p53 in polyoma virus transformed REF52 cells

Abstract

While cellular transformation by small DNA tumour viruses usually involves targeting the product of the p53 tumour suppressor gene by a virally encoded protein, none of the three polyoma virus (Py) speci®ed T antigens have been observed to interact with p53. We show that primary mouse embryo ®broblasts and REF52 cells, which resemble primary cells in requiring co-operating oncogenes for transformation, cannot be transformed by the Py oncogene, middle T-antigen (PyMT), alone. These cells can be transformed by the complete Py early region, which encodes the Py large, middle and small Tantigens. We ®nd that PyMT can transform rodent cells lacking a functional p53 protein (p53 null mouse embryo ®broblasts and DN-REF52 cells which contain a dominant negative p53). In Py transformed REF52 cells (Py-REF52) there is no signi®cant accumulation of p53 protein, as opposed to SV40 transformed REF52 cells (SV-REF52) in which the amount of steady state p53 protein is elevated. However accumulation of p53 is observed following exposure of Py-REF52 cells to u.v. Treatment of Py-REF52 cells with X-rays results in a rapid increase in the levels of the p53-induced proteins p21/WAF1 and MDM2. In untransformed REF52 cells, X-irradiation causes p53 activation, which results in induction of both G1/S and G2/M blocks. In SV-REF52 and DN-REF52 cells, p53 abrogation results in the absence of both the G1/S and G2/M blocks. Only the absence of a G1/S block is observed in Py-REF52 cells exposed to X-irradiation. Together these results indicate that in contrast to most other DNA tumour viruses, Py does not appear to interefere with the DNA damage induced transactivation activities of the p53 protein but absence of a functional p53 protein can mediate transformation by the PyMT oncogene in the absence of other co-operating oncogenes. Possible modes of transformation by Py are discussed.

Polyoma virus disrupts ARF signaling to p53

“…Which Py protein(s) is required to cooperate with the PyMT oncogene for the neoplastic transformation of primary rodent cells remains to be elucidated. We have recently shown that the Py early region, encoding all three Py T-antigens, will transform REF52 cells, a line of rat ®broblasts that resemble primary cells in terms of their sensitivity to cooperating oncogenes (Mor et al, 1997). PyMT alone will not transform REF52 cells or primary mouse embryo ®broblasts but both cell types become sensitive to transformation by PyMT if p53 function is inactivated, indicating that inhibition of p53 activity may play a role in Py-mediated transformation (Mor et al, 1997).…”

Chromosomal instability at a mutational hotspot in polyoma middle T-antigen affects its ability to activate the ARF-p53 tumor suppressor pathway

“…Cell culture REF52 and DNp53REF52 have been previously described (Mor et al, 1997;Lomax and Fried, 2001). The DNp53REF52 cell line was established after infection of REF52 cells with pBabehygro-p53(302-390) (Mor et al, 1997).…”

“…The DNp53REF52 cell line was established after infection of REF52 cells with pBabehygro-p53(302-390) (Mor et al, 1997). All cell lines were routinely cultured in Dulbecco's modified Eagles media supplemented with 10% fetal calf serum, 100 mg/ml penicillin and 100 mg/ml streptomycin.…”

“…They have a high cloning efficiency at low cell density and a very low spontaneous transformation rate (McClure et al, 1984). REF52 cells are unique in that they are similar to primary normal cells in containing an intact ARF-p53 tumor suppressor pathway (Lomax and Fried, 2001) and in requiring oncogene cooperation for their transformation (Franza et al, 1986;Kohl and Ruley, 1987;Hicks et al, 1991;Ragozzino et al, 1991;Mor et al, 1997;Lomax and Fried, 2001). Most other established cell lines contain defects in their ARF-p53 tumor suppressor pathway and can be easily transformed by single activated oncogenes.…”

“…Most other established cell lines contain defects in their ARF-p53 tumor suppressor pathway and can be easily transformed by single activated oncogenes. Expression of PyMT can cause REF52 cells to stop dividing or die via the activation of the ARF-p53 tumor suppressor pathway (Mor et al, 1997;Lomax and Fried, 2001;O'Shea and Fried, 2005).…”