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Expression of C-terminal deleted p53 isoforms in neuroblastoma.

ABSTRACT: The tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and functional analysis of p53 expressed in eight cell lines: three wild-type cell lines and five cell lines harboring mutations. We identified two transcription-inactive p53 variants truncated in the C-terminus, one of which corresponded to the p53beta isoform recently identified in normal tissue by Bourdon et al. [J. C. Bourdon, K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D. P. Xirodimas, M. K. Saville and D. P. Lane (2005) Genes Dev., 19, 2122-2137]. Our results show, for the first time, that the p53beta isoform is the only p53 species to be endogenously expressed in the human NB cell line SK-N-AS, suggesting that the C-terminus truncated p53 isoforms may play an important role in NB tumor development.

SUBMITTER: Goldschneider D

PROVIDER: S-EPMC1636465 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Expression of C-terminal deleted p53 isoforms in neuroblastoma.

Goldschneider David D Horvilleur Emilie E Plassa Louis-François LF Guillaud-Bataille Marine M Million Karine K Wittmer-Dupret Evelyne E Danglot Gisèle G de Thé Hughes H Bénard Jean J May Evelyne E Douc-Rasy Sétha S

Nucleic acids research 20061005 19

The tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and function ...[more]

PMID: 17028100

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Project description:BACKGROUND:TP53 mutations occur in only about 3% of primary and 10-20% of relapse B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). However, alternative mechanisms may contribute to functionally impairing the p53 pathway in the absence of a mutation. Candidate mechanisms include overexpression of p53 mRNA variants encoding either dominant-negative p53 protein isoforms such as Delta40p53 and Delta133p53, or modulatory isoforms such as p53beta, which counteract the effects of Delta133p53 on replicative senescence in T-lymphocytes. METHODS:We used semi-quantitative reverse-transcriptase PCR (RT-PCR) and Western blot to investigate the expression of full length p53 (TAp53), Delta40p53, Delta133p53 or p53beta in diagnostic marrow from a clinical cohort of 50 BCP-ALL patients without TP53 mutation (29 males and 21 females, age range 2-14 years) and in the bone marrow cells of 4 healthy donors (used as controls). RESULTS:Irrespective of isoforms, levels of p53 mRNA were low in controls but were increased by 2 to 20-fold in primary or relapse BCP-ALL. TAp53 was increased in primary BCP-ALL, Delta40p53 was elevated in relapse BCP-ALL, whereas Delta133p53 and p53beta were increased in both. Next, mRNA levels were used as a basis to infer the ratio between protein isoform levels. This inference suggested that, in primary BCP-ALL, p53 was predominantly in active oligomeric conformations dominated by TAp53. In contrast, p53 mostly existed in inactive quaternary conformations containing ≥2 Delta40 or Delta133p53 in relapse BCP-ALL. Western blot analysis of blasts from BCP-ALL showed a complex pattern of N-terminally truncated p53 isoforms, whereas TAp53beta was detected as a major isoform. The hypothesis that p53 is in an active form in primary B-ALL was consistent with elevated level of p53 target genes CDKN1A and MDM2 in primary cases, whereas in relapse BCP-ALL, only CDKN1A was increased as compared to controls. CONCLUSION:Expression of p53 isoforms is deregulated in BCP-ALL in the absence of TP53 mutation, with increased expression of alternative isoforms in relapse BCP-ALL. Variations in isoform expression may contribute to functional deregulation of the p53 pathway in BCP-ALL, specifically contributing to its down-regulation in relapse forms.

Project description:The p53 protein family coordinates stress responses of cells and organisms. Alternative promoter usage and/or splicing of p53 mRNA gives rise to at least nine mammalian p53 proteins with distinct N- and C-termini which are differentially expressed in normal and malignant cells. The human N-terminal p53 variants contain either the full-length (FL), or a truncated (?N/?40) or no transactivation domain (?133) altogether. The functional consequences of coexpression of the different p53 isoforms are poorly defined. Here we investigated functional aspects of the zebrafish ?Np53 ortholog in the context of FLp53 and the zebrafish ?133p53 ortholog (?113p53) coexpressed in the developing embryo.We cloned the zebrafish ?Np53 isoform and determined that ionizing radiation increased expression of steady-state ?Np53 and ?113p53 mRNA levels in zebrafish embryos. Ectopic ?Np53 expression by mRNA injection caused hypoplasia and malformation of the head, eyes and somites, yet partially counteracted lethal effects caused by concomitant expression of FLp53. FLp53 expression was required for developmental aberrations caused by ?Np53 and for ?Np53-dependent expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A, p21, Cip1, WAF1). Knockdown of p21 expression markedly reduced the severity of developmental malformations associated with ?Np53 overexpression. By contrast, forced ?113p53 expression had little effect on ?Np53-dependent embryonal phenotypes. These functional attributes were shared between zebrafish and human ?Np53 orthologs ectopically expressed in zebrafish embryos. All 3 zebrafish isoforms could be coimmunoprecipitated with each other after transfection into Saos2 cells.Both alternative N-terminal p53 isoforms were expressed in developing zebrafish in response to cell stress and antagonized lethal effects of FLp53 to different degrees. However, in contrast to ?113p53, forced ?Np53 expression itself led to developmental defects which depended, in part, on p21 transactivation. In contrast to FLp53, the developmental abnormalities caused by ?Np53 were not counteracted by concomitant expression of ?113p53.

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Expression of C-terminal deleted p53 isoforms in neuroblastoma.

Publications

Expression of C-terminal deleted p53 isoforms in neuroblastoma.

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