Composition of a Medium for Serum-free Culture of an Adipose-derived Stem Cell Line Established with a Simian Virus 40 T Antigen

ABSTRACT Serum-free mouse embryo (SFME) cells are a neural stem cell line that is dependent upon epidermal growth factor (EGF) for survival. Removal of EGF results in the G1 arrest and apoptosis of SFME cells. We have shown that the expression of simian virus 40 large T antigen in SFME cells blocks apoptosis and allows cell survival and division in the absence of EGF. Therefore the presence of T antigen abrogates the EGF requirement. The steady-state levels of p53, p21, and mdm-2 do not increase as SFME cells undergo apoptosis upon EGF withdrawal. Furthermore, the amino-terminal 136 amino acids (N136) of T antigen are sufficient to block death and to promote proliferation in the absence of EGF, while the carboxy-terminal fragment (C251–708), which contains the p53 binding site, is unable to block death. Taken together, these data suggest that SFME cells deprived of EGF undergo p53-independent apoptosis. Mutations that disrupt either the J domain or Rb family binding abolish the ability of T antigen to block SFME cell apoptosis and to promote cell growth. We conclude that T antigen must act on one or more members of the Rb family to inhibit SFME cell apoptosis.

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Cyclic AMP regulates PDGF-stimulated signal transduction and differentiation of an immortalized optic-nerve-derived cell line

Journal of Experimental Biology ◽

10.1242/jeb.202.4.461 ◽

1999 ◽

Vol 202 (4) ◽

pp. 461-473

Author(s):

R.I. Cohen ◽

R. Mckay ◽

G. Almazan

Keyword(s):

Signal Transduction ◽

Optic Nerve ◽

Growth Factor ◽

Cyclic Amp ◽

Cell Line ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Temperature Sensitive ◽

Permissive Temperature

To facilitate the study of the molecular events underlying the development of optic-nerve-derived oligodendrocytes and their growth-factor-related signal transduction events, we immortalized perinatal rat optic nerve cells with a temperature-sensitive simian virus 40 large T-antigen, carrying the tsA58 and U19 mutations, via a retrovirus vector. The line, tsU19-9, was selected on the basis of the expression of the neural precursor marker nestin. At the permissive temperature, 33 degreesC, tsU19-9 cells had a flat epithelial morphology. In contrast, following exposure to platelet-derived growth factor (PDGF), a factor important in the lineage progression of oligodendrocytes, or in the presence of dibutyryl cyclic AMP at 39 degreesC (the non-permissive temperature), the cells underwent morphological and antigenic differentiation to cells characteristic of the oligodendrocyte lineage. We used this cell line to investigate the binding characteristics of PDGF and related signalling cascades. Competition binding, phosphoinositide hydrolysis and intracellular Ca2+ mobilization assays all demonstrated that the three different isoforms of PDGF (AA, AB and BB) bound to and acted on the cell line. Overnight exposure to forskolin, a treatment that initiated morphological and phenotypic progression into an oligodendrocyte lineage, decreased PDGF-BB-induced intracellular Ca2+ mobilization and inhibited basal and PDGF-stimulated [3H]thymidine incorporation. Our results demonstrate that tsU19-9 may serve as a resource to study early optic-nerve oligodendrocyte development.

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Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6664-6673.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6664-6673

Author(s):

T E Riley ◽

A Follin ◽

N C Jones ◽

P S Jat

Keyword(s):

Cell Line ◽

Growth Arrest ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Growth Defect ◽

Primary Cells ◽

Nonpermissive Temperature ◽

Large T Antigen ◽

Adenovirus E1a

Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.

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Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6664 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6664-6673 ◽

Cited By ~ 5

Author(s):

T E Riley ◽

A Follin ◽

N C Jones ◽

P S Jat

Keyword(s):

Cell Line ◽

Growth Arrest ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Growth Defect ◽

Primary Cells ◽

Nonpermissive Temperature ◽

Large T Antigen ◽

Adenovirus E1a

Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.

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Immortalization of Rat Eustachian Tube Epithelial Cells by Adenovirus 12-Simian Virus 40 Hybrid Virus

Annals of Otology Rhinology & Laryngology ◽

10.1177/000348940211101011 ◽

2002 ◽

Vol 111 (10) ◽

pp. 919-925 ◽

Cited By ~ 2

Author(s):

Sunji Jin ◽

Shigehiro Ueyama ◽

Sung-Kyun Moon ◽

Johng S. Rhim ◽

Xin-Xing Gu ◽

...

Keyword(s):

Epithelial Cells ◽

Cell Line ◽

Otitis Media ◽

Eustachian Tube ◽

Primary Cultures ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Male Rat ◽

Positive Staining

The eustachian tube epithelial cells play an important role in the initial pathogenesis of otitis media. In order to study the role of the eustachian tube epithelial cells in the pathogenesis of otitis media, we have established a rat eustachian tube epithelial cell line. The cell line was derived by infecting primary cultures of eustachian tube epithelial cells with the adenovirus 12-simian virus 40 (Ad12-SV40) hybrid virus. The immortalized cells have retained the morphological characteristics of the parental cells and show positive staining with anti-cytokeratin antibodies (a marker for epithelial cells), but not with anti-vimentin antibodies (a fibroblast marker). The cells have been in continuous culture for more than 10 months and have undergone 38 passages. Western blotting and cell staining have confirmed the expression of the SV40 T antigen and p53. Chromosomal analysis indicates that the cell line is aneuploid and derived from male rat epithelial cells. Together, our results suggest that the cell line originated from eustachian tube epithelial cells from a male rat and was successfully immortalized by the Ad12-SV40 virus.

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DEVELOPMENT OF THE CONDITIONALLY IMMORTALIZED TESTICULAR SERTOLI CELL LINE TTE3 EXPRESSING SERTOLI CELL SPECIFIC GENES FROM MICE TRANSGENIC FOR TEMPERATURE SENSITIVE SIMIAN VIRUS 40 LARGE T ANTIGEN GENE

The Journal of Urology ◽

10.1097/00005392-200203000-00094 ◽

2002 ◽

pp. 1538-1545

Author(s):

YOSHIAKI TABUCHI ◽

SHOICHIRO OHTA ◽

NOBUAKI YANAI ◽

MASUO OBINATA ◽

TAKASHI KONDO ◽

...

Keyword(s):

Cell Line ◽

Sertoli Cell ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Temperature Sensitive ◽

Large T Antigen ◽

Antigen Gene ◽

Sertoli Cell Line

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Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3864-3871.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3864-3871

Author(s):

D A Williams ◽

M F Rosenblatt ◽

D R Beier ◽

R D Cone

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Cell Lines ◽

Stromal Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Hematopoietic Stem ◽

Stem Cell Proliferation

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.

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The growth-stimulatory effect of simian virus 40 T antigen requires the interaction of insulinlike growth factor 1 with its receptor

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5069-5077.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5069-5077

Author(s):

P Porcu ◽

A Ferber ◽

Z Pietrzkowski ◽

C T Roberts ◽

M Adamo ◽

...

Keyword(s):

Growth Factor ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Serum Free Medium ◽

Free Medium ◽

3T3 Cells ◽

Sv40 T Antigen ◽

Serum Free ◽

Insulinlike Growth Factor

We have used a plasmid expressing a temperature-sensitive (ts) mutant of simian virus 40 (SV40) T antigen, stably transfected into 3T3 cells, to study the role of insulinlike growth factor 1 (IGF-1) and its receptor in T-antigen-mediated growth. While 3T3 cells do not grow in serum-free medium, in 1% serum, or with the sole addition of either platelet-derived growth factor (PDGF) or IGF-1, cells expressing the tsA T antigen (BALB 58 cells) grow at 34 degrees C in either PDGF or 1% serum but not in IGF-1. At the restrictive temperature (39.6 degrees C), these cells can only grow in 10% serum. We show that BALB 58 cells, at 34 degrees C, have a markedly increased expression of IGF-1 and IGF-1 mRNA and that their growth in 1% serum (at 34 degrees C) is inhibited by an antisense oligodeoxynucleotide to the IGF-1 receptor RNA. When this tsA plasmid is stably transfected into cells constitutively overexpressing the human IGF-1 receptor cDNA, the resulting cell lines show a constitutively phosphorylated IGF-1 receptor and grow in serum-free medium at 34 degrees C (but not at 39.6 degrees C). A functional SV40 T antigen also increases the expression of a plasmid in which the reporter luciferase gene is under the control of a rat IGF-1 promoter. We conclude (i) that the SV40 T antigen induces the expression of IGF-1 and IGF-1 mRNA, at least in part by a transcriptional mechanism, thus altering the growth factors requirements, and (ii) that, in BALB/c3t3 cells, the SV40 T antigen necessitates a functional IGF-1 receptor for its growth-stimulating effect in low serum (or PDGF).

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