Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells

Chinghai Kao, Paul Hauser, William S. Reznikoff, Catherine A. Reznikoff

Research output: Contribution to journalArticle

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Abstract

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1′Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.

Original languageEnglish (US)
Pages (from-to)1987-1995
Number of pages9
JournalJournal of Virology
Volume67
Issue number4
StatePublished - Apr 1993
Externally publishedYes

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Neoplastic Cell Transformation
Simian virus 40
replication origin
Replication Origin
Viral Tumor Antigens
antigens
mutation
Mutation
chemical derivatives
cells
Cell Line
cell lines
Genes
plasmids
Plasmids
genes
transfection
Transfection
neoplastic cell transformation
DNA-binding domains

ASJC Scopus subject areas

  • Immunology

Cite this

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells. / Kao, Chinghai; Hauser, Paul; Reznikoff, William S.; Reznikoff, Catherine A.

In: Journal of Virology, Vol. 67, No. 4, 04.1993, p. 1987-1995.

Research output: Contribution to journalArticle

Kao, Chinghai ; Hauser, Paul ; Reznikoff, William S. ; Reznikoff, Catherine A. / Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells. In: Journal of Virology. 1993 ; Vol. 67, No. 4. pp. 1987-1995.
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