Evidence for free and metabolically stable p53 protein in nuclear subfractions of simian virus 40-transformed cells.

Abstract

To determine functional subcellular loci of p53, a cellular protein associated with cellular transformation, we analyzed the nucleoplasmic, chromatin, and nuclear matrix fractions from normal mouse 3T3 cells, from methylcholanthren-transformed mouse (MethA) cells, and from various simian virus 40 (SV40)-transformed cells for the presence of p53. In 3T3 and MethA cells, p53 was present in all nuclear subfractions, suggesting an association of p53 with different structural components of the nucleus. In 3T3 cells, p53 was rapidly turned over, whereas in MethA cells, p53 was metabolically stable. In SV40-transformed cells, p53 complexed to large tumor antigen (large T) was found in the nucleoplasmic and nuclear matrix fractions, as described previously (M. Staufenbiel and W. Deppert, Cell 33:173-181, 1983). In addition, however, metabolically stable p53 not complexed to large T (free p53) was also found in the chromatin and nuclear matrix fractions of these cells. This free p53 did not arise by dissocation of large T-p53 complexes, suggesting that stabilization of p53 in SV40-transformed cells can also occur by means other than formation of a complex with large T. p53 is a cellular protein which is associated with cellular transformation in a variety of systems (for reviews, see references 3, 18, and 26). In normal cells, the closely restricted expression of p53 during cell proliferation strongly suggests an important role for p53 in the regulation of this process, especially in controlling initiation of cellular DNA synthesis (6,(14)(15)(16)(17)24). In these cells, p53 exhibits a very short half-life (5 to 20 min, depending on the cell system analyzed) and is present in very low quantities (for reviews, see references 3, 18, and 26). Many transformed cells, in contrast, express a p53 with a strongly increased half-life which is present in grossly elevated levels (for reviews, see references 3, 18, and 26). Thus, it seems likely that expression of elevated levels of p53 or metabolic stabilization of this protein or both are causally related to the process of transformation (for reviews, see references 3, 18, and 26).So far, no distinct biochemical activity has been attributed to p53. Thus, a likely mode of p53 action in both normal and transformed cells is a regulatory interaction with cellular processes leading to cell proliferation. Because these complex processes require the participation of various structural systems of the cell, it may be assumed that p53 associates, at least temporarily, with various structural systems of the cell involved in cell proliferation. We have previously described a method which allows the analysis of various nuclear subfractions for the presence of regulatory proteins (30). Application of this method to determine the subnuclear distribution of the transforming protein of simian virus 40 (SV40), the SV40 large tumor antigen (large T), led to the identification and characterization of three distinct nuclear subclasses of large T: large T in the nucleoplasm, large T at the cellular chr...

Modulation of p53 protein expression during cellular transformation with simian virus 40.

“…This view was supported by experiments described by Linzer et al (17) which demonstrated that metabolic stabilization of p53 depends on the expression of a functional large T. However, formation of a p53 complex with a transforming protein could not explain the increase in metabolic stability of p53 in many other transformed cells: except for formation of complexes with large T of SV40 and several other papovaviruses (for a review, see reference 3) and with the adenovirus Elb 58K tumor antigen (33), no other complexes of p53 with viral or cellular transforming proteins have been reported. Furthermore, we have provided evidence that SV40-transformed cells also contain p53 molecules which are not complexed to large T (free p53) but are metabolically stable, like p53 in non-SV40-transformed cells, e.g., MethA cells (5). This finding suggested that at least the free p53 in SV40-transformed cells might become stabilized by a cellular process.…”

“…A detailed description of the cell fractionation procedure, as well as a characterization of extracts and structures, has been given elsewhere (11,(34)(35)(36). Briefly, cells grown on petri dishes (9 cm) were washed with KM buffer [10 mM morpholinepropanesulfonic acid (MOPS; pH 6.8), 10 mM NaCl, 1.5 mM MgCl2, 1 mM ethylene glycol-bis(,B-aminoethyl ether)-N,N, N',N'-tetraacetic acid (EGTA), 5 (5,35).…”

Stabilization of the p53 transformation-related protein in mouse fibrosarcoma cell lines: effects of protein sequence and intracellular environment.

“…Similarly, p53 in complex with the adenovirus 58-kilodalton (kDa) Elb protein is also very stable (38,44). Nevertheless, there is growing evidence that at least part of this stabilization may be due to indirect effects of the viral transformation rather than simply to the tight association between the proteins (10,11,44).…”

Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis.

“…First, p53 turnover decreases in adenovirus type 12-transformed cells, even though the protein does not form a stable, immunoprecipitable complex with Adl2 p55 ~m (Zantema et al 1985). Second, the half-lives of free and T antigen-associated p53 are similar in SV40-transformed cells (Deppert and Haug 1986). In both cases, the effect of the tumor antigen on p53 turnover was attributed to metabolic changes associated with cell transformation, although neither study excluded the possibility of physical interactions between the tumor antigen and p53.…”