Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.

Abstract

The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts2O, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme El corrects the temperature sensitivity defect in ts2O and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitinmediated proteolytic pathway.The nuclear phosphoprotein p53 is a negative regulator of cell proliferation and transformation (2,11,14). In many established cell lines and human cancers, the gene is mutated. Such mutations commonly result in a p53 which has increased stability and markedly altered function and have been the subject of several studies (30,31,38,57). Genetic instability indicated by gene amplification and impaired control of the G1-S transition in the cell cycle are exhibited by cells homozygous for p53 mutations (reviewed in reference 23). A role for p53 in transcriptional control is shown by sequence-specific DNA binding (4,15,26,28). Other studies have shown that wild-type p53 can affect expression of several genes and that specific binding of p53 to transcription factors, TATA-binding protein, and CCAAT-binding factor results in the altered transcription from the test promoters (1,51). An association between mdm2 protein and p53 has been established, and the mdm2 gene is known to undergo amplification in murine transformed cells and many human sarcomas (13,40,42). Transgenic mice with a complete loss of p53 protein develop normally but have a high frequency of tumors postnatally (10). Humans heterozygous for germ line p53 mutations have a high susceptibility to carcinogenesis (34).The mechanism by which p53 is regulated intracellularly is not completely understood, although it is widely accepted that turnover of the protein is an important aspect. Normally, p53 is a short-lived protein with a half-life of about 30 min (16,43,46). DNA tumor viruses express gene products which bind the normal p53 and alter its properties. In the case of human papillomavirus type 16, for example, the half-life of p53 is decreased in complexes with E6 and p53 (50), whereas complexes with simian virus 40 (SV40) large T protein and adenovirus ElB p55 result in an increased half-life of p53 (46,49). Mutations in SV40 large T protein which affect binding of p53 decrease the efficiency of SV40-mediated transformation of susceptible cells (32,35,56,63 (50) have shown that E6-p53 complexes generated in vitro are more rapidly degraded in reticulocyte extracts and that ubiquitinated intermediates can be detected. Under their experimental conditions, however, free p53 wa...

Human mammary epithelial cells exhibit a differential p53-mediated response following exposure to ionizing radiation or UV light

“…In human and rodent ®broblasts, the halflife of p53 is approximately 30 min (Reich et al, 1983;Rogel et al, 1985), while it is greater than 3 h in HMEC (Delmolino et al, 1993). Because p53 is targeted for degradation by a ubiquitin-dependent proteolytic pathway (Chowdary et al, 1994), this suggests that there are also di erences in this pathway between the two tissue types. The extended half-life of p53 in HMEC most likely accounts for the elevated endogenous levels of p53 found in HMEC relative to ®broblasts (Meyer and Leadon, unpublished results; Lehman et al, 1993).…”

Defective induction but normal activation and function of p53 in mouse cells lacking poly-ADP-ribose polymerase

“…Various stimuli induce a dramatic increase in the level of p53 protein, due primarily to an increase in its half-life (Kastan et al, 1991;Matlzman and Czyzyk, 1984). Recent evidence suggests that p53 is degraded by a ubiquitinmediated proteolytic pathway (Chowdary et al, 1994). p53 protein levels may also be regulated at the translational level through negative autoregulation, although this mechanism has only been shown to apply to induction of p53 when G 0 -arrested cells are stimulated with serum and not yet in response to DNA damage (Mosner et al, 1995).…”

Endoplasmic reticulum stress-induced cell death mediated by the proteasome

“…The temperature-sensitive ts20 Balb/C 3T3 clone A31 fibroblast cell line and its E1 corrected H38-5 derivative were cultured in DMEM with 10% FCS, 4500 mg/l glucose, 60 mg/ml penicillin, and 100 mg/ml streptomycin. 38 Cells were maintained at 341C. Where indicated, they were shifted to 401C for 30 h before treatment with brefeldin A (BFA), or 36 h for treatment with tunicamycin or thapsigargin, with or without cycloheximide (CHX).…”