scholarly journals Proteolysis by calpains: a possible contribution to degradation of p53.

1997 ◽  
Vol 17 (5) ◽  
pp. 2806-2815 ◽  
Author(s):  
M Pariat ◽  
S Carillo ◽  
M Molinari ◽  
C Salvat ◽  
L Debüssche ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Tertiary Structure ◽  
P53 Protein ◽  
Calcium Ionophore ◽  
Expression Vectors ◽  
Wild Type ◽  
Experimental Conditions ◽  
Null Cell ◽  
Wide Range ◽  
Wild Type P53

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

scholarly journals p73 Function Is Inhibited by Tumor-Derived p53 Mutants in Mammalian Cells

1999 ◽  
Vol 19 (2) ◽  
pp. 1438-1449 ◽  
Author(s):  
Charles J. Di Como ◽  
Christian Gaiddon ◽  
Carol Prives
Keyword(s):  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Hot Spot ◽  
P53 Protein ◽  
Wild Type ◽  
Null Cell ◽  
Broad Array ◽  
Wild Type P53 ◽  
Mutant Forms ◽  
P53 Tumor Suppressor Protein

ABSTRACT The p53 tumor suppressor protein, found mutated in over 50% of all human tumors, is a sequence-specific transcriptional activator. Recent studies have identified a p53 relative, termed p73. We were interested in determining the relative abilities of wild-type and mutant forms of p53 and p73α and -β isoforms to transactivate various p53-responsive promoters. We show that both p73α and p73β activate the transcription of reporters containing a number of p53-responsive promoters in the p53-null cell line H1299. However, a number of significant differences were observed between p53 and p73 and even between p73α and p73β. Additionally, a Saccharomyces cerevisiae-based reporter assay revealed a broad array of transcriptional transactivation abilities by both p73 isoforms at 37°C. Recent data have shown that p73 can associate with p53 by the yeast two-hybrid assay. When we examined complex formation in transfected mammalian cells, we found that p73α coprecipitates with mutant but not wild-type p53. Since many tumor-derived p53 mutants are capable of inhibiting transactivation by wild-type p53, we tested the effects of two representative hot-spot mutants (R175H and R248W) on p73. By cotransfecting p73α along with either p53 mutant and a p53-responsive reporter, we found that both R175H and R248W reduces the transcriptional activity of p73α. This decrease in transcriptional activity is correlated with the reduced ability of p73α to promote apoptosis in the presence of tumor-derived p53 mutants. Our data suggest the possibility that in some tumor cells, an outcome of the expression of mutant p53 protein may be to interfere with the endogenous p73 protein.

Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae

1992 ◽  
Vol 12 (3) ◽  
pp. 1357-1365
Author(s):  
J M Nigro ◽  
R Sikorski ◽  
S I Reed ◽  
B Vogelstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Inhibition ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Inducible Promoter ◽  
Mutant P53 ◽  
Wild Type ◽  
Growth Inhibitory Activity ◽  
Wild Type P53 ◽  
P53 Genes

Human wild-type and mutant p53 genes were expressed under the control of a galactose-inducible promoter in Saccharomyces cerevisiae. The growth rate of the yeast was reduced in cells expressing wild-type p53, whereas cells transformed with mutant p53 genes derived from human tumors were less affected. Coexpression of the normal p53 protein with the human cell cycle-regulated protein kinase CDC2Hs resulted in much more pronounced growth inhibition that for p53 alone. Cells expressing p53 and CDC2Hs were partially arrested in G1, as determined by morphological analysis and flow cytometry. p53 was phosphorylated when expressed in the yeast, but differences in phosphorylation did not explain the growth inhibition attributable to coexpression of p53 and CDC2Hs. These results suggest that wild-type p53 has a growth-inhibitory activity in S. cerevisiae similar to that observed in mammalian cells and suggests that this yeast may provide a useful model for defining the pathways through which p53 acts.

scholarly journals Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (3) ◽  
pp. 1357-1365 ◽  
Author(s):  
J M Nigro ◽  
R Sikorski ◽  
S I Reed ◽  
B Vogelstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Inhibition ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Inducible Promoter ◽  
Mutant P53 ◽  
Wild Type ◽  
Growth Inhibitory Activity ◽  
Wild Type P53 ◽  
P53 Genes

Human wild-type and mutant p53 genes were expressed under the control of a galactose-inducible promoter in Saccharomyces cerevisiae. The growth rate of the yeast was reduced in cells expressing wild-type p53, whereas cells transformed with mutant p53 genes derived from human tumors were less affected. Coexpression of the normal p53 protein with the human cell cycle-regulated protein kinase CDC2Hs resulted in much more pronounced growth inhibition that for p53 alone. Cells expressing p53 and CDC2Hs were partially arrested in G1, as determined by morphological analysis and flow cytometry. p53 was phosphorylated when expressed in the yeast, but differences in phosphorylation did not explain the growth inhibition attributable to coexpression of p53 and CDC2Hs. These results suggest that wild-type p53 has a growth-inhibitory activity in S. cerevisiae similar to that observed in mammalian cells and suggests that this yeast may provide a useful model for defining the pathways through which p53 acts.

scholarly journals Tumor Eradication by Wild-type p53-specific Cytotoxic T Lymphocytes

1997 ◽  
Vol 186 (5) ◽  
pp. 695-704 ◽  
Author(s):  
Michel P.M. Vierboom ◽  
Hans W. Nijman ◽  
Rienk Offringa ◽  
Ellen I.H. van der Voort ◽  
Thorbald van Hall ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Tumor Cells ◽  
Cytotoxic T Lymphocytes ◽  
Normal Tissue ◽  
P53 Protein ◽  
P53 Gene ◽  
Wild Type ◽  
Wild Type P53 ◽  
Tumor Outgrowth ◽  
Tumor Eradication

The tumor suppressor protein p53 is overexpressed in close to 50% of all human malignancies. The p53 protein is therefore an attractive target for immunotherapy. Cytotoxic T lymphocytes (CTLs) recognizing a murine wild-type p53 peptide, presented by the major histocompatibility complex class I molecule H-2Kb, were generated by immunizing p53 gene deficient (p53 −/−) C57BL/6 mice with syngeneic p53-overexpressing tumor cells. Adoptive transfer of these CTLs into tumor-bearing p53 +/+ nude mice caused complete and permanent tumor eradication. Importantly, this occurred in the absence of any demonstrable damage to normal tissue. When transferred into p53 +/+ immunocompetent C57BL/6 mice, the CTLs persisted for weeks in the absence of immunopathology and were capable of preventing tumor outgrowth. Wild-type p53-specific CTLs can apparently discriminate between p53-overexpressing tumor cells and normal tissue, indicating that widely expressed autologous molecules such as p53 can serve as a target for CTL-mediated immunotherapy of tumors.

scholarly journals The mdm-2 oncogene can overcome wild-type p53 suppression of transformed cell growth

1993 ◽  
Vol 13 (1) ◽  
pp. 301-306 ◽  
Author(s):  
C A Finlay
Keyword(s):  
P53 Protein ◽  
Mutant P53 ◽  
Wild Type ◽  
Rat Embryo ◽  
Ras Gene ◽  
Double Minute ◽  
Murine Double Minute ◽  
Low Levels ◽  
Wild Type P53 ◽  
P53 Genes

Expression of a p53-associated protein, Mdm-2 (murine double minute-2), can inhibit p53-mediated transactivation. In this study, overexpression of the Mdm-2 protein was found to result in the immortalization of primary rat embryo fibroblasts (REFs) and, in conjunction with an activated ras gene, in the transformation of REFs. The effect of wild-type p53 on the transforming properties of mdm-2 was determined by transfecting REFs with ras, mdm-2, and normal p53 genes. Transfection with ras plus mdm-2 plus wild-type p53 resulted in a 50% reduction in the number of transformed foci (relative to the level for ras plus mdm-2); however, more than half (9 of 17) of the cell lines derived from these foci expressed low levels of a murine p53 protein with the characteristics of a wild-type p53. These results are in contrast to previous studies which demonstrated that even minimal levels of wild-type p53 are not tolerated in cells transformed by ras plus myc, E1A, or mutant p53. The mdm-2 oncogene can overcome the previously demonstrated growth-suppressive properties of p53.

scholarly journals TRRAP is essential for regulating the accumulation of mutant and wild-type p53 in lymphoma

Blood ◽  
2018 ◽  
Vol 131 (25) ◽  
pp. 2789-2802 ◽  
Author(s):  
Alexander Jethwa ◽  
Mikołaj Słabicki ◽  
Jennifer Hüllein ◽  
Marius Jentzsch ◽  
Vineet Dalal ◽  
...  
Keyword(s):  
Protein Stability ◽  
P53 Protein ◽  
Mutant P53 ◽  
Wild Type ◽  
Therapeutic Targeting ◽  
Key Points ◽  
Wild Type P53

Key Points The HAT complex member TRRAP is vital for maintaining high p53 levels by shielding it against the natural p53 degradation machinery. Acetylation-modifying complexes regulate p53 protein stability, which may provide a basis for therapeutic targeting of mutant p53.

Role of P53 Mutations in the Radiosensitivity Status of Tumor Cells

1998 ◽  
Vol 84 (5) ◽  
pp. 517-520 ◽  
Author(s):  
Vincenzo Chiarugi ◽  
Lucia Magnelli ◽  
Marina Cinelli
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
P53 Protein ◽  
Cell Cycle Control ◽  
P53 Mutations ◽  
Wild Type ◽  
Bladder Tumors ◽  
Variable Effect ◽  
Wild Type P53

Wild-type p53 is involved in cellular response to DNA damage including cell cycle control, DNA repair and activation of apoptosis. Accumulation of p53 protein following DNA damage may initiate the apoptotic process, resulting in cell death. DNA damage induced by radiation is an example of apoptotic stimulus involving p53. Regulation of apoptosis by p53 can occur through transcriptional regulation of pro-apoptotic (e.g. bax) and anti-apoptotic (e.g. bel-2) factors. Although wild-type p53 usually sensitizes cells to radiation therapy, p53 mutations have a variable effect on radiation response. For example p53 mutations in bone or breast tumors have been found to be associated with resistance to chemotherapeutic drugs or ionizing radiation. Mutated p53 has has been reported to increase sensitivity to radiation and drugs in colorectal and bladder tumors. The present brief commentary tries to find an explanation at molecular level of these conflicting results.

Wild-Type p53 Overexpression and Its Correlation With MDM2 and p14ARF Alterations: An Alternative Pathway to Non–Small-Cell Lung Cancer

2005 ◽  
Vol 23 (1) ◽  
pp. 154-164 ◽  
Author(s):  
Yu-Chien Wang ◽  
Ruo-Kai Lin ◽  
Yi-Hung Tan ◽  
Jung-Ta Chen ◽  
Chih-Yi Chen ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
P53 Protein ◽  
Small Cell ◽  
Sequencing Analysis ◽  
Nuclear Staining ◽  
Wild Type ◽  
Small Cell Lung ◽  
Wild Type P53

Purpose We found a relatively reduced frequency of p53 mutation with a much greater frequency of p53 protein overexpression, which reflected stabilization of p53 protein in the absence of p53 gene mutation. Therefore, we investigated the possibility of alternative mechanisms leading to p53 protein stabilization. Patients and Methods We performed gene and protein alteration studies on p53 and its upstream effectors, MDM2 and p14ARF, in tumors from 94 non–small-cell lung cancer (NSCLC) patients. Results Immunohistochemical and sequencing analyses indicated that 37 tumors showed overexpression of wild-type p53. An absence of nuclear staining of MDM2 protein was found in 95% of these tumors (35 of 37; P < .001). The tumors with negative MDM2 staining showed a significantly high concordance of loss of Akt activity and low MDM2 mRNA expression (P < .001). Sequencing analysis revealed five distinct MDM2 splicing variants disrupting the conserved p53 binding domain. Corresponding variant proteins were detected in three lung cancer cell lines using the Western blot analysis. Our results also indicated that among the tumors with overexpression of the wild-type p53, 92% (34 of 37) showed immunoreactivity to p14ARF (P = .001). In addition, the deregulation of p53 and MDM2 genes was significantly associated with squamous lung cancer (P < .05) and was correlated with advanced stages (P < .05) and poor prognosis (P < .05). Conclusion Our data suggest that immunopositivity of p14ARF together with a low expression of MDM2 contributes to accumulation of the wild-type p53, and that deregulation of the p53-MDM2-p14ARF pathway is important in the pathogenesis and outcome of a subset of NSCLC.

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