Mutant p53 elicits context-dependent pro-tumorigenic phenotypes

A mutation in p53 is frequently reported in nearly 50% of all of human cancers arising from DNA-binding core domain of p53. DNA-contact mutant R273H rendered p53 at dysfunctional state due to the substitution of single residue Arg273 for His273. Here, natural bioactive compounds curcumin, alpinetin and flavokawain B were investigated for possible stabilisation of wild-type p53 expression in vitro using HT-29 cells harbouring R273H rendered p53. Accordingly, all the bioactive compounds were able to induce the expression of wild-type p53 both at the levels of gene and protein expression. A dose-dependent induction of p53 was evident at 12.5, 25 and 50 μM concentration. The present study has shown that the bioactive compounds may have restored the wild-type p53 functional activity in tumour cells expressing R273H mutant p53. Keywords: Curcumin, Alpinetin, Flavokawain B, p53, R273H

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Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5581 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5581-5592 ◽

Cited By ~ 224

Author(s):

E Shaulian ◽

A Zauberman ◽

D Ginsberg ◽

M Oren

Keyword(s):

Dna Binding ◽

Point Mutation ◽

P53 Gene ◽

Dominant Negative ◽

Mutant P53 ◽

Wild Type ◽

Transforming Activity ◽

Wild Type P53 ◽

Negative Dominance

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

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High p53 Protein Expression LEVEL Independent Of Mutational Status Is An Adverse Prognostic Factor For Survival In ACUTE Myeloid Leukemia

Blood ◽

10.1182/blood.v122.21.1330.1330 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1330-1330

Author(s):

Alfonso Quintas-Cardama ◽

Sean M. Post ◽

Kensuke Kojima ◽

Yi Hua Qiu ◽

Michael Andreeff ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Mutant P53 ◽

P53 Mutations ◽

Wild Type ◽

P53 Pathway ◽

P53 Expression ◽

Mutational Status ◽

Wild Type P53 ◽

Acute Myeloid

Abstract Background The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML), particularly in cases with high-risk cytogenetics. It has been shown that p53 stabilization, which frequently occurs when the protein is mutated, can compromise its function. We have shown that p53 stabilization, regardless of the presence of mutations, suggesting alterations of other components in the p53 pathway. Methodology p53 expression was determined using high-throughput reverse phase protein array (RPPA) technology in 719 samples from 511 pts. Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status of p53 alleles was assessed by Sanger sequencing of exons 5 through 9. Expression of components of the p53 pathway was determined using standard immunohistochemical techniques. Nutlin-3a was used in in vitro culture experiments. Results Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). p53 expression correlated directly with CD34 (p=0.001) and inversely correlated with WBC (p=0.007), PB and BM blast burden (p=0.0001), and survival (p=0.01). High p53 (p53high) expression was more associated with unfavorable cytogenetics, particularly -5 (p=0.00001). p53high resulted in lower complete remission (CR) rates (51% vs 56%; p=??), higher relapsed rates (82% vs 62%; p=??), and shorter median overall survival (OS; 29.8 vs. 51 wks, p=0.009) compared to p53low pts. Most cases with p53high had unfavorable cytogenetics. We next correlated p53 stabilization with the presence of p53 mutations in 68 pts. p53 mutations were detected in 20/54 (37%) p53high pts and in 0/14 (0%) pts with p53low. p53high, either in the presence (29 wks) or in the absence (24 wks) of p53 mutations (p=1.0), was associated with significantly shorter OS compared with p53low pts (56 wks; p=0.05). Multivariate analysis revealed p53 expression to be an independent risk factor for survival in AML (p=0.02). p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), BAD (p=0.0001), cleaved PARP (p=0.002), and cleaved PARP (p=0.01), and negatively with p21 (p=0.01), and MDM2 (p=0.001).Given the similar OS in p53high pts carrying mutant or wild-type p53, we scored the immunohistochemical expression of MDM2, MDM4, and p21 in 30 p53high pts (9 p53 mutated, 21 wild-type p53). Overexpression of MDM2 was observed in 44% vs 48% pts with mutant vs wild-type p53, respectively, whereas rates were 67% vs 62% for MDM4, and 0% vs 19% for p21, for each respective genotype. Overall, of the 21 p53high pts carrying wild-type p53, 15 (71%) had overexpression of MDM2 and/or MDM4, whereas 81% had no p21 expression, indicating deficient activation of the p53 pathway similar to those cases carrying mutant p53. We are currently assessing response to nutlin-3a therapy in 24 primary AML samples (4 mutant p53, 20 wild-type p53). Results showing the impact of p53 mutation and/or stabilization, and expression levels of MDM2, MDM4, and p21 on nutlin-3a therapy will be presented. Conclusions p53 stabilization (p53high) is a powerful predictive and prognostic factor in AML, which is independent of the presence of mutant p53 alleles. Poor outcomes in pts with p53high lacking p53 mutations are very frequently associated with overexpression of negative regulators of p53 such as MDM2 and/or MDM4 and p21 downregulation, indicating a functionally altered p53 pathway. These findings may have implications for therapies targeting the MDM2/p53 axis in AML. Disclosures: No relevant conflicts of interest to declare.

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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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Targeting Aggregation of Wilde-Type p53 and Mutant p53 with ReACp53 As a Novel Therapeutic Concept for AML

Blood ◽

10.1182/blood.v128.22.3944.3944 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3944-3944 ◽

Cited By ~ 1

Author(s):

Jianfang Zeng ◽

Alice Soragni ◽

Jo Ishizawa ◽

Vivian Ruvolo ◽

Christopher B. Benton ◽

...

Keyword(s):

Cell Lines ◽

Term Treatment ◽

Mutant P53 ◽

Amyloid Aggregation ◽

Wild Type ◽

Short Term ◽

Short Term Treatment ◽

P53 Expression ◽

Hematopoietic Malignancies ◽

Wild Type P53

Abstract Background: The tumor suppressor p53 is a master regulator of apoptosis, autophagy, cell cycle, and senescence. It is inactivated via mutation in approximately 50% of solid tumors, but only in 15% of hematopoietic malignancies including acute myeloid leukemia (AML). A recently proposed mechanism has linked loss of p53 function with its amyloid aggregation. Conceptually, certain p53 mutations can favor partial unfolding of the protein and expose a natively buried aggregation-prone segment. This can result in amyloidogenic aggregation and prevent p53 transcriptional activity and anti-tumor functions. The cell-permeable peptide ReACp53, has been recently developed to block p53 aggregation and restore its transcriptional function in the nucleus as well as its ubiquitination by MDM2. ReACp53 showed significant cytotoxicity in ovarian cancer but no toxicity to normal hematopoietic cells in animal experiment. We sought to determine the anti-tumor activity of ReACp53 in hematopoietic malignancies. Results: We examined the p53 status in 23 malignant hematopoietic cell lines by PCR, Sanger sequencing, and immunoblotting. Two cell lines were null for p53 expression, one harbored frame shift mutations, 11 cell lines expressed various missense p53 mutations, one cell line had an in frame deletion of p53, and eight cell lines expressed wild-type p53. Additionally, immunofluorescence staining (IF) with the conformation-specific PAb240 antibody revealed high levels of cytoplasmic, partially unfolded p53 in the cells expressing mutant p53. In p53 wild-type cells, p53 protein was mainly localized in the nucleus and was negative for PAb240. The p53 null and frame shift-mutant cells showed no p53 expression. All the cells were treated short-term with various concentrations of ReACp53, or a scrambled peptide, and assessed for apoptosis by flow cytometry. We found that ReACp53 was cytotoxic not only to the p53-mutant cells, but also to the wild-type p53 lines. In fact, all p53 wild type AML cell lines were highly sensitive. The p53 negative cell lines were seemingly resistant to short-term exposure to ReACp53. DeltaNp73, an isoform of p73 that antagonizes p53 and TAp73, is expressed in most AML cells and also has a similar aggregation-prone segment. We examined the levels of DeltaNp73 and total p73 in 12 AML cell lines by PCR, immunoblotting, and IF. Both proteins were overexpressed in all five wild-type p53 cell lines, and DeltaNp73 was predominately localized in the cytoplasm of these cells. After short-term treatment with ReACp53, DeltaNp73 expression and localization didn't change in wild type p53 AML cells. Over-expressing DeltaNp73 in HEK293T cells enhanced their level of Thioflavin T staining indicating amyloid aggregation of the protein. Compared to controls, the DeltaNp73 overexpressing HEK293T cells were more prone to apoptosis following ReACp53 treatment. Absent of transactivation domain, DeltaNp73 is not expected to be restored to function like TAp73. Mutant p53 is known to cross-aggregate p73 and p63 because of their highly similar aggregation-prone segments, therefore, we hypothesize that DeltaNp73 cross-aggregated p53 and p73 and ReACp53 inhibited the aggregation as to restore p53 and TAp73 function and exposure to MDM2. We chose two wild-type p53 AML cell lines, OCI-AML3 and MOLM-14, which express MDM2 and are sensitive to the MDM2 inhibitor DS3032b. After short-term treatment with ReACp53, p53 and p73 (also a MDM2 target) expression decreased significantly in both cells. We tested the anti-leukemia efficacy of the DS3032b and ReACp53 combinatorial treatment in these cells and found that DS3032b synergized with ReACp53 to efficiently kill the cells compared to the cytotoxic activity of DS3032b or ReACp53 treatment alone. Conclusions: We demonstrate a new mechanism of DeltaNp73 inhibition of wild-type p53 and TAp73 mediated by induction of amyloid aggregation. ReACp53 showed apoptogenic efficacy in malignant hematopoietic cells, both in cells expressing wild-type p53 as well as mutant p53. In the wild-type AML cells where p73 and DeltaNp73 were overexpressed, sensitivity to ReACp53 increased. ReACp53 also exhibited synergistic activity when combined with the MDM2 inhibitor DS3032b in wild-type p53 cells. Together, our data suggest a novel mechanism of p53 inactivation by amyloid formation, that can be corrected in acute myeloid leukemia carrying either wild-type or mutant p53. Disclosures No relevant conflicts of interest to declare.

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Wild-type p53-induced phosphatase 1 (WIP1) regulates the proliferation of swine Sertoli cells through P53

Reproduction Fertility and Development ◽

10.1071/rd20215 ◽

2020 ◽

Vol 32 (18) ◽

pp. 1350

Author(s):

Bingyuan Wang ◽

Mingrui Zhang ◽

Jingjing Che ◽

Kui Li ◽

Yulian Mu ◽

...

Keyword(s):

Cell Proliferation ◽

Sertoli Cell ◽

Sertoli Cells ◽

Male Reproduction ◽

Wild Type ◽

P53 Expression ◽

Cancer Types ◽

Wild Type P53 ◽

Oncogenic Function ◽

Sertoli Cell Line

Wild-type p53-induced phosphatase 1 (WIP1) plays an oncogenic function by increasing cell proliferation in various cancer types. Deficiency in WIP1 expression leads to male infertility, possibly by impairing the blood–testis barrier and spermatogenesis. However, how WIP1 functions in the Sertoli cells to affect male reproduction remains unclear. Thus, in the present study we used a swine Sertoli cell line to investigate whether WIP1 regulated the proliferation of Sertoli cells to participate in male reproduction. The WIP1 inhibitor GSK2830371, WIP1-short interference (si) RNAs and an upstream microRNA (miR-16) were used to inhibit the expression of WIP1, after which the proliferation of swine Sertoli cells, P53 expression and the levels of P53 phosphorylation were determined. Inhibiting WIP1 expression suppressed swine Sertoli cell proliferation, increased P53 expression and increased levels of P53 phosphorylation. In addition, overexpression of miR-16 in swine Sertoli cells resulted in a decrease in WIP1 expression and increases in both P53 expression and P53 phosphorylation. Together, these findings suggest that WIP1 positively regulates the proliferation of swine Sertoli cells by inhibiting P53 phosphorylation, and the miR-16 is likely also involved by targeting WIP1.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1344-1352.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1344-1352

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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Identification of a minimal transforming domain of p53: negative dominance through abrogation of sequence-specific DNA binding

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5581-5592.1992 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5581-5592 ◽

Cited By ~ 3

Author(s):

E Shaulian ◽

A Zauberman ◽

D Ginsberg ◽

M Oren

Keyword(s):

Dna Binding ◽

Point Mutation ◽

P53 Gene ◽

Dominant Negative ◽

Mutant P53 ◽

Wild Type ◽

Transforming Activity ◽

Wild Type P53 ◽

Negative Dominance

Mutations in the p53 gene are most frequent in cancer. Many p53 mutants possess transforming activity in vitro. In cells transformed by such mutants, the mutant protein is oligomerized with endogenous cell p53. To determine the relevance of oligomerization for transformation, miniproteins containing C-terminal portions of p53 were generated. These miniproteins, although carrying no point mutation, transformed at least as efficiently as full-length mutant p53. Transforming activity was coupled with the ability to oligomerize with wild-type p53, as well as with the ability to abrogate sequence-specific DNA binding by coexpressed wild-type p53. These findings suggest that p53-mediated transformation may operate through a dominant negative mechanism, involving the generation of DNA binding-incompetent oligomers.

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