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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.301-306.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 301-306 ◽

Cited By ~ 1

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.301 ◽

1993 ◽

Vol 13 (1) ◽

pp. 301-306 ◽

Cited By ~ 195

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.

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Semi-Synthesis of Small Molecules of Aminocarbazoles: Tumor Growth Inhibition and Potential Impact on p53

Molecules ◽

10.3390/molecules26061637 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1637

Author(s):

Solida Long ◽

Joana B. Loureiro ◽

Carla Carvalho ◽

Luís Gales ◽

Lucília Saraiva ◽

...

Keyword(s):

Growth Inhibition ◽

Small Molecules ◽

Tumor Cells ◽

Mutant P53 ◽

Wild Type ◽

Naturally Occurring ◽

Growth Inhibitory ◽

Growth Inhibitory Activity ◽

Amino Derivatives ◽

Carbazole Alkaloids

The tumor suppressor p53 is inactivated by mutation in approximately 50% of human cancers. Small molecules that bind and stabilize those mutants may represent effective anticancer drugs. Herein, we report the tumor cell growth inhibitory activity of carbazole alkaloids and amino derivatives, as well as their potential activation of p53. Twelve aminocarbazole alkaloids were semi-synthesized from heptaphylline (1), 7-methoxy heptaphylline (2), and 7-methoxymukonal (3), isolated from Clausena harmandiana, using a reductive amination protocol. Naturally-occurring carbazoles 1–3 and their amino derivatives were evaluated for their potential effect on wild-type and mutant p53 activity using a yeast screening assay and on human tumor cell lines. Naturally-occurring carbazoles 1–3 showed the most potent growth inhibitory effects on wild-type p53-expressing cells, being heptaphylline (1) the most promising in all the investigated cell lines. However, compound 1 also showed growth inhibition against non-tumor cells. Conversely, semi-synthetic aminocarbazole 1d showed an interesting growth inhibitory activity in tumor cells expressing both wild-type and mutant p53, exhibiting low growth inhibition on non-tumor cells. The yeast assay showed a potential reactivation of mutant p53 by heptaphylline derivatives, including compound 1d. The results obtained indicate that carbazole alkaloids may represent a promising starting point to search for new mutp53-reactivating agents with promising applications in cancer therapy.

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

Blood ◽

10.1182/blood-2017-09-806679 ◽

2018 ◽

Vol 131 (25) ◽

pp. 2789-2802 ◽

Cited By ~ 9

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.

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CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

Journal of Biological Chemistry ◽

10.1074/jbc.m401854200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45887-45896 ◽

Cited By ~ 45

Author(s):

Mark J. Demma ◽

Serena Wong ◽

Eugene Maxwell ◽

Bimalendu Dasmahapatra

Keyword(s):

Dna Binding ◽

Mammalian Cells ◽

P53 Protein ◽

Binding Activity ◽

Mutant P53 ◽

Dependent Manner ◽

Wild Type ◽

Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

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

Molecular and Cellular Biology ◽

10.1128/mcb.17.5.2806 ◽

1997 ◽

Vol 17 (5) ◽

pp. 2806-2815 ◽

Cited By ~ 116

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.

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Mutant p53 tumor suppressor alleles release ras-induced cell cycle growth arrest.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1344 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1344-1352 ◽

Cited By ~ 34

Author(s):

G G Hicks ◽

S E Egan ◽

A H Greenberg ◽

M Mowat

Keyword(s):

Cell Cycle ◽

Tumor Suppressor ◽

Growth Regulation ◽

Growth Arrest ◽

Mutant P53 ◽

Wild Type ◽

Type Activity ◽

Wild Type P53 ◽

Negative Growth ◽

P53 Genes

Overexpression of an activated ras gene in the rat embryo fibroblast line REF52 results in growth arrest at either the G1/S or G2/M boundary of the cell cycle. Both the DNA tumor virus proteins simian virus 40 large T antigen and adenovirus 5 E1a are able to rescue ras induced lethality and cooperate with ras to fully transform REF52 cells. In this report, we present evidence that the wild-type activity of the tumor suppressor gene p53 is involved in the negative growth regulation of this model system. p53 genes encoding either a p53Val-135 or p53Pro-193 mutation express a highly stable p53 protein with a conformation-dependent loss of wild-type activity and the ability to eliminate any endogenous wild-type p53 activity in a dominant negative manner. In cotransfection assays, these mutant p53 genes are able to rescue REF52 cells from ras-induced growth arrest, resulting in established cell lines which express elevated levels of the ras oncoprotein and show morphological transformation. Full transformation, as assayed by tumor formation in nude mice, is found only in the p53Pro-193-plus-ras transfectants. These cells express higher levels of the ras protein than do the p53Val-135-plus-ras-transfected cells. Transfection of REF52 cells with ras alone or a full-length genomic wild-type p53 plus ras results in growth arrest and lethality. Therefore, the selective event for p53 inactivation or loss during tumor progression may be to overcome a cell cycle restriction induced by oncogene overexpression (ras). These results suggest that a normal function of p53 may be to mediate negative growth regulation in response to ras or other proliferative inducing signals.

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.2.1438 ◽

1999 ◽

Vol 19 (2) ◽

pp. 1438-1449 ◽

Cited By ~ 279

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.

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