scholarly journals Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen

2001 ◽  
Vol 171 (1) ◽  
pp. 45-55 ◽  
Author(s):  
TM Thway ◽  
CM Clay ◽  
JK Maher ◽  
DK Reed ◽  
KJ McDowell ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Lineage ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Alpha Subunit ◽  
Subunit Gene ◽  
Immortalized Cell Lines ◽  
Subunit Expression ◽  
Immortalized Cell

Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a chorionic girdle cell lineage was verified by immunological detection of cell-surface antigens specific to equine invasive trophoblasts. The cell lines differed, however, from mature chorionic girdle cells or endometrial cup cells in that they did not produce eCG and did express MHC class I molecules. Thus, these cell lines appear to have been arrested at a stage of development prior to final differentiation into endometrial cup cells. It was also determined that some of these cell lines as well as endometrial cups express the estrogen receptor-related receptor beta gene, but not the glial cell missing gene (GCMa) both of which are expressed in the murine and human placenta. Among these cell lines, three (eCG 50.5, 100.6 and 500.1) express eCG alpha mRNA. Since regulation of eCG alpha subunit gene is largely unknown, we investigated the signal transduction pathways regulating the eCG alpha subunit gene. Both activators of protein kinase A (PKA) and protein kinase C (PKC) induced the expression of eCG alpha subunit expression 3.2 (P<0.05)- and 1.9 (P<0.05)-fold respectively, in the eCG 500.1 cell line. However, activation of these pathways failed to induce eCG beta subunit expression. In conclusion, lines of equine trophoblast cells have been immortalized that display markers characteristic of those with the equine chorionic girdle and endometrial cup cell lineage. A subset of these cells expresses the eCG alpha subunit gene which is responsive to activators of the PKA and PKC signal transduction pathways.

scholarly journals Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

1985 ◽  
Vol 5 (4) ◽  
pp. 642-648 ◽  
Author(s):  
J A Small ◽  
D G Blair ◽  
S D Showalter ◽  
G A Scangos
Keyword(s):  
Cell Lines ◽  
Choroid Plexus Papilloma ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Primary Cell ◽  
Tissue Samples ◽  
Primary Cell Lines

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens

1986 ◽  
Vol 6 (4) ◽  
pp. 1204-1217
Author(s):  
P S Jat ◽  
C L Cepko ◽  
R C Mulligan ◽  
P A Sharp
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Vector System ◽  
Large T Antigen ◽  
T Antigens ◽  
Sv40 Large T Antigen ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.

scholarly journals Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

1989 ◽  
Vol 9 (7) ◽  
pp. 3093-3096 ◽  
Author(s):  
R L Radna ◽  
Y Caton ◽  
K K Jha ◽  
P Kaplan ◽  
G Li ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Cellular Aging ◽  
Cellular Growth ◽  
Viral Gene ◽  
Large T Antigen ◽  
Temperature Dependent

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.

scholarly journals Hormonal regulation of the transformation phenotype in simian virus 40-transformed rat embryonic preadipose cell lines.

1984 ◽  
Vol 4 (4) ◽  
pp. 712-721 ◽  
Author(s):  
S Yasumoto
Keyword(s):  
Cell Density ◽  
Cell Lines ◽  
Hormonal Regulation ◽  
Growth Regulation ◽  
High Cell Density ◽  
Calf Serum ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
High Cell

The regulation of transformed phenotypes was studied in newly isolated preadipose cell lines which were established after infection with simian virus 40 tsA58 dl2009. The clonal cell lines isolated exhibited most of the characteristics typical of transformed cells. The transformants, however, were able to differentiate into adipocytes in the presence of low calf serum (0.5%) and a combination of several hormones, including hydrocortisone and insulin. Treatment with insulin alone stimulated the growth of these cells but did not induce lipid accumulation without added hydrocortisone. The effect of hydrocortisone was accompanied by a restoration of growth control in the transformants after they reached high cell density. The blot hybridization analysis of cellular DNAs digested by restriction enzymes revealed that simian virus 40 genomes were integrated at multiple separate sites at which a head-to-tail oligomeric insertion took place. Large T antigen was synthesized in growing cells but was regulated at high cell density when cells were committed to differentiate by glucocorticoids. These results suggest that the glucocorticoid hydrocortisone is capable of restoring growth regulation at high cell densities to simian virus 40-transformed preadipose cell lines.

scholarly journals Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

1985 ◽  
Vol 5 (5) ◽  
pp. 1043-1050 ◽  
Author(s):  
R E Lanford ◽  
C Wong ◽  
J S Butel
Keyword(s):  
Cell Lines ◽  
Mouse Embryo ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Mouse Embryo Fibroblasts ◽  
Established Cell Lines ◽  
Established Cell ◽  
Embryo Fibroblasts

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

Gastric epithelial morphogenesis in normal and transgenic mice

1997 ◽  
Vol 272 (5) ◽  
pp. G1209-G1220 ◽  
Author(s):  
S. M. Karam ◽  
Q. Li ◽  
J. I. Gordon
Keyword(s):  
Parietal Cell ◽  
Cell Lineage ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Parietal Cells ◽  
Cellular Migration ◽  
Multipotent Stem Cells ◽  
Proliferative Zone ◽  
Beta Subunit Gene

The epithelium located in the corpus of the adult mouse stomach forms mucosal invaginations known as gastric units. Gastric units are populated by members of the pit, parietal, and neck-zymogenic cell lineages all of which are derived from multipotent stem cells. Gastric unit morphogenesis was examined in normal embryonic day 18 (E18) to postnatal day 28 (P28) FVB/N mice with electron microscopy and multilabel immunohistochemistry. E18 units appear as short, solid infoldings (primordial buds), 92% of whose cells represent pit, parietal, and neck cell precursors. Although the total number of cells per bud does not change from P1 to P7, immature cells decrease to 22% as differentiated pit, neck, and parietal cells appear. From P7 to P15, lineage precursors and their differentiated progeny increase and buds elongate. Between P15 and P21 the multipotent stem cell and its descendants are assembled into a distinct proliferative zone (isthmus) located in the midportion of each unit, and cellular migration-differentiation programs become compartmentalized. To examine the role of parietal cells in regulating gastric unit morphogenesis, nucleotides -1035 to +24 of the mouse H(+)-K(+)-adenosinetriphosphatase beta-subunit gene were used to express simian virus 40 large T antigen (SV40 TAg) exclusively in this lineage. SV40 TAg amplified the normally rare pre-parietal cell and disclosed a pre-parietal cell precursor. Pre-parietal cells and their precursors were the predominant cells in E18-P1 transgenic buds. At later stages of development (P1-P28) there was a block in differentiation of pre-parietal to mature parietal cells, a decrease in neck cells, and a marked depletion of zymogenic cells. These findings suggest that members of the parietal cell lineage are the source of instructions that affect the neck-zymogenic cell lineage, even before the gastric unit is compartmentalized into its anatomically distinct pit, isthmus, neck, and base regions.

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