin caused a DNA damage response characterized by increased appearance pDNA-PKcs foci in cell nuclei and elevated levels of pATM S1981 and pCHK2 T68. Additionally, evolving structural chromosomal aberrations in U251-MG cells transduced with Survivin indicated a DNA-repair by non-homologous end joining recombination. Subcutaneous transplantation of U251-MG cells overexpressing Survivin and mycN instead of mycN oncogene alone generated tumors with shortened latency and decreased apoptosis. Subsequent SKY-analysis of Survivin/mycN-tumors revealed an increase in structural chromosomal aberrations in cells when compared to mycN-tumors.ConclusionsOur data suggest that increased Survivin levels promote adaptive evolution of tumors through combining induction of genetic heterogeneity with inhibition of apoptosis.

Viral MicroRNA Effects on Pathogenesis of Polyomavirus SV40 Infections in Syrian Golden Hamsters

Effects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×107 plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.

Abstract C269: Targeting casein kinase 1 delta and epsilon (CK1δ/ϵ) in breast cancer therapeutics.

Abstract The Caesin Kinase 1 (CK1) family consists of 7 ubiquitously expressed serine/threonine kinases that play diverse roles in a number of cellular processes. Interestingly, CK1δ and ϵ are highly expressed in some cancer types where they have been shown to promote cell growth, survival, and tumorigenicity in vivo. For example, exogenous expression of kinase dead mutants of CK1δ block SV40 induced cell transformation as well as mammary carcinogenesis in xenograft models. Furthermore, the effect of CK1δ/ϵ shRNAs on breast cancer cell growth has been shown to correlate with high levels of nuclear β-catenin, a molecular phenotype known to be associated with the invasive TNBC subtype and poor clinical outcome. Therefore, we investigated whether inhibition of CK1 δ/ϵ could represent a viable strategy for the treatment of TNBC, for which there are currently no targeted therapies. Here we report the development of novel small molecule, ATP competitive inhibitors of CK1δ/ϵ, that are highly selective for CK1δ and ϵ and have low nanomolar biochemical and anti-breast cancer cell potency. In particular, our top leads (such as SR-3029) show selectivity toward cancer cells over non-tumorigenic cells and screening of the NCI-60 panel of tumor cell lines suggest potency against a number of additional malignancies, including GBM and melanoma. We show, using genetic tools, that SR-3029 is on-target and functions to promote apoptotic cell death in TNBC cells through negative regulation of β-catenin. Furthermore, SR-3029 is highly efficacious in vivo, promoting regression of human MDA-MB-231 tumors established in the mouse mammary fat pad. Our studies demonstrate pre-clinical proof of concept that targeting of CK1δ/ϵ is an effective anti-TNBC strategy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C269. Citation Format: Laura H. Rosenberg, Weimin Chen, Shima Nakanishi, Mathieu Bibian, Michael D. Cameron, John L. Cleveland, William R. Roush, Derek R. Duckett. Targeting casein kinase 1 delta and epsilon (CK1δ/ϵ) in breast cancer therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C269.

Clonal Immortalized Human Glial Cell Lines Support Varying Levels of JC Virus Infection due to Differences in Cellular Gene Expression

JC virus (JCV) is a ubiquitous human polyomavirus that causes the demyelinating disease Progressive Multifocal Leukoencephalopathy (PML). JCV replicates in limited cell types in culture, predominantly in human glial cells. Following introduction of a replication defective SV40 mutant that expressed large T protein into a heterogeneous culture of human fetal brain cells, multiple phenotypes became immortalized (SVG cells). A subset of SVG cells could support JCV replication. In the current study, clonal cell lines were selected from the original SVG cell culture. The 5F4 clone showed low levels of viral growth. The 10B1 clone was highly permissive for JCV DNA replication and gene expression and supported persistent and stable JCV infection over months in culture. Microarray analysis revealed that viral infection did not significantly change gene expression in these cells. More resistant 5F4 cells expressed high levels of transcription factors known to inhibit JCV transcription. Interestingly, 5F4 cells expressed high levels of RNA of markers of radial glia and 10B1 cells had high expression of markers of immature glial cells and activation of transcription regulators important for stem/progenitor cell self-renewal. These SVG-derived clonal cell lines provide a biologically relevant model to investigate cell type differences in JCV host range and pathogenesis, as well as neural development. Several transcription regulators were identified which may be targets for therapeutic modulation of expression to abrogate JCV replication in PML patients. Additionally, these clonal cell lines can provide a consistent culture platform for testing therapies against JCV infection of the central nervous system.

Infection by agnoprotein-negative mutants of polyomavirus JC and SV40 results in the release of virions that are mostly deficient in DNA content

BackgroundHuman polyomavirus JC (JCV) is the etiologic agent of a brain disease, known as progressive multifocal leukoencephalopathy (PML). The JCV genome encodes a small multifunctional phospho-protein, agnoprotein, from the late coding region of the virus, whose regulatory functions in viral replication cycle remain elusive. In this work, the functional role of JCV and SV40 agnoproteins in virion release was investigated using a point mutant (Pt) of each virus, where the ATG codon of agnoprotein was mutated to abrogate its expression.ResultsAnalysis of both viral protein expression and replication using Pt mutant of each virus revealed that both processes were substantially down-regulated in the absence of agnoprotein compared to wild-type (WT) virus. Complementation studies in cells, which are constitutively expressing JCV agnoprotein and transfected with the JCV Pt mutant genome, showed an elevation in the level of viral DNA replication near to that observed for WT. Constitutive expression of large T antigen was found to be not sufficient to compensate the loss of agnoprotein for efficient replication of neither JCV nor SV40 in vivo. Examination of the viral release process for both JCV and SV40 Pt mutants showed that viral particles are efficiently released from the infected cells in the absence of agnoprotein but were found to be mostly deficient in viral DNA content.ConclusionsThe results of this study provide evidence that agnoprotein plays an important role in the polyomavirus JC and SV40 life cycle. Infection by agnoprotein-negative mutants of both viruses results in the release of virions that are mostly deficient in DNA content.

Calcium Bridge Triggers Capsid Disassembly in the Cell Entry Process of Simian Virus 40

The calcium bridge between the pentamers of polyoma viruses maintains capsid metastability. It has been shown that viral infection is profoundly inhibited by the substitution of lysine for glutamate in one calcium-binding residue of the SV40 capsid protein, VP1. However, it is unclear how the calcium bridge affects SV40 infectivity. In this in vitro study, we analyzed the influence of host cell components on SV40 capsid stability. We used an SV40 mutant capsid (E330K) in which lysine had been substituted for glutamate 330 in protein VP1. The mutant capsid retained the ability to interact with the SV40 cellular receptor GM1, and the internalized mutant capsid accumulated in caveolin-1-mediated endocytic vesicles and was then translocated to the endoplasmic reticulum (ER) region. However, when placed in ER-rich microsome, the mutant capsid retained its spherical structure in contrast to the wild type, which disassembled. Structural analysis of the mutant capsid with cryo-electron microscopy and image reconstruction revealed altered pentamer coordination, possibly as a result of electrostatic interaction, although its overall structure resembled that of the wild type. These results indicate that the calcium ion serves as a trigger at the pentamer interface, which switches on capsid disassembly, and that the failure of the E330K mutant capsid to disassemble is attributable to an inadequate triggering system. Our data also indicate that calcium depletion-induced SV40 capsid disassembly may occur in the ER region and that this is essential for successful SV40 infection.

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

A Functional Genetic Screen Identifies TFE3 as a Gene That Confers Resistance to the Anti-proliferative Effects of the Retinoblastoma Protein and Transforming Growth Factor-β

Reorganization of RNA polymerase II on the SV40 genome occurs coordinately with the early to late transcriptional switch

The pattern of organization of RNA polymerase II (RNAPII) in wild-type and mutant cs1085 SV40 chromosomes isolated between 30 min and 48 h post-infection was determined using a combination of chromatin immunoprecipitation (ChIP) techniques. During the course of a wild-type infection, we observed a slow but significant decline in the relative occupancy of RNAPII at the early region and a corresponding increase in occupation in the late region. In the promoter, occupancy began high, decreased to a minimum at 8 h post-infection, and then increased to a high level by 48 h post-infection. In the mutant cs1085, which does not down-regulate early transcription, we observed high occupancy of the early region and the promoter throughout the infection. The changing organization of RNAPII on the wild-type SV40 but not the mutant cs1085 genome appears to be a result of the switch from early to late transcription.

Hepatocyte CYP2E1 Overexpression and Steatohepatitis Lead to Impaired Hepatic Insulin Signaling

Insulin resistance and increased cytochrome P450 2E1 (CYP2E1) expression are both associated with and mechanistically implicated in the development of nonalcoholic fatty liver disease. Although currently viewed as distinct factors, insulin resistance and CYP2E1 expression may be interrelated through the ability of CYP2E1-induced oxidant stress to impair hepatic insulin signaling. To test this possibility, the effects of in vitro and in vivo CYP2E1 overexpression on hepatocyte insulin signaling were examined. CYP2E1 overexpression in a hepatocyte cell line decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 in response to insulin. CYP2E1 overexpression was also associated with increased inhibitory serine 307 and 636/639 IRS-1 phosphorylation. In parallel, the effects of insulin on Akt activation, glycogen synthase kinase 3, and FoxO1a phosphorylation, and glucose secretion were all significantly decreased in CYP2E1 overexpressing cells. This inhibition of insulin signaling by CYP2E1 overexpression was partially c-Jun N-terminal kinase dependent. In the methionine- and choline-deficient diet mouse model of steatohepatitis with CYP2E1 overexpression, insulin-induced IRS-1, IRS-2, and Akt phosphorylation were similarly decreased. These findings indicate that increased hepatocyte CYP2E1 expression and the presence of steatohepatitis result in the down-regulation of insulin signaling, potentially contributing to the insulin resistance associated with nonalcoholic fatty liver disease.

Temperature-Sensitive Sv40-Immortalized Rat Middle Ear Epithelial Cells

The proliferation and differentiation of middle ear epithelial cells are essential in both normal and diseased middle ears. The normal situation involves physiologic growth and renewal of the epithelium, and the diseased situation involves pathological changes of the epithelium such as mucous cell metaplasia and ciliated cell proliferation in otitis media. In this study, we used a temperature-sensitive large T antigen (the SV40 mutant) to transduce and immortalize the primary culture of middle ear epithelial cells. SV40-immortalized middle ear epithelial cells have been cultured for more than 50 passages and are stable morphologically. Their nonimmortalized parent cells died at the second passage. Immortalized middle ear epithelial cells carrying the SV40 mutant show a monolayer, cobblestonelike morphology. The cell line expresses characteristic middle ear mucosal molecules such as mucins, keratins, and collagens. It also responds to temperature changes; namely, cells proliferate at 33 degrees C, when the SV40 antigen is active, and differentiate at 39 degrees C, when the SV40 antigen is inactive. Therefore, we conclude that a temperature-sensitive middle ear epithelial cell line has successfully been established.

Conditional transformation of mouse pancreatic epithelial cells: an in vitro model for analysis of genetic events in pancreatocarcinogenesis

Pancreatic ductal adenocarcinomas arise through the accumulation of certain genetic alterations including ras, p16, p53, and DPC4. We found that activation of ras and inactivation of p53 could cooperatively induce in vitro tumorigenicity in conditionally immortalized pancreatic epithelial (IMPE) cells. IMPE cells were established from transgenic mice bearing a temperature-sensitive mutant SV40 Large T (LT) antigen. IMPE cells grew continuously under permissive conditions (33°C with interferon-γ), but rapidly suffered growth arrest under non-permissive conditions (39°C without interferon-γ). The cells showed strong expression of E-cadherin and β-catenin as epithelial markers, and cytokeratin 19, a specific ductal cell marker. Cell proliferation under permissive conditions was associated with down-regulation of p21 expression through inactivation of p53 after overexpression of LT antigen. Intriguingly, the shift from the permissive to non-permissive culture conditions caused G2/M arrest of IMPE cells. Although the cells did not form colonies when cultured in soft agar without activation of ras, cells with ras activation via an adenovirus vector formed colonies under permissive conditions. These findings suggest that activation of ras and inactivation of p53 can cooperatively induce anchorage-independent growth of IMPE cells. This cell line might be useful for studying the processes involved in pancreatocarcinogenesis.

Conditional transformation of mouse pancreatic epithelial cells: an in vitro model for analysis of genetic events in pancreatocarcinogenesis

Pancreatic ductal adenocarcinomas arise through the accumulation of certain genetic alterations including ras, p16, p53, and DPC4. We found that activation of ras and inactivation of p53 could cooperatively induce in vitro tumorigenicity in conditionally immortalized pancreatic epithelial (IMPE) cells. IMPE cells were established from transgenic mice bearing a temperature-sensitive mutant SV40 Large T (LT) antigen. IMPE cells grew continuously under permissive conditions (33 °C with interferon-γ), but rapidly suffered growth arrest under non-permissive conditions (39 °C without interferon-γ). The cells showed strong expression of E-cadherin and β-catenin as epithelial markers, and cytokeratin 19, a specific ductal cell marker. Cell proliferation under permissive conditions was associated with down-regulation of p21 expression through inactivation of p53 after overexpression of LT antigen. Intriguingly, the shift from the permissive to non-permissive culture conditions caused G2/M arrest of IMPE cells. Although the cells did not form colonies when cultured in soft agar without activation of ras, cells with ras activation via an adenovirus vector formed colonies under permissive conditions. These findings suggest that activation of ras and inactivation of p53 can cooperatively induce anchorage-independent growth of IMPE cells. This cell line might be useful for studying the processes involved in pancreatocarcinogenesis.

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.

Expression Analysis of Several Lymphoma-specific Genes in Human Fibroblasts Immortalized with SV40

Subtraction hybridization was earlier used to obtain cDNA clones corresponding to human genes upregulated in HIV-associated centroblast lymphoma (CL) as compared with HIV-associated immunoblast lymphoma (IL). With inverse subtraction hybridization, clones were isolated that correspond to genes upregulated in IL compared with CL. In addition to cDNAs characterized earlier, the resulting clones contained several (seven CL-specific and three IL-specific) sequences of unknown function. To identify the lymphoma-specific genes that are overexpressed in early carcinogenesis, Northern blotting was used to assess the level of gene transcription in two human fibroblast lines and in their derivatives immortalized with either a temperature-sensitive mutant of SV-40 or with pSV3neo carrying the SV-40 А gene, considering the latter as a model of early cell malignant transformation. Increased expression in at least one immortalized line compared with normal fibroblasts was observed for set, a-myb, ND1, ND2, ND4 (NADH dehydrogenase subunits 1, 2, and 4), COX2, COX3 (cytochrome c oxidase subunits 2 and 3), KIAA0129, and the gene corresponding to hss2-1-7-10 cDNA. High expression of these genes was assumed to be associated not only with lymphomogenesis, but also with early transformation (immortalization) of other, nonlymphoid cells. Expression of the calpain gene and the gene corresponding to hss2-2-9-5 cDNA proved to be lower in immortalized than in normal fibroblasts. This was indicative of an alternative mechanism of fibroblast transformation or of different processes regulating the expression of these genes in early and late carcinogenesis.

The involvement of apoptotic regulators during in vitro decidualization.

The uterus responds to an implanting blastocyst by undergoing extensive tIssue modification leading to decidualization. This modification includes differentiation and apoptosis of epithelial as well as stromal cell compartments. It is generally accepted that the decidual cell regression pattern is similar to the pattern of initial differentiation, suggesting that decidual cell death is the end point of timed differentiation. However, the molecular mechanisms controlling these events are not understood clearly. Therefore, we aimed to investigate the involvement of apoptotic factors using an in vitro cell culture system. In order to assess the role of apoptotic factors during decidualization, we used a decidual cell line (GG-AD) that had been transformed with a temperature-sensitive SV-40 mutant. At the non-permissive temperature (39 degrees C), these cells showed the characteristics of differentiated decidual cells. They dedifferentiated into stromal cells when the temperature was shifted back to 33 degrees C. We performed Northern blot analysis for bax, bcl-x(L) and bcl-2 at both temperatures. The onset of apoptosis was examined by Annexin V staining. The expression of p53 protein was also determined by Western blot. We found an increase in the expression of bax when GG-AD cells were grown at 39 degrees C. We also showed apoptosis with Annexin V staining at 39 degrees C. The p53 protein expression was also similar to that of the animal models, suggesting that the programmed cell death of the decidual cells occurred in a p53-independent manner. These data indicate that a parallelism exists between the increased expression of pro-apoptotic genes and decidual cell death, similar to animal models. Therefore, an in vitro model of GG-AD cells can be used to assess directly the relationship between apoptotic regulators and decidualization and could be used to study the mechanism of decidual cell regression.

The response of adult rat sertoli cells, immortalized by a temperature-sensitive mutant of SV40, to 1,2-dinitrobenzene, 1,3-dinitrobenzene, 2,4-dinitrotoluene, 3,4-dinitrotoluene, and cadmium.

In this study we test the hypothesis that immortalized adult rat Sertoli cells respond to known testicular toxins in a similar manner to Sertoli cells tested in vivo and in primary culture. This cell line was developed by immortalizing adult rat Sertoli cells with the temperature-sensitive mutant of SV40, ts255, such that the cells proliferate at the permissive temperature of 33 degrees C but express differentiated characteristics at the nonpermissive temperature of 40 degrees C. Confluent monolayers, grown at 33 degrees C or 40 degrees C, were exposed to a range of concentrations of dinitrobenzene (DNB) or dinitrotoluene (DNT) isomers or to cadmium chloride. Cellular response was assessed by neutral-red cell viability assay and ultrastructural changes. Cells grown at 40 degrees C were sensitive to lower concentrations of each toxicant than were cells grown at 33 degrees C. 1,2-DNB was more toxic than 1,3-DNB, and 3,4-DNT was more toxic than 2,4-DNT, as judged by the neutral-red cell viability assay. Ultrastructurally, cells treated with 1,2-DNB or 2,4-DNT showed increased numbers of autophagic vesicles compared to controls. Intercellular penetration of ruthenium red demonstrated breached tight junctions in 1,2-DNB and cadmium-treated cells. From these observations, we conclude that this cell line can serve as a model for studying toxic mechanisms in adult Sertoli cells.

Priming polyvalent immunity by DNA vaccines expressing chimeric antigens with a stress protein-capturing, viral J-domain.

The N-terminal domain of large tumor antigens (T-Ag) of polyomaviruses forms a DnaJ-like structure with a conserved J domain that associates with constitutively expressed stress protein heat shock protein (hsp)73. Mutant (but not wild-type) SV40 T-Ag show stable, ATP-dependent binding to the stress protein hsp73 when expressed in cells from different vertebrate tissues. Intracellular T/hsp73 complexes accumulate to high steady-state levels. From this observation, we designed a vector system that supports stable expression of a large variety of hsp73-capturing, chimeric antigens containing an N-terminal, T-Ag-derived domain, and different C-terminal antigenic domains from unrelated antigens. Most antigenic domains tested could be stably expressed only in eukaryotic cells as fusion protein/hsp73 complexes. The N-terminal 77 residues representing the J domain of T-Ag were required for stable hsp73 binding and efficient expression of chimeric antigens. Hsp73-bound chimeric antigens expressed by DNA vaccines showed strikingly enhanced immunogenicity evident in humoral (antibody) and cellular cytolytic T lymphocytes (CTL) responses. The described system supports efficient expression of chimeric, polyvalent antigens and their codelivery with hsp73 as a "natural adjuvant" for enhanced immunogenicity for T and B cells.

A Role for p53 in Maintaining and Establishing the Quiescence Growth Arrest in Human Cells

The p53 tumor suppressor protein induces transient growth arrest or apoptosis in response to genotoxic stress and mediates the irreversible growth arrest of cellular senescence. We present evidence here that p53 also contributes to the reversible, growth factor-dependent arrest of quiescence (G(0)). Microinjection of expression vectors encoding either MDM2 or a pRb-binding mutant of SV40 T antigen, both of which abrogate p53 function, stimulated quiescent normal human fibroblasts to initiate DNA synthesis and were 40-70% as effective as wild-type T antigen. Electrophoretic mobility shift and p53 transactivation assays showed that p53 activity was higher in quiescent and senescent cells compared with proliferating cells. As proliferating cells entered G(0) after growth factor withdrawal, the p53 mRNA level increased, followed by transient accumulation of the protein. Shortly thereafter, the expression (mRNA and protein) of p21, a p53 target gene and effector of cell cycle arrest, increased. Finally, stable expression of the HPV16 E6 oncogene or dominant negative p53 peptide, GSE-22, both of which inhibit p53 function, delayed entry into quiescence following growth factor withdrawal. Our data indicate that p53 is activated during both quiescence and senescence. They further suggest that p53 activity contributes, albeit not exclusively, to the quiescent growth arrest.