Simian virus 40 T antigen induces p53-independent apoptosis but does not suppress erbB2/neu gene expression in immortalized human epithelial cells

ABSTRACT Human keratinocytes immortalized by full-length or early-region simian virus 40 (SV40) DNA grow in agarose and form tumors in nude mice, in contrast to keratinocytes immortalized by the E6/E7 genes of human papillomaviruses. To determine the molecular basis for this biological difference in growth, we have used the individual SV40 oncogenes (large T antigen [LT] and small t antigen [st]) and human papillomavirus oncogenes (E6/E7) to study the progression of human epithelial cells from the nonimmortal to the immortal state as well as from the immortal to the anchorage-independent state. Transfection of primary human foreskin keratinocytes with LT did not immortalize cells but did extend the in vitro life span and produced cells that were resistant to calcium- and serum-induced terminal differentiation. Cells transfected with st alone did not passage beyond vector-transfected keratinocytes. The simultaneous expression of LT- and st-immortalized keratinocytes occurred without evidence of crisis and, as anticipated, these immortal cells were anchorage- independent for growth. Interestingly, we found that keratinocytes expressing both LT and st, but not keratinocytes with LT alone, exhibited increased phosphorylation of the protein kinase AKT. In addition, AKT activation was paralleled by an increase in telomerase activity. Addition of st to anchorage-dependent keratinocytes, expressing either LT (nonimmortal) or E6/E7 (immortal), converted the cells to anchorage independence, with similar accompanying increases in AKT phosphorylation and telomerase activity. However, it was not possible to induce keratinocyte growth in agarose with activated AKT and/or overexpressed hTERT, indicating that these newly defined st-induced activities are not sufficient for progression to the anchorage-independent state.

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Strontium phosphate transfection of human cells in primary culture: stable expression of the simian virus 40 large-T-antigen gene in primary human bronchial epithelial cells

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.2031-2034.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 2031-2034

Author(s):

D E Brash ◽

R R Reddel ◽

M Quanrud ◽

K Yang ◽

M P Farrell ◽

...

Keyword(s):

Epithelial Cells ◽

Primary Culture ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Bronchial Epithelial Cells ◽

Large T Antigen ◽

Bronchial Epithelial ◽

Antigen Gene ◽

Strontium Phosphate

Strontium ion formed DNA-phosphate precipitates analogous to those formed by calcium but lacking the lethal and differentiation-inducing effects of calcium on many epithelial cell types in primary culture. Human primary bronchial epithelial cells were transiently and stably transfected by using strontium phosphate; the frequency of stable transformation with a plasmid carrying the simian virus 40 large-T-antigen gene was greater than 10(-4).

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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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Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.1034-1042.1985 ◽

1985 ◽

Vol 5 (5) ◽

pp. 1034-1042

Author(s):

J C Alwine

Keyword(s):

Gene Expression ◽

High Activity ◽

Plasmid Dna ◽

Promoter Activity ◽

Transient Gene Expression ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

293 Cells ◽

E1a Protein

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.

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Deletion of the carboxy terminus of simian virus 40 large T antigen affects viral late gene expression.

10.1349/ddlp.2888 ◽

1990 ◽

Author(s):

Terryl Stacy

Keyword(s):

Gene Expression ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Late Gene ◽

Large T Antigen ◽

Carboxy Terminus ◽

Late Gene Expression

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Methylation of single sites within the herpes simplex virus tk coding region and the simian virus 40 T-antigen intron causes gene inactivation.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.2004 ◽

1994 ◽

Vol 14 (3) ◽

pp. 2004-2010 ◽

Cited By ~ 28

Author(s):

A Graessmann ◽

G Sandberg ◽

E Guhl ◽

M Graessmann

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Dna Synthesis ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Coding Region ◽

Simplex Virus

In order to determine whether partial methylation of the herpes simplex virus (HSV) tk gene prevents tk gene expression, the HSV tk gene was cloned as single-stranded DNA. By in vitro second-strand DNA synthesis, specific HSV tk gene segments were methylated, and the hemimethylated DNA molecules were microinjected into thymidine kinase-negative rat2 cells. Conversion of the hemimethylated DNA into symmetrical methylated DNA and integration into the host genome occurred early after gene transfer, before the cells entered into the S phase. HSV tk gene expression was inhibited either by promoter methylation or by methylation of the coding region. Using the HindIII-SphI HSV tk DNA fragment as a primer for in vitro DNA synthesis, all cytosine residues within the coding region, from +499 to +1309, were selectively methylated. This specific methylation pattern caused inactivation of the HSV tk gene, while methylation of the cytosine residues within the nucleotide sequence from +811 to +1309 had no effect on HSV tk gene activity. We also methylated single HpaII sites within the HSV tk gene using a specific methylated primer for in vitro DNA synthesis. We found that of the 16 HSV tk HpaII sites, methylation of 6 single sites caused HSV tk inactivation. All six of these "methylation-sensitive" sites are within the coding region, including the HpaII-6 site, which is 571 bp downstream from the transcription start site. The sites HpaII-7 to HpaII-16 were all methylation insensitive. We further inserted separately the methylation-sensitive HSV tk HpaII-6 site and the methylation-insensitive HpaII-13 site as DNA segments (32-mer) into the intron region of the simian virus 40 T antigen (TaqI site). Methylation of these HpaII sites caused inhibition of simian virus 40 T-antigen synthesis.

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