Immunodetection of SV40 large T antigen in human central nervous system tumours

BACKGROUND/AIMS: DNA sequences from Simian virus 40 (SV40) have been previously isolated from various human tumours of the central nervous system (CNS). This study aimed to investigate a series of tumours of the CNS for the expression of the SV40 large T antigen (Tag), which is an oncogenic protein of the virus. METHODS: A French series of 82 CNS tumours was investigated for Tag expression using a monoclonal antibody and immunohistochemistry. A Tag positive hepatocellular carcinoma cell line from transgenic mice and a kidney biopsy from a patient infected by SV40 were used as positive controls. RESULTS: None of the tumours (20 ependymomas, 20 glioblastomas, 12 oligodendrogliomas, three plexus choroid adenomas, two plexus choroid carcinomas, 15 meningiomas, and 10 medulloblastomas) contained SV40 Tag positive cells. CONCLUSIONS: The lack of SV40 Tag in 82 CNS tumours of various types is at variance with previous studies from different countries, and suggests that the virus may not be an important factor in CNS tumorigenesis, at least in French cases.     

Co-expression of CXCR4 and CXCR7 in human endometrial stromal cells is modulated by steroid hormones

Endometrium modulated by estrogen (E) and progesterone (P) is important for implantation and pregnancy. The present study compared the expression of chemokine CXCL12 and chemokine receptor CXCR4 and CXCR7 between human cycling and early pregnant endometria by immunohistochemistry (IHC). Then the modulation of E and P on expression of CXCL12, CXCR4 and CXCR7 in human endometrial stromal cells (ESCs) was explored at both mRNA and protein level. The result of IHC showed that human ESCs of the menstrual period did not express CXCL12, CXCR4 or CXCR7 protein, however, the expression of CXCR4 and CXCR7 but not CXCL12 in ESCs increased in the proliferative and secretory phase, and the expression intensity for CXCR4 and CXCR7 in ESCs was the highest in the first trimester. Moreover, E and P were able to up-regulate the mRNA and protein expression of CXCR4 and protein expression of CXCR7 in ESCs (P<0.01). Thus, ESCs spatiotemporally co-express CXCR4 and CXCR7 rather than CXCL12, and E and P are able to regulate the expression of CXCR4 and CXCR7 in ESCs, suggesting the modulation of steroid hormones on chemokine receptor expression in ESCs.     

SV40 17KT antigen complements dnaj mutations in large T antigen to restore transformation of primary human fibroblasts

Transformation of human cells requires both SV40 large T and small t antigens. Plasmids that contained mutations in the amino-terminal dnaJ domain of the early region fail to transform human diploid fibroblasts. However, large T dnaJ mutants can be rescued by plasmids that express early region products other than large T antigen. The protein found to be responsible for such complementation was the third early region product, 17KT. Similar to large T, this protein reduces levels of the retinoblastoma-related protein, p130, and stimulates cell-cycle progression of quiescent fibroblasts, two activities of large T that are disrupted by dnaJ mutations.     

Decidualization of human endometrial stromal cells in vitro: effects of progestin and relaxin on the ultrastructure and production of decidual secretory proteins

Decidual cells arise by proliferation and differentiation ofendometrial stromal cells of the uterus after appropriate stimulationby ovarian hormones. Previously we have shown that progestinand relaxin stimulate the secretion of several decidual-cell-specificsecretory proteins in a long-term primary cell culture system.We now report the effects of progestin and relaxin on the morphologyof stromal cells in association with the production rate oftwo major decidual secretory proteins, prolaction and insulin-likegrowth factor binding protein-1 (IGFBP-1). Stromal cells werecultured in RPMI 1640 and 2% fetal bovine serum for 22 daysunder control conditions (no hormone), with relaxin or medroxyprogesteroneacetate (MPA), or MPA plus relaxin. Cells treated with MPA aloneor MPA plus porcine relaxin grew to a high density with manyareas of heaping while control cells and cells grown in mediumcontaining relaxin alone formed discontinuous layers. The cytoplasmwas distinguished by aggregates of rough endoplasmic reticulumand secretory granules. Surfaces of cells treated with MPA plusrelaxin had clusters of short blunt processes containing secretorygranules. The processes were rarely seen in cells exposed toMPA alone and completely absent in control cells or cells exposedto relaxin alone. Intercellular space was greatly widened incells treated with MPA alone or MPA plus relaxin. There weremany extended gap junctions in MPA and relaxin-treated cellsin contrast to controls. The production rates of prolactin andIGFBP-1 measured by radioimmunoassay and enzyme-linked immunosorbentassay respectively increased from 0.004 to 0.7 µg forprolactin and 0.01 to 44 µg for IGFBP-1 per 10⁶ cells/dayafter 20 days incubation with MPA and relaxin, in agreementwith the previous findings. The parallel biochemical eventsand morphological changes are similar to those of decidualizationof endometrium from the secretory phase to the early gestationin vivo.     

Roles of p38 and c-jun in the differentiation, proliferation and immortalization of normal human endometrial cells

BACKGROUND: It has been reported that p38 and c-jun operateas mediators of cell proliferation and differentiation. Therefore,by studying the roles of c-jun and p38 in the proliferationand differentiation of normal human endometrial cells, we canbetter understand the mechanism of these processes in endometrialcells. METHODS: Separation of glandular and stromal componentswas based on a modification of the work of Satyaswaroop et al.To confirm the purification of the endometrial cells and theexpression of the transfected SV40 large T antigen, immunocytochemicalanalysis and western blot analysis were performed. RESULTS:There were polygonal shapes in the stromal cells in the earlypassage 1–2, while the aged endometrial stromal cellswere spindle shaped. To investigate passage-dependent molecularevents in endometrial cells, the c-jun and pp38 levels wereexamined. Both c-jun and pp38 were significantly reduced withcellular aging and passages. To understand the role of c-jun,endometrial stromal cells were treated with SP600125 which isa specific inhibitor of c-jun. SP600125 induced morphologicalchanges of young endometrial stromal cells with polygonal shape;the young cells appeared as aged endometrial cells with spindleshape. In addition, an immortalized endometrial cell line wasestablished and shown to express activated c-jun, similiar tonormal endometrial cells. CONCLUSIONS: These results suggestthat the modulation of p38 and c-jun may play an important rolein the differentiation and proliferation of human endometrialcells.     

Expression of a secretory product by microvillous and ciliated cells of the human endometrial epithelium in vivo and in vitro

A monoclonal antibody which identifies a component of post-ovulatoryendometrial secretions is now shown to be expressed within thecytoplasm and on the cell surface of both microvillous and ciliatedepithelial cells. A glandular explantatlon model was developedin order to study the ‘carry over’ of this secretionto the regenerative phase endometriwn. A loss of cytoplasmicantigen was observed in vitro. However, it was retained on thecell surface in a fashion consistent with its expression atthe time of explantation. Mosaicism of expression of this secretorycomponent occurs throughout the secretory-phase and is particularlypronounced at the time of transition from proliferative to secretoryphase. It is conduded that both ciliated and microvillous epithelialcells produce a post-ovulatory secretory component which maybe retained on the cell surface in the absence of hormonal stimulation.     

Fluorescent laser scanning microscopy of F-actin disruption in human endometrial stromal cells expressing the SV40 large T antigen and the EJ ras oncogene

To attempt to understand the effects of the SV40 large T antigen and an activated EJ ras oncogene on F-actin organization, we compared normal human endometrial stromal cells (HESC; proliferating, short life span) to cells transfected with the SV40 large T antigen either alone or in combination with the EJ ras oncogene. Normal HESC displayed numerous bundles of actin filaments (stress fibers) evenly distributed throughout the cell. In HESC transfected with a plasmid containing the gene for a temperature-sensitive SV40 large T antigen, stress fibers were disrupted and the remaining F-actin was also disrupted and clumped near the plasma membrane. Cells expressing both the SV40 large T antigen and the EJ ras oncogene sometimes appeared rounded, with stress fibers organized mainly near the cell periphery. Under restrictive temperature conditions for the function of the SV40 large T antigen, cells with or without the EJ ras oncogene reorganize actin stress fibers to resemble those of normal HESC. Therefore, the EJ ras oncogene alone does not disrupt F-actin organization. When operating in cooperation with the SV40 large T antigen, however, it leads to the reorganization of F-actin at the cell periphery and confers a rounded structure on the cells.     

Effects of the SV40 large T antigen and EJ ras oncogene on fibronectin localization in human endometrial cells as viewed by confocal laser scanning microscopy.

We utilized confocal laser scanning microscopy to examine the localization of fibronectin deposition in cultures of human endometrial stromal cells. We found that fibronectin in normal human endometrial stromal cell cultures was both intracellular, occurring in rough endoplasmic reticulum and in perinuclear regions, and extracellular, occurring diffusely over the entire cell surface. Endometrial stromal cells were transfected with a plasmid containing an origin-defective Simian Virus 40 (SV40) which codes for a temperature-sensitive large T antigen. When these cells were placed under temperature-restrictive conditions for large T-antigen function, they exhibited staining patterns similar to normal endometrial cells. Fibronectin deposition in cultures of partially or fully transformed endometrial cells was not intracellular as in normal cells, but was localized primarily between cells. Cells expressing the SV40 large T antigen deposited fibronectin mainly in parallel clumps between cells. Cells expressing both the SV40 large T antigen and the EJ ras oncogene, at high cell density, displayed networks of fibronectin arranged in matrix-like patterns between cells. The malignant cell line examined, sarcoma cells, also exhibited fibronectin networks between cells. Cell density affected fibronectin deposition in endometrial stromal cells expressing the EJ ras oncogene. At low density, cells expressing the SV40 large T antigen and the EJ ras oncogene displayed diffuse fibronectin patterns and, at high density, these cells formed colonies with networks of fibronectin between cells.     

Endothelial cell proliferation in the endometrium of women with menorrhagia and in women following endometrial ablation

Local endometrial aberrations are thought to be the major contributingfactor to essential menorrhagia. Here we have examined the roleof endometrial angiogenesis, the growth of new blood vessels,in essential menorrhagia. Our study tested two hypotheses: firstlythat angiogenesis is disturbed in the endometrium of women withmenorrhagia; and secondly that when menstrual blood loss isdecreased following endometrial ablation, an endometrial environmentfavouring normal angiogenesis has returned. Angio-genesis wasmeasured by endothelial cell proliferation. Proliferating endothelialcells were identified by an immunohistochemical double stainingtechnique. A total of 57 women participated in this study, ofwhom 19 were controls, 20 had menorrhagia and 18 were 3–6months post-ablation. There was a significant increase in endothelialcell proliferation in the endometrium of patients with menorrhagiacompared with the control endometrium. Conversely, post-ablationendometrium showed a nonsignificant decrease in endothelialcell proliferation. The increased endothelial cell proliferationin the endometrium of patients with menorrhagia was not theresult of a general increase in endometrial cellular proliferationand did not result in a change in endothelial cell concentrationcompared with control endometrium. These results support thehypothesis that angiogenesis is disturbed in the endo-metriumof patients with menorrhagia and normalized in post-ablationendometrium.     

Induction of a polarized micro-environment by human T cells and interferon-gamma in three-dimensional spheroid cultures of human endometrial epithelial cells

Expression of human leukocyte antigen (HLA)-DR molecules andproliferation of epithelium in human endometrium are polarized.We have suggested that the induction of such a polarized micro-environmentis T cell and interferon (IFN)-gamma dependent. The presentstudy was designed to demonstrate the induction of such a micro-environmentaround T cells and around the source of IFN-gamma. Spheroidsreminiscent of endometrial glands were formed by allowing three-dimensionalaggregation of endometrial epithelial cells of a cloned HLA-DRnegative endometrial carcinoma cell line (ECC1) over agarose.Both HLA-DR expression and inhibition of proliferation werefound to be directly dependent on the dose of IFN-gamma thatwas allowed to diffuse in the agarose beneath the spheroids.To show that the interaction of the epithelial cells with activatedT cells also induces HLA-DR molecules in a paracrine fashionin the epithelial cells, ECC1 spheroids were co-cultured withincreasing numbers of allogeneic peripheral blood T cells forvarious time-intervals. T cells bound to the ECC1 cells, andbecame activated as indicated by the expression of interleukin(IL)-2 receptor and HLA-DR molecules. A focal HLA-DR expressionbecame apparent in the ECC1 cells adjacent to the T cells. Asthe number of T cells added to spheroid cultures was increased,a concomitant increase in the number of HLA-DR positive ECC1cells occurred and HLA-DR immunoreactivity was enhanced in eachcell. There was a corresponding decrease in the proliferationof the ECC1 cells in T cell-ECC1 spheroid co-cultures. Basedon these data, we suggest that activation of T cells is associatedwith the induction of HLA-DR expression and inhibition of proliferationin a paracrine fashion in the epithelial cells and may be responsiblefor the creation of a polarized micro-environment in vivo.     

Initiation of DNA synthesis and uptake of T antigen by chick erythrocyte nuclei in heterokaryons with SV40-transformed human cells

Nonsynchronized and hydroxyurea (HU)-synchronized SV40-transformed human cells (W₉₈VaD) were fused with chick embryo erythrocytes (CE). The uptake of T antigen by CE nuclei was compared with initiation of chick nuclear DNA synthesis. Uptake of T antigen by CE nuclei occurred at about the same timeafter fusion with asynchronous as with HU-synchronized cells. CE nuclei rapidly became T antigen-positive between 16 h and 28 h after fusion and usually almost all CE nuclei were T antigen-positive by 48 h after fusion. In contrast, initiation of chick nuclear DNA synthesis occurred as a function of timeafter reversal of the HU block, when the host cell nuclei were also synthesizing DNA. Chick nuclear DNA synthesis occurred in many heterokaryons before the CE nuclei became T antigen-positive by immunofluorescence.     

Selection of fetal bovine serum for use in the C3H/10T1/2 CL8 cell transformation assay system

The purpose of this communication is to report our experience concerning the variation in cloning efficiency and transformation frequency utilizing C3H/10T1/2 CL8 cells with 23 different lots of fetal bovine sera. These sera were purchased from five different commercial sources. The standard cell transformation assay using 1,000 cells per dish and 3-methylcholanthrene (7.5 μg/ml) as the transforming agent was performed. The chemical exposure period was 3 days. The cloning efficiency was determined in parallel toxicity tests using 200 cells per dish. Only three out of 23 serum lots supported a strong response in cell transformation. The results indicated that variation in the ability of sera to support cell transformation was not supplier dependent. In addition, our results showed that serum lots exhibiting the best cloning efficiencies did not necessarily support cell transformation. It is apparent that reliance on cloning efficiency alone would be inadequate as a means of selecting a serum lot. We therefore recommend that a complete cell transformation assay be performed when selecting fetal bovine serum for use in this assay.     

Influence of amino acids encoded in the 3′ open reading frame of the SV40 early region on transformation and antigenicity of large T antigen

The mutant dlA2414 bears a frame-shift deletion of nucleotides 2936-2927 in the coding sequence for the simian virus 40 (SV40) large T antigen. Based on its nucleotide sequence, this mutant should produce a T antigen containing the first 627 authentic large T antigen amino acids followed by 97 amino acids encoded in the alternate open reading frame at the 3' end of the early region. This protein resembles the hypothetical T* protein that would be translated from an early SV40 mRNA if it were spliced to permit utilization of the open reading frame. We show that stable mouse cell lines can be generated that express the T antigen produced by dlA2414 and that this T antigen has an altered carboxy terminus. In addition, the expected tryptic peptides were missing from the large T antigen and replaced by more hydrophobic peptides. The T*-like protein produced by dlA2414 was apparently less stable than wild-type T antigen and did not stably complex with the cellular phosphoprotein p53. This protein retained the ability to immunize mice against a challenge of syngeneic SV40-tumor cells. The dlA2414 T antigen was expressed at the surface of cells as shown by in vitro lymphocyte-mediated cytotoxicity assay. The results presented here also showed that the expression of a T*-like protein at the cell surface is not likely to be essential for tumorigenesis of cells transformed by SV40.     

Enhanced sensitivity of the C3H/10T1/2 cell transformation system to alkylating and chemotherapeutic agents by treatment with 12-O-tetradecanoylphorbol-13-acetate

The failure of the C3H/10T1/2 cell transformation system to respond to numerous known carcinogens has limited its applications for the detection and study of cancer-causing substances. Recent studies have found, however, that some carcinogens function as initiating agents for the process of transformation in these cells. Treatment with such agents is generally not sufficient to transform low-density asynchronous cultures of C3H/101/2 cells, but morphologic transformation will occur if such cultures are subsequently exposed to the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, the ability of TPA to enhance transformation was examined in cultures treated with a variety of chemical agents. The addition of TPA after chemical treatment enhanced the transformation of these cells by methylmethanesulfonate, ethylmethanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, N-nitrosomethylurea, N-nitrosoethylurea, mitomycin C, 5-fluorodeoxyuridine, and 5-azacytidine. Treatment with amethopterin or benzo(e)pyrene did not produce significant numbers of foci in the presence or absence of TPA. TPA inhibited transformation by high concentrations of 3-methylcholanthrene and benzo(a)pyrene. Thus, numerous carcinogens function as initiating agents for these cells and the presence of TPA can dramatically increase the sensitivity of this cell transformation system.     

Induction of apoptosis in the LNCaP human prostate carcinoma cell line and prostate adenocarcinomas of SV40T antigen transgenic rats by the Bowman–Birk inhibitor

The soybean-derived serine protease inhibitor, Bowman–Birk inhibitor (BBI), has been reported as a potent chemoprevention agent against several types of tumors. The present study was undertaken to evaluate the effects of BBI on androgen-sensitive/dependent prostate cancers using a human prostate cancer cell (LNCaP) and the transgenic rats developing adenocarcinoma of the prostate (TRAP) model. Treatment of LNCaP prostate cancer cells with 500 µg/mL BBI resulted in inhibition of viability measured on WST-1 assays, with induction of connexin 43 (C×43) and cleaved caspase-3 protein expression. Feeding of 3% roughly prepared BBI (BBIC) to TRAP from the age 3 weeks to 13 weeks resulted in significant reduction of the relative epithelial areas within the acinus and multiplicity of the adenocarcinomas in the lateral prostate lobes. C×43- and terminal deoxynucleotidyl transferase mediated dUTP-biotin end labeling of fragmented DNA (TUNEL)-positive apoptotic cancer cells were more frequently observed in the lateral prostates treated with BBIC than in the controls. These in vivo and in vitro results suggest that BBI possesses chemopreventive activity associated with induction of C×43 expression and apoptosis.     

Aberrant expression of the monocyte/macrophage phenotype in a human T cell line immortalized by HTLV-I and an adult T cell leukemia/lymphoma cell line

An HTLV-I-immortalized human T cell line (JP-2), a N -methyl- N '-nitro- N -nitrosoguanidine-treated JP-2 line (JP-2T), and an adult T cell leukemia cell line (ATL-1T) were examined morphologically and phenotypically. All of these cell lines expressed some T cell markers, including CD4, and showed rearrangement of T cell receptor (TCR) genes, but they lacked CD3 and TCR antigens and expressed some myelomonocytic markers (CD68, HL-21, CD15, CD16). JP-2 cells grew in suspension, but JP-2T and ATL-1T cells, which mostly adhered to the surface of culture vessels, showed macrophage-like morphological features and expressed more monocyte/macrophage markers (lysozyme, α₁-antitrypsin) and fibronectin. ATL-1T cells transplanted into SCID mice lost the macrophage features. These results suggest that HTLV-I infected T cells can express some macrophage features and that these cells may provide a model that will be useful in elucidating the phenotypic variability of T cell lymphomas.     

Cytotoxic T lymphocyte antigen 4 immunoglobulin modified dendritic cells attenuate allergic airway inflammation and hyperresponsiveness by regulating the development of T helper type 1 (Th1)/Th2 and Th2/regulatory T cell subsets in a murine model of asthma

T helper type 2 (Th2) and regulatory T cells (T(reg) ) have been postulated to have critical roles in the pathogenesis of allergic asthma. Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) gene-modified dendritic cells (DC-CTLA4Ig) have the potential to reduce Th2 cells and induce T(reg) cells. In the present study, we evaluated the therapeutic effects and potential mechanisms of the adoptive transfer of DC-CTLA4Ig into mice in an experimental model of asthma. BALB/c mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA for 7 days. Just prior to the first challenge, DC-CTLA4Ig, DCs or DCs infected with DC-green fluorescent protein (GFP) were injected intravenously into mice. The administration of DC-CTLA4Ig reduced airway hyperresponsiveness, relieved asthmatic airway inflammation and decreased the numbers of esosinophils in the BALF in OVA-sensitized/challenged mice. In addition, DC-CTLA4Ig altered the balance of Th1/Th2 cytokine production in the lungs with increased interferon (IFN)-γ levels and decreased interleukin (IL)-4 levels, decreased the percentage of Th2 and increased both the percentage of Th1 and T(reg) cells in the lungs of OVA-sensitized/challenged mice. This research demonstrates that DC-CTL4Ig reduces airway hyperresponsiveness effectively and prevents airway inflammation in OVA-sensitized/challenged mice, which is due most probably to attenuated secretion of Th2 cytokines and increased secretion of Th1 cytokines in the local airway, and the correction of the pulmonary imbalance between Th1/Th2 cells and Th2/T(reg) cells.