Characterization of doxycycline-dependent inducible Simian Virus 40 large T antigen immortalized human conjunctival epithelial cell line

An ion-transporting human epithelial cell line, NCL-SG3, has been established by simian virus 40 (SV40) infection of primary cultures from eccrine sweat glands. The line has been passaged 38 times (over 100 population doublings), has an aneuploid karyotype but has not undergone any ‘crisis’. The cells have retained epithelial morphology and expression of cytokeratin, the intermediate filament characteristic of epithelial cells. Approximately 85% of the population shows at least weak co-expression of vimentin, an intermediate filament associated with mesenchymal and some other non-epithelial cell types in vivo. In addition, SV40 large T-antigen is present, in a predominantly nuclear localization. Electrically resistant cell sheets are formed on dialysis tubing and cellulose-ester permeable supports. Electrogenic ion transport can be stimulated by the beta-adrenergic agonist isoproterenol (10(−6) M) and by lysylbradykinin (10(−7) M) but not by the cholinergic agonist carbachol at 10(−6) M).

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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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