Primary rat cells expressing a hybrid polyomavirus-simian virus 40 large T antigen have altered growth properties.
Expression of simian virus 40 (SV40) large T antigen efficiently immortalizes and transforms primary cells. We previously reported that a hybrid polyomavirus-SV40 large T antigen, PyT1-521-SVT336-708, binds to both p53 and pRb but does not transform an established rat cell line (J. J. Manfredi and C. Prives, J. Virol. 64:5250-5259, 1990). Here we show that this hybrid large T antigen is capable of immortalizing primary rat cells. Plasmids that express resistance to G418 sulfate and either SV40 large T antigen or PyT1-521-SVT336-708 were transfected into primary rat embryo fibroblasts, and cell lines were established. The cell lines that expressed PyT1-521-SVT336-708 were not fully transformed but did exhibit altered growth properties. Although these PyT1-521-SVT336-708-expressing lines did not form foci, they did grow in low serum and grew to a high saturation density; these cell lines also formed colonies in soft agar, but their colonies were much smaller than those seen with an SV40 large-T-antigen-expressing line. PyT1-521-SVT336-708 also demonstrated the ability to cooperate with activated Ha-ras to form foci on primary rat embryo fibroblasts. Surprisingly, two types of morphologies in such lines were observed: refractile and spindle shaped. Although there was no correlation between T-antigen level and morphology, all lines that displayed refractile morphology expressed high levels of p21ras. Since the p53 binding activity of PyT1-521-SVT336-708 appears to be intact, these results suggest that there are functions residing in the amino end of SV40 large T antigen which are necessary for full transformation that are missing from the amino end of polyomavirus large T antigen. Conversely, conferring the ability to bind to p53 on an amino-terminal fragment of polyomavirus large T antigen, although not enough to allow full transformation function, does increase its oncogenic activity in saturation density and soft agar growth assays.
Manfredi
J J
JJ
Department of Biological Sciences, Columbia University, New York, New York 10027.
Prives
C
C
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis* / Q000235:genetics; D002455:Cell Division / Q000502:physiology*; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology; D008979:Moloney murine leukemia virus / Q000235:genetics; D010957:Plasmids; D051381:Rats; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming