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1.
Clin J Am Soc Nephrol ; 15(7): 1015-1023, 2020 07 01.
Article in English | MEDLINE | ID: mdl-32601093

ABSTRACT

BACKGROUND AND OBJECTIVES: The published tissue adequacy requirement of kidney medulla for BK virus allograft nephropathy diagnosis lacks systematic verification and competes against potential increased procedural risks from deeper sampling. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We evaluated whether the presence of kidney medulla improved the diagnostic rate of BK nephropathy in 2244 consecutive biopsy samples from 856 kidney transplants with detailed histologic and virologic results. RESULTS: Medulla was present in 821 samples (37%) and correlated with maximal core length (r=0.35; P<0.001). BK virus allograft nephropathy occurred in 74 (3% overall) but increased to 5% (42 of 821) with medulla compared with 2% (32 of 1423) for cortical samples (P<0.001). Biopsy medulla was associated with infection after comprehensive multivariable adjustment of confounders, including core length, glomerular number, and number of cores (adjusted odds ratio, 1.81; 95% confidence interval, 1.02 to 3.21; P=0.04). In viremic cases (n=275), medulla was associated with BK virus nephropathy diagnosis (39% versus 19% for cortex; P<0.001) and tissue polyomavirus load (Banff polyomavirus score 0.64±0.96 versus 0.33±1.00; P=0.006). Biopsy medulla was associated with BK virus allograft nephropathy using generalized estimating equation (odds ratio, 2.04; 95% confidence interval, 1.05 to 3.96; n=275) and propensity matched score comparison (odds ratio, 2.24; 95% confidence interval, 1.11 to 4.54; P=0.03 for 156 balanced pairs). Morphometric evaluation of Simian virus 40 large T immunohistochemistry found maximal infected tubules within the inner cortex and medullary regions (P<0.001 versus outer cortex). CONCLUSIONS: Active BK virus replication concentrated around the corticomedullary junction can explain the higher detection rates for BK virus allograft nephropathy with deep sampling. The current adequacy requirement specifying targeting medulla can be justified to minimize a missed diagnosis from undersampling.


Subject(s)
BK Virus , Kidney Diseases/diagnosis , Kidney Diseases/pathology , Kidney Medulla/pathology , Polyomavirus Infections/complications , Tumor Virus Infections/complications , Adult , Allografts/pathology , Antigens, Polyomavirus Transforming/analysis , Biopsy/standards , Female , Humans , Kidney Cortex/pathology , Kidney Cortex/virology , Kidney Diseases/virology , Kidney Medulla/virology , Kidney Transplantation , Male , Middle Aged , Prospective Studies , Viral Load
2.
Scand J Gastroenterol ; 49(6): 705-14, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24730442

ABSTRACT

We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.


Subject(s)
Antigens, Polyomavirus Transforming/analysis , Cell Differentiation , Cell Line , Fetal Stem Cells/cytology , Hepatocytes/cytology , Phenotype , Albumins/analysis , Albumins/genetics , Animals , Antigens, CD/analysis , Antigens, Neoplasm/analysis , Antigens, Polyomavirus Transforming/genetics , Aryl Hydrocarbon Hydroxylases/analysis , Aryl Hydrocarbon Hydroxylases/genetics , Biomarkers, Tumor/analysis , Cell Adhesion Molecules/analysis , Cytochrome P-450 CYP3A/analysis , Cytochrome P-450 CYP3A/genetics , Epithelial Cell Adhesion Molecule , Fetal Stem Cells/chemistry , Fetal Stem Cells/transplantation , Gene Expression , Hepatocyte Nuclear Factor 1-alpha/analysis , Hepatocyte Nuclear Factor 4/analysis , Hepatocyte Nuclear Factor 4/genetics , Hepatocytes/chemistry , Hepatocytes/transplantation , Humans , Keratins/analysis , Male , Mice , Mice, Inbred BALB C , Plasmids , RNA, Messenger/analysis , Simian virus 40 , Transfection , Tumor Suppressor Protein p53/analysis
3.
Hum Pathol ; 45(1): 119-26, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24321520

ABSTRACT

Langerhans cell histiocytosis (LCH) is a group of granulomatous disorders in which abnormal Langerhans cells proliferate as either a localized lesion in a single bone or disseminated disease involving two or more organs or systems. Because the different LCH forms exhibit significantly elevated levels of inflammatory molecules, including pro-inflammatory cytokines and tissue-degrading enzymes, we investigated for a possible viral trigger in LCH pathogenesis. We looked for Merkel cell polyomavirus (MCPyV) in peripheral blood cells and tissues using quantitative real-time PCR and immunohistochemistry staining with anti-MCPyV large T-antigen antibody. Our findings revealed elevated amounts of MCPyV DNA in the peripheral blood cells of 2 of 3 patients affected by LCH with high-risk organ involvement (RO+) and absence of MCPyV DNA in the blood cells in all 12 LCH-RO- patients (P = .029). With lower viral loads (0.002-0.033 copies/cell), an elevated number of MCPyV DNA sequences was detected in 12 LCH tissues in comparison with control tissues obtained from patients with reactive lymphoid hyperplasia (0/5; P = .0007), skin diseases not related to LCH in children younger than 2 years (0/11; P = .0007), or dermatopathic lymphadenopathy (5/20; P = .0002). The data, including frequent but lower viral loads and low large-T antigen expression rate (2/13 LCH tissues), suggest that development of LCH as a reactive rather than a neoplastic process may be related to MCPyV infection.


Subject(s)
DNA, Viral/analysis , Histiocytosis, Langerhans-Cell/virology , Merkel cell polyomavirus , Polyomavirus Infections/complications , Polyomavirus Infections/virology , Antigens, Polyomavirus Transforming/analysis , Child , Child, Preschool , Female , Humans , Immunohistochemistry , Infant , Infant, Newborn , Laser Capture Microdissection , Male , Real-Time Polymerase Chain Reaction , Viral Load
4.
Enzyme Microb Technol ; 52(3): 196-8, 2013 Mar 05.
Article in English | MEDLINE | ID: mdl-23410932

ABSTRACT

Here we describe a non-radioactive assay that exploits the fluorescent dye SYBR Green to measure the helicase enzyme activity. SYBR Green I emits fluorescence upon intercalation with double-stranded DNA or RNA. The fluorescence is lost proportionally as the nucleic acid is converted to single strands by a helicase, and this decrease in fluorescence intensity can be used to measure the activity of the helicase enzyme. The reaction was prepared by mixing a double-stranded substrate with the helicase enzyme, buffer, ATP and SYBR Green I. After completion, the reaction was terminated by EDTA and fluorescence was measured. Using this technique, a linear increase in substrate release was observed with increasing time and helicase concentrations. The assay described here is speedy, efficient and economical; it holds promise for use in large-scale screening of drugs that target helicases.


Subject(s)
Antigens, Polyomavirus Transforming/analysis , DNA Helicases/analysis , Fluorescent Dyes/analysis , Fluorometry/methods , Organic Chemicals/analysis , Viral Nonstructural Proteins/analysis , Adenosine Triphosphate/metabolism , Antigens, Polyomavirus Transforming/metabolism , Benzothiazoles , Ciprofloxacin/pharmacology , DNA Helicases/metabolism , Diamines , Hepacivirus/enzymology , Nucleic Acid Renaturation , Oligonucleotides/metabolism , Quinolines , Simian virus 40/enzymology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
5.
Carcinogenesis ; 33(12): 2586-92, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22976928

ABSTRACT

Plumbagin (PL), 5-hydroxy-2-methyl-1,4-naphthoquinone, is a quinoid constituent isolated from the roots of the medicinal plant Plumbago zeylanica L. (also known as chitrak). PL has also been found in Juglans regia (English Walnut), Juglans cinerea (whitenut) and Juglans nigra (blacknut). The roots of P. zeylanica have been used in Indian and Chinese systems of medicine for more than 2500 years for the treatment of various types of ailments. We were the first to report that PL inhibits the growth and invasion of hormone refractory prostate cancer (PCa) cells [Aziz,M.H. et al. (2008) Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res., 68, 9024-9032.]. Now, we present that PL inhibits in vivo PCa development in the transgenic adenocarcinoma of mouse prostate (TRAMP). PL treatment (2 mg/kg body weight i.p. in 0.2 ml phosphate-buffered saline, 5 days a week) to FVB-TRAMP resulted in a significant (P < 0.01) decrease in prostate tumor size and urogenital apparatus weights at 13 and 20 weeks. Histopathological analysis revealed that PL treatment inhibited progression of prostatic intraepithelial neoplasia (PIN) to poorly differentiated carcinoma (PDC). No animal exhibited diffuse tumor formation in PL-treated group at 13 weeks, whereas 75% of the vehicle-treated mice elicited diffuse PIN and large PDC at this stage. At 20 weeks, 25% of the PL-treated animals demonstrated diffuse PIN and 75% developed small PDC, whereas 100% of the vehicle-treated mice showed large PDC. PL treatment inhibited expression of protein kinase C epsilon (PKCε), signal transducers and activators of transcription 3 phosphorylation, proliferating cell nuclear antigen and neuroendocrine markers (synaptophysin and chromogranin-A) in excised prostate tumor tissues. Taken together, these results further suggest PL could be a novel chemopreventive agent against PCa.


Subject(s)
Adenocarcinoma/prevention & control , Anticarcinogenic Agents/therapeutic use , Chromogranin A/antagonists & inhibitors , Naphthoquinones/therapeutic use , Prostatic Neoplasms/prevention & control , Protein Kinase C-epsilon/antagonists & inhibitors , STAT3 Transcription Factor/antagonists & inhibitors , Synaptophysin/antagonists & inhibitors , Adenocarcinoma/pathology , Animals , Antigens, Polyomavirus Transforming/analysis , Disease Models, Animal , Male , Mice , Mice, Transgenic , Phosphorylation , Proliferating Cell Nuclear Antigen/analysis , Prostatic Neoplasms/pathology
6.
Mol Carcinog ; 50(2): 100-12, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21229607

ABSTRACT

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol, which is present in cruciferous vegetables and has been reported to possess anti-carcinogenic properties. In the present study, we examined whether DIM inhibits the development of prostate cancer using the transgenic adenocarcinoma mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen and proliferating cell nuclear antigen, and increased the number of terminal dUTP nick-end labeling-positive cells in the dorsolateral lobes of the prostate. Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate cancer cells were cultured with various concentrations of DIM. DIM induced a substantial reduction in the numbers of viable cells and induced apoptosis in LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly (ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability and the translocation of cytochrome c and Smac/Diablo from the mitochondria. Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid. These results indicate that DIM inhibits prostate carcinogenesis via induction of apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in prostate cancer cells via the mitochondria- and death receptor-mediated pathways.


Subject(s)
Adenocarcinoma/drug therapy , Anticarcinogenic Agents/therapeutic use , Cell Transformation, Neoplastic/drug effects , Indoles/therapeutic use , Prostatic Neoplasms/drug therapy , Animals , Antigens, Polyomavirus Transforming/analysis , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Carrier Proteins/analysis , Caspases/analysis , Cell Line, Tumor , Cyclin A/analysis , Cyclin-Dependent Kinase 2/analysis , Cyclin-Dependent Kinase 4/analysis , Cyclin-Dependent Kinase Inhibitor p27/analysis , Cytochromes c/analysis , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondrial Membranes/drug effects , Mitochondrial Proteins/analysis , Oligopeptides/therapeutic use , Peptides , Permeability/drug effects , Poly(ADP-ribose) Polymerases/analysis , Proliferating Cell Nuclear Antigen/analysis , Protein Transport/drug effects , bcl-X Protein/analysis
7.
Blood ; 116(24): 5280-4, 2010 Dec 09.
Article in English | MEDLINE | ID: mdl-20817850

ABSTRACT

Merkel cell polyomavirus (MCPyV) is detected in approximately 80% of Merkel cell carcinomas (MCC). Yet, clonal integration and truncating mutations of the large T antigen (LTAg) of MCPyV are restricted to MCC. We tested the presence and mutations of MCPyV in highly purified leukemic cells of 70 chronic lymphocytic leukemia (CLL) patients. MCPyV was detected in 27.1% (n = 19) of these CLL cases. In contrast, MCPyV was detected only in 13.4% of normal controls (P < .036) in which no LTAg mutations were found. Mutational analyses revealed a novel 246bp LTAg deletion in the helicase gene in 6 of 19 MCPyV-positive CLL cases. 2 CLL cases showed concomitant mutated and wild-type MCPyV. Immunohistochemistry revealed protein expression of the LTAg in MCPyV-positive CLL cases. The detection of MCPyV, including LTAg deletions and LTAg expression in CLL cells argues for a potential role of MCPyV in a significant subset of CLL cases.


Subject(s)
Antigens, Polyomavirus Transforming/analysis , Antigens, Polyomavirus Transforming/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/virology , Polyomavirus/pathogenicity , Sequence Deletion , Adult , Aged , Aged, 80 and over , Carcinoma, Merkel Cell/virology , DNA Mutational Analysis , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Merkel Cells , Middle Aged , Polyomavirus/genetics , Polyomavirus/immunology , Tumor Virus Infections
8.
Cancer Genet Cytogenet ; 200(2): 79-99, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20620590

ABSTRACT

Cancers are clones of autonomous cells defined by individual karyotypes, much like species. Despite such karyotypic evidence for causality, three to six synergistic mutations, termed oncogenes, are generally thought to cause cancer. To test single oncogenes, they are artificially activated with heterologous promoters and spliced into the germ line of mice to initiate cancers with collaborating spontaneous oncogenes. Because such cancers are studied as models for the treatment of natural cancers with related oncogenes, the following must be answered: 1) which oncogenes collaborate with the transgenes in cancers; 2) how do single transgenic oncogenes induce diverse cancers and hyperplasias; 3) what maintains cancers that lose initiating transgenes; 4) why are cancers aneuploid, over- and underexpressing thousands of normal genes? Here we try to answer these questions with the theory that carcinogenesis is a form of speciation. We postulate that transgenic oncogenes initiate carcinogenesis by inducing aneuploidy. Aneuploidy destabilizes the karyotype by unbalancing teams of mitosis genes. This instability thus catalyzes the evolution of new cancer species with individual karyotypes. Depending on their degree of aneuploidy, these cancers then evolve new subspecies. To test this theory, we have analyzed the karyotypes and phenotypes of mammary carcinomas of mice with transgenic SV40 tumor virus- and hepatitis B virus-derived oncogenes. We found that (1) a given transgene induced diverse carcinomas with individual karyotypes and phenotypes; (2) these karyotypes coevolved with newly acquired phenotypes such as drug resistance; (3) 8 of 12 carcinomas were transgene negative. Having found one-to-one correlations between individual karyotypes and phenotypes and consistent coevolutions of karyotypes and phenotypes, we conclude that carcinogenesis is a form of speciation and that individual karyotypes maintain cancers as they maintain species. Because activated oncogenes destabilize karyotypes and are dispensable in cancers, we conclude that they function indirectly, like carcinogens. Such oncogenes would thus not be valid models for the treatment of cancers.


Subject(s)
Antigens, Polyomavirus Transforming/genetics , Mammary Neoplasms, Experimental/genetics , Oncogenes , Aneuploidy , Animals , Antigens, Polyomavirus Transforming/analysis , Cell Line, Tumor , Drug Resistance, Neoplasm , Female , Karyotyping , Mammary Neoplasms, Experimental/etiology , Mice , Phenotype , Trans-Activators/genetics , Transgenes , Viral Regulatory and Accessory Proteins
9.
J Virol Methods ; 164(1-2): 145-7, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20005903

ABSTRACT

SV40 titer is determined traditionally by the conventional plaque assay. Plaques appear after several rounds of infection and the assay takes around two weeks, which may delay research. A simpler assay was developed, based on detection of T-antigen in the infected cells by flow cytometry. Cells grown in 6-well plates are infected with serial dilutions of the viral stock, harvested 48h post-infection, stained and analyzed for T-antigen using a flow cytometer. The viral titer is calculated based on the percentage of T-antigen positive cells. The procedure is accomplished in 2 days. Unexpectedly we found that titers on different permissive African Green Monkey kidney cell lines were consistently different, suggesting variable susceptibility to SV40 infection. The method described, optimized for SV40 titration, may be adapted readily to other viruses.


Subject(s)
Flow Cytometry/methods , Simian virus 40/isolation & purification , Viral Load/methods , Animals , Antigens, Polyomavirus Transforming/analysis , Cell Line , Chlorocebus aethiops , Time Factors
10.
Odontology ; 97(1): 18-31, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19184294

ABSTRACT

Most in vitro studies of oral carcinogenesis in human cells are carried out with oral keratinocytes immortalized by human papillomavirus type 16 DNA. However, because various etiological factors for oral cancer are known, it is important to establish new human keratinocyte cell lines useful for studying the mechanism of oral carcinogenesis. Normal human gingival keratinocytes in secondary cultures grown in serum-free medium were either transfected with origin (-) SV40 DNA or sequentially transfected with origin (-) SV40 DNA and human c-fos. The transfected cells were continually passaged and analyzed for cytological and cytogenetic characterizations. Four immortal cell lines were grown for over 1100 days in culture and maintained a vigorous growth for over 250 population doublings. They expressed SV40 T antigen, cytokeratins 8 and 18, and E-cadherin, and overexpressed the c-Fos protein. The immortal cell lines had telomerase activity but lacked transformed phenotypes on soft agar or in nude mice. Each cell line had nonrandom chromosomal abnormalities and minisatellite alterations. One of the immortal cell lines, NDUSD-1, retained the capability to deposit calcium, which was also demonstrated in normal human gingival keratinocytes by alizarin red staining, indicating the possibility that NDUSD-1 cells may retain some natural characteristics of normal gingival keratinocytes. Because the oral ectoderm plays an important role in tooth development, these immortal cell lines may be useful in various experimental models for investigations of oral biology and oral carcinogenesis.


Subject(s)
Cell Transformation, Neoplastic/pathology , Gingiva/cytology , Keratinocytes/cytology , Animals , Anthraquinones , Antigens, Polyomavirus Transforming/analysis , Cadherins/analysis , Calcium/metabolism , Cell Line , Cell Line, Transformed , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Viral/genetics , Chromosome Aberrations , Coloring Agents , DNA, Viral/genetics , Genes, fos/genetics , Humans , Karyotyping , Keratin-18/analysis , Keratin-8/analysis , Keratinocytes/pathology , Mice , Mice, Nude , Minisatellite Repeats/genetics , Phenotype , Proto-Oncogene Proteins c-fos/analysis , Simian virus 40/genetics , Simian virus 40/immunology , Telomerase/metabolism , Time Factors , Transfection/methods
11.
Indian J Pathol Microbiol ; 52(1): 42-5, 2009.
Article in English | MEDLINE | ID: mdl-19136778

ABSTRACT

BACKGROUND: The human polyoma virus, also known as the JC virus (JCV), replicates predominantly in the oligodendrocytes, the myelin producing cells in the central nervous system and results in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) especially in immunosuppressed patients with AIDS. Several investigators have also documented the presence of the viral genome and early and late antigens in a variety of brain tumors particularly in medulloblastomas, gliomas and ependymomas. Reports also indicate the presence of JCV in patients with colon cancer. The T antigen of JCV has been postulated to have oncogenic potential as substantiated by animal experiments. Although JCV infects 80% of the population, there are scant epidemiological studies regarding JCV from India. There are also reports of the low prevalence of PML in patients with AIDS from India and Africa. AIM: This study was undertaken to investigate if Indian children with medulloblastomas also show evidence of JCV. METHODS: Twenty-two consecutive cases of medulloblastomas were investigated for the presence of T antigen and agnoprotein of JCV in biopsy specimens by immunohistochemistry. Antibodies to the agnoprotein antigen raised in rabbits and a monoclonal antibody against SV40 T antigen raised in mice that cross-reacts with JCV T antigen were used. RESULTS: Out of 22 patients, 4 had desmoplastic tumors while the rest had classical tumors. All children were below the age of 10. Results indicate that while PML tissues showed consistent immunostaining both with antibody to T antigen and agnoprotein antibody, none of the tumors showed any positive staining for JC viral antigens. CONCLUSION: JCV antigens could not be detected by immunohistochemistry in the tumor tissues of Indian children with medulloblastomas.


Subject(s)
Antigens, Polyomavirus Transforming/analysis , JC Virus/chemistry , Medulloblastoma/virology , Viral Regulatory and Accessory Proteins/analysis , Animals , Antibodies, Monoclonal , Antibodies, Viral , Biopsy , Brain/pathology , Child , Child, Preschool , Humans , Immunohistochemistry , India , Mice , Rabbits
12.
Cancer Res ; 68(22): 9488-96, 2008 Nov 15.
Article in English | MEDLINE | ID: mdl-19010924

ABSTRACT

Suppression of the late gene expression, usually by integration of the viral DNA into the host genome, is a critical step in DNA tumor virus carcinogenesis. SV40 induces high rates of transformation in infected primary human mesothelial cells in tissue culture, leading to the formation of immortal cell lines (SV40-transformed human mesothelial cell lines, S-HML). The studies described here were designed to elucidate the unusual susceptibility of primary human mesothelial cells to SV40 carcinogenesis. We found that S-HML contained wild-type, mostly episomal SV40 DNA. In these cells, the early genes that code for the viral oncogenes are expressed; at the same time, the synthesis of the late genes, capsid proteins, is suppressed and S-HML are not lysed. Late gene suppression is achieved through the production of antisense RNA molecules. These antisense RNA molecules originate in the early region of the SV40 circular chromosome and proceed in antisense orientation into the late gene region, leading to the formation of highly unstable double-strand RNA, which is rapidly degraded. Our results reveal a novel biological mechanism responsible for the suppression of late viral gene products, an important step in viral carcinogenesis in humans.


Subject(s)
Cell Transformation, Neoplastic , Gene Silencing , Simian virus 40/genetics , Antigens, Polyomavirus Transforming/analysis , Blotting, Northern , Capsid Proteins/analysis , Capsid Proteins/genetics , Cell Line, Transformed , Endothelial Cells/pathology , Humans , Promoter Regions, Genetic , RNA, Antisense/genetics , RNA, Messenger/analysis , Sequence Analysis, DNA , Virus Integration
13.
Neoplasia ; 9(11): 938-50, 2007 Nov.
Article in English | MEDLINE | ID: mdl-18030362

ABSTRACT

Following castration, the transgenic adenocarcinoma of mouse prostate (TRAMP) model demonstrates rapid development of SV40-Tag-driven poorly differentiated tumors that express neuroendocrine cell markers. The cell population dynamics within the prostates of castrated TRAMP mice were characterized by analyzing the incorporation of 5-bromodeoxyuridine (BrdUrd) and the expression of SV40-Tag, synaptophysin, and androgen receptor (AR). Fourteen days postcastration, the remaining epithelial cells and adenocarcinoma cells were nonproliferative and lacked detectable SV40-Tag or synaptophysin expression. In contrast, morphologically distinct intraglandular foci were identified which expressed SV40-Tag, synaptophysin, and Ki67, but that lacked AR expression. These proliferative SV40-Tag and synaptophysin-expressing intraglandular foci were associated with the rare BrdUrd-retaining cells. These foci expanded rapidly in the postcastration prostate environment, in contrast to the AR- and SV40-Tag-expressing adenocarcinoma cells that lost SV40-Tag expression and underwent apoptosis after castration. Intraglandular foci of synaptophysin-expressing cells were also observed in the prostates of intact TRAMP mice at a comparable frequency; however, they did not progress to rapidly expanding tumors until much later in the life of the mice. This suggests that the foci of neuroendocrine-like cells that express SV40-Tag and synaptophysin, but lack AR, arise independent of androgen-deprivation and represent the source of the poorly differentiated tumors that are the lethal phenotype in the TRAMP model.


Subject(s)
Adenocarcinoma/pathology , Androgens/physiology , Prostatic Neoplasms/pathology , Animals , Antigens, Polyomavirus Transforming/analysis , Antigens, Polyomavirus Transforming/genetics , Apoptosis , Bromodeoxyuridine/metabolism , Cell Proliferation , Disease Models, Animal , Hepatocyte Nuclear Factor 3-beta/analysis , Lymphatic Metastasis , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Orchiectomy , Prostatic Neoplasms/blood supply , Prostatic Neoplasms/metabolism , Receptors, Androgen/analysis , Simian virus 40/immunology , Synaptophysin/analysis
14.
J Biosci Bioeng ; 104(1): 14-21, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17697978

ABSTRACT

A line of double-transgenic mice that develop neoplasms arising primarily in the pancreas was established. In these mice, the oncogene SV40 T antigen (Tag) was detected in the pancreas with and without the control of Tet-on system. The transgenic mice that developed pancreatic tumors as early as 20 weeks of age showed hypoglycemia on a blood glucose test. Pathological and immunohistochemical characterizations demonstrated that the tumors belonged to neuroendocrine neoplasms arising from pancreatic islets. A change in IGFs/IGF-1R signaling pathway was detected using real-time PCR analysis. A potential association between the IGFs/IGF-1R system and SV40Tag was studied to further explain the cancerogenesis of the double-transgenic mice by Western blot analysis and immunoprecipitation experiments. The results suggest that a Tag transgenic mice model could be used to study the molecular mechanism of the tumorigenesis of islets.


Subject(s)
Disease Models, Animal , Insulinoma/genetics , Mice, Transgenic , Mice , Neuroendocrine Tumors/genetics , Pancreatic Neoplasms/genetics , Animals , Antigens, Polyomavirus Transforming/analysis , Antigens, Polyomavirus Transforming/metabolism , Blood Glucose/metabolism , Insulin Receptor Substrate Proteins , Insulinoma/chemistry , Insulinoma/pathology , Neuroendocrine Tumors/chemistry , Neuroendocrine Tumors/pathology , Pancreatic Neoplasms/chemistry , Pancreatic Neoplasms/pathology , Phosphoproteins/metabolism , Somatomedins/metabolism , Tetracycline/pharmacology
15.
Cancer Invest ; 24(3): 223-8, 2006.
Article in English | MEDLINE | ID: mdl-16809147

ABSTRACT

Recent studies have linked simian virus 40 (SV40) to non-Hodgkin's lymphoma (NHL), especially in countries in which people were exposed to contaminated polio vaccines prior to 1963. In Taiwan, nearly all children were not exposed to contaminated polio vaccine during this period; the relationship between SV40 infection and hematological malignancies is unclear and deserves to be studied. Using PCR amplification of SV40 large T antigen DNA, confirmed by Southern blot hybridization and sequence analysis, 91 frozen lymph nodes from NHL patients were examined. Thirteen (14.3 percent) showed positive for SV40. All other test samples, including diagnostic bone marrow from patients with acute leukemia, peripheral blood from 10 relatives of SV40 positive-patients and 91 age-matched normal volunteers, and 5 reactive hyperplastic lymphoid tissues, showed negative. These results may reflect that human-to-human transmission of SV40 is independent of contaminated polio vaccines; and SV40 is possibly associated with the development of NHL in Taiwan (p = 0.0001). Prospective studies are needed to determine the prevalence of SV40 infections in our and other human populations and to explore the means of transmission of the virus.


Subject(s)
Drug Contamination , Lymphoma, Non-Hodgkin/virology , Poliovirus Vaccines/adverse effects , Polyomavirus Infections/transmission , Simian virus 40 , Acute Disease , Adolescent , Adult , Aged , Aged, 80 and over , Antigens, Polyomavirus Transforming/analysis , Blotting, Southern , Bone Marrow Cells/virology , Female , Humans , Leukemia/virology , Lymph Nodes/virology , Male , Middle Aged , Polymerase Chain Reaction , Polyomavirus Infections/epidemiology , Prevalence , Taiwan , Tumor Virus Infections/epidemiology
16.
Cancer ; 107(3): 481-8, 2006 Aug 01.
Article in English | MEDLINE | ID: mdl-16795066

ABSTRACT

BACKGROUND: JC virus (JCV) is a polyomavirus that commonly infects humans and is the causative agent of progressive multifocal leukoencephalopathy in immune-compromised patients. An association between JCV and human cancers long has been suspected, because this virus induces brain tumors in several animal models. The oncogenic potential of JCV is mediated by a transforming protein, the T-antigen (T-Ag), which is a multifunctional protein that transforms cells through interactions with various growth-regulatory genes, including p53 and pRb, and by stabilizing beta-catenin. Previously, the laboratory at the authors' institution demonstrated that JCV is present frequently in the human gastrointestinal tract and may play a role in colorectal carcinogenesis. However, to date, no studies have determined whether JCV sequences are present specifically in gastric cancers. The current study was designed to investigate whether JCV sequences and expression are found in human gastric cancers. METHODS: DNA was extracted from 23 paraffin embedded and 14 frozen gastric cancer specimens. For the detection of JCV gene sequences, polymerase chain reaction amplifications were performed using gene-specific primers for T-Ag, VP-1 (a JCV capsid gene), and the viral regulatory region (or transcriptional control region). Immunohistochemical staining was performed with an anti-T-Ag monoclonal antibody to detect protein expression. RESULTS: Twenty-one of 37 gastric cancers (57%) harbored JCV T-Ag sequences, and 13 of 37 gastric cancers (30%) contained VP-1 sequences. T-Ag sequences also were found in adjacent nonneoplastic mucosa. In addition, JCV regulatory region sequences were present frequently in gastric cancers and adjacent nonneoplastic mucosa. T-Ag protein expression was found in 9 of 23 gastric cancers (39%), whereas no expression was observed in any of the nonneoplastic tissues. CONCLUSIONS: To the authors' knowledge, this is the first demonstration of the presence of JCV T-Ag expression in human gastric cancers. These findings suggest a possible role for this polyomavirus in gastric carcinogenesis.


Subject(s)
Antigens, Polyomavirus Transforming/genetics , JC Virus/genetics , Stomach Neoplasms/virology , Antigens, Polyomavirus Transforming/analysis , Antigens, Polyomavirus Transforming/immunology , Antigens, Polyomavirus Transforming/metabolism , Chromosomal Instability , Humans , JC Virus/immunology , Polymerase Chain Reaction , Stomach Neoplasms/chemistry , Stomach Neoplasms/genetics
17.
Int J Urol ; 13(4): 439-41, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16734867

ABSTRACT

Polyoma virus nephropathy (PVN) is a significant cause of renal allograft dysfunction in transplant patients. A 58-year-old male received a cadaveric renal transplant and 12 weeks later presented with fever, diarrhea, and dysuria. He was diagnosed with a polyoma virus infection of the bladder by a transurethral bladder biopsy. One year post-transplant, he presented with renal allograft dysfunction and was diagnosed by biopsy with PVN of the non-native kidney. The diagnosis of a polyoma virus infection was confirmed by immunoreactivity to the polyoma T-antigen. We suggest that polyoma virus infection of the bladder be included in the differential diagnosis of urinary dysfunction in post-transplant patients, as such infections might be an under-recognized comorbidity in individuals with PVN.


Subject(s)
Cystitis/virology , Kidney Transplantation/adverse effects , Nephritis/virology , Polyomavirus Infections/virology , Polyomavirus/immunology , Tumor Virus Infections/virology , Antigens, Polyomavirus Transforming/analysis , Biopsy , Cystitis/etiology , Cystitis/pathology , Diabetic Nephropathies/surgery , Diagnosis, Differential , Follow-Up Studies , Humans , Male , Middle Aged , Nephritis/etiology , Nephritis/pathology , Polyomavirus Infections/etiology , Polyomavirus Infections/pathology , Tumor Virus Infections/etiology , Tumor Virus Infections/pathology
18.
World J Gastroenterol ; 12(17): 2785-8, 2006 May 07.
Article in English | MEDLINE | ID: mdl-16718771

ABSTRACT

AIM: To generate a SV40Tag transgenic tumor animal model and to study the mechanism underlying tumorigenesis. METHODS: A mammary gland expression vector containing SV40Tag DNA was generated. Transgene fragments were microinjeted into fertilized eggs of FVB mice. The genetically manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. PCR and Northern blot analysis were used for genotype analysis of F1 and F2 mice. Transgene expression was detected by RT-PCR and immunohistochemistry. RESULTS: SV40Tag gene was detected in two lines of transgenic mice. One of them delivered the transgene to F1 and a tumor was found in the pancreas of these mice. RT-PCR and immunohistochemistry showed that SV40Tag gene was expressed in the tumor. Pathological characterization of the transgenic mice demonstrated that the tumor belonged to pancreatic cystic neoplasm. CONCLUSION: SV40Tag transgenic mouse model can be successfully established. The transgenic mice develop a pancreatic tumor, which can be used for investigation of the molecular mechanism of tumorigenesis in vivo.


Subject(s)
Disease Models, Animal , Mice, Transgenic/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Animals , Antigens, Polyomavirus Transforming/analysis , Antigens, Polyomavirus Transforming/genetics , DNA, Neoplasm/genetics , DNA, Viral , Female , Gene Expression Regulation, Neoplastic , Gene Expression Regulation, Viral , Genetic Vectors , Immunohistochemistry , Male , Mice , Pancreatic Neoplasms/chemistry , Pancreatic Neoplasms/physiopathology , Pregnancy , Pregnancy, Animal , Pseudopregnancy , Reverse Transcriptase Polymerase Chain Reaction , Simian virus 40/immunology , Transgenes/genetics
20.
Haematologica ; 90(12): 1635-42, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16330436

ABSTRACT

BACKGROUND AND OBJECTIVES: Simian virus 40 (SV40) is an oncogenic DNA virus implicated in some human malignancies, including lymphomas. In the present masked case-control study, we investigated the prevalence of SV40 sequences and the expression of the viral oncoprotein, large tumor antigen (T-ag), in lymphomas and control specimens from patients negative for the human immunodeficiency virus in Costa Rica. DESIGN AND METHODS: Coded specimens were anlyzed by polymerase chain reaction for SV40 and Epstein-Barr virus (EBV). SV40 sequences were confirmed by Southern blot and DNA sequence analysis. Immunohistochemistry was used to detect the expression of SV40 T-ag in coded samples and to immunophenotype the lymphomas. RESULTS: When samples were decoded, SV40 DNA sequences were detected significantly more often in lymphomas than in control samples (30/125, 24% vs. 0/91, 0%; p=0.001). SV40 DNA was detected in 26% and 10% of non-Hodgkin's and Hodgkin's lymphomas, respectively. EBV DNA was detected in 10% of lymphomas and 33% of control specimens. None of the lymphomas was positive for both SV40 and EBV. Expression of SV40 T-ag was detected in 64% of B-cell lymphomas that contained T-ag DNA sequences and in none of the samples negative for viral DNA. Not all cells in a positive tumor expressed T-ag and the reactions were relatively low intensity. A germinal center B-cell-like profile was frequently associated with SV40-positive lymphomas. Of note, 20% of patients with SV40-related lymphomas were born in the 1970s and 1980s. INTERPRETATION AND CONCLUSIONS: These results indicate that SV40 is significantly associated with some B-cell neoplasms in Costa Rica today.


Subject(s)
Antigens, Polyomavirus Transforming/analysis , Lymphoma/virology , Poliovirus Vaccine, Inactivated/adverse effects , Polyomavirus Infections/epidemiology , Simian virus 40/pathogenicity , Tumor Virus Infections/epidemiology , Adult , Case-Control Studies , Comorbidity , Costa Rica/epidemiology , DNA, Viral/analysis , Drug Contamination , Epstein-Barr Virus Infections/epidemiology , Female , Germinal Center/virology , HIV Seronegativity , Herpesvirus 4, Human/isolation & purification , Herpesvirus 4, Human/pathogenicity , Hodgkin Disease/epidemiology , Hodgkin Disease/virology , Humans , Liver Neoplasms/epidemiology , Liver Neoplasms/virology , Lymph Nodes/virology , Lymphoma/epidemiology , Lymphoma, B-Cell/epidemiology , Lymphoma, B-Cell/virology , Lymphoma, Non-Hodgkin/epidemiology , Lymphoma, Non-Hodgkin/virology , Male , Middle Aged , Palatine Tonsil/virology , Polyomavirus Infections/virology , Pseudolymphoma/epidemiology , Pseudolymphoma/virology , Simian virus 40/isolation & purification , Stomach Neoplasms/epidemiology , Stomach Neoplasms/virology , Tumor Virus Infections/virology
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