Article

Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function.

Department of Biology and Center for Cancer Research, and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 04/2002; 99(5):2948-53. DOI: 10.1073/pnas.052713099
Source: PubMed

ABSTRACT The p53 tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line p53 mutations cause a familial predisposition for cancer. Germ-line or sporadic p53 mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to gamma irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in p53-dependent apoptotic pathways after treatment with gamma irradiation or doxorubicin, whereas p53-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated p53 can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.

0 Bookmarks
 · 
83 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear receptor (NR) subfamily 4 group A (NR4A) is a family of three highly homologous orphan nuclear receptors that have multiple physiological and pathological roles, including some in cancer. These NRs are reportedly dysregulated in multiple cancer types, with many studies demonstrating pro-oncogenic roles for NR4A1 (Nur77) and NR4A2 (Nurr1). Additionally, NR4A1 and NR4A3 (Nor-1) are described as tumor suppressors in leukemia. The dysregulation and functions of the NR4A members are due to many factors, including transcriptional regulation, protein-protein interactions, and post-translational modifications. These various levels of intracellular regulation result from the signaling cross-talk of the NR4A members with various signaling pathways, many of which are relevant to cancer and likely explain the family members' functions in oncogenesis and tumor suppression. In this review, we discuss the multiple functions of the NR4A receptors in cancer and summarize a growing body of scientific literature that describes the interconnectedness of the NR4A receptors with various oncogene and tumor suppressor pathways. Copyright © 2014. Published by Elsevier Inc.
    Cellular Signalling 11/2014; 27(2). DOI:10.1016/j.cellsig.2014.11.009 · 4.47 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In pluripotent stem cells, DNA damage triggers repair mechanisms, loss of pluripotency, and apoptosis, which acts as a safeguard to exclude cells containing damaged DNA from the lineage. The DNA damage response (DDR) signaling network that ensures that the response is proportional to the severity of the damage remains unresolved. Here, an RNAi screen targeting all kinases, phosphatases, and transcription factors was combined with global transcriptomics and phosphoproteomics to map the DDR in embryonic stem cells (ESC) treated with the DNA cross linker, cisplatin (CP). Integrated signaling networks were derived from shared overrepresented canonical pathways implicated in DNA damage repair, cell cycle & survival, and differentiation. Experimental probing of these networks identified a novel mode of DNA-damage induced Wnt/β-catenin signaling that constrains apoptosis. In contrast to loss-of-pluripotency and apoptosis, which are mediated by p53; silencing or deletion of the p53 gene demonstrates that genotoxic stress elicits Wnt signaling in a p53-independent manner. Instead, the latter occurs through downregulation of suppressors of Wnt/β-catenin signaling, including Csnk1a1 (CK1α). Our findings reveal a balance between p53-signaling that triggers elimination of stem cells and Wnt/β-catenin signaling that attenuates this response to tune the outcome of the DDR.
    19th MDO and 12th European Regional International society for the study of xenobiotics Meeting, The Netherlands; 06/2012
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Osteosarcoma (OS) is the most common primary malignant bone tumor and prevalently occurs in the second decade of life. Etoposide, a chemotherapeutic agent used in combined treatments of recurrent human OS, belongs to the topoisomerase inhibitor family and causes DNA breakage. In this study we evaluated the cascade of events determined by etoposide-induced DNA damage in OS cell lines with different p53 status focusing on methylation status and expression of miR-34a that modulate tumor cell growth and cell cycle progression. Wild-type p53 U2-OS cells and U2-OS cells expressing dominant-negative form of p53 (U2- OS175) were more sensitive to etoposide than p53-deficient MG63 and Saos-2 cells, showing increased levels of unmethylated miR-34a, reduced expression of CDK4 and cell cycle arrest in G1 phase. In contrast, MG63 and Saos-2 cell lines presented aberrant methylation of miR-34a promoter gene with no miR-34a induction after etoposide treatment, underlining the close connection between p53 expression and miR-34a methylation status. Consistently, in p53siRNA transfected U2-OS cells we observed loss of miR-34a induction after etoposide exposure associated with a partial gain of gene methylation and cell cycle progress towards G2/M phase. Our results suggest that the open and unmethylated conformation of the miR-34a gene may be regulated by p53 able to bind the gene promoter. In conclusion, cell response to etoposide-induced DNA damage was not compromised in cells with dominant-negative p53 expression.
    PLoS ONE 12/2014; 9(12):e114757. DOI:10.1371/journal.pone.0114757 · 3.53 Impact Factor

Preview

Download
0 Downloads
Available from