Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation.

Ory, Katherine; Legros, Yann; Auguin, C; Soussi, Thierry

doi:10.1002/j.1460-2075.1994.tb06656.x

Cited by 217 publications

(199 citation statements)

References 51 publications

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“…Interestingly, both substitutions have a higher relative frequency of occurrence in the aforementioned secondary structures than Arg (Creighton, 1984). Three additional reports by Chen et al (1993), Ory et al (1994 and Kawamura et al (1996) provide evidence that could support both sides. These groups studied the biological properties of several p53 mutants and observed a diversity in their functions making the picture of mutants p53 behaviour much more complex.…”

Section: Discussionmentioning

confidence: 91%

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene

Gorgoulis

Zacharatos²,

Manolis³

et al. 1998

Br J Cancer

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Summary The present study represents a continuation of previous works in which we observed that lung carcinomas co-expressing MDM2 protein and p53 mutants (mt p53) exhibited more aggressive behaviour. In the above studies, we suggested a 'gain of function' mechanism of mt p53 proteins based on the fact that the MDM2 gene possesses a p53-responsive element (MDM2-p53RE). In this study, to prove our hypothesis, we selected 12 cases from a series of 51 bronchogenic carcinomas. In these 12 cases, we examined the ability of the expressed mt p53 to bind the MDM2-p53RE and correlated the findings with MDM2 expression. Furthermore, we constructed four of these p53 mutants and studied their transactivation properties by co-transfecting them with a reporter plasmid carrying MDM2-p53RE in the p53 null non-smallcell lung carcinoma cell line (NSCLC) H1299. We observed mutant p53 protein DNA-binding activity, which depended on the nature and the position of the amino acid substitution. The fact that the cases with DNA-binding activity were accompanied with MDM2 protein isoforms' overexpression is indicative of a 'gain of function' phenotype. This hypothesis was enforced by the findings of the transfection experiments, which revealed that certain p53 mutants enhanced the expression of the luciferase reporter gene either directly or indirectly via a dominant positive effect on the wild-type p53. In conclusion, this work is one first attempt to examine if the deregulation of the p53/MDM2 autoregulatory feedback loop is due to novel properties of certain p53 mutants in the specific environment of a subset of bronchogenic carcinomas.Keywords: p53 mutations; p53-responsive element; MDM2 gene; MDM2 isoforms; lung cancer The p53 oncosuppressor gene is mapped to chromosomal region 17pl3 and encodes a 393 amino acid (aa), 53-kDa nuclear phosphoprotein. The protein is divided into three main structural and functional domains. The first 42 amino acids at the N-terminus constitute the transactivation domain, the residues between amino acids 120 and 290 make up the sequence-specific DNA binding domain, whereas residues 310-393 at the C-terminus contain the nuclear localization signals, the tetramerization domain and the extreme carboxy-terminus that allosterically regulates p53 specific DNA binding. p53 protein is involved in vital aspects of the cell life, such as control of cell cycle checkpoints G, and G2, maintaining genomic integrity, DNA repair, replication, transcription, programmed cell death (apoptosis) and differentiation. Functional loss of p53, mostly via mutations, is considered the most common genetic lesion in human cancer (Greenblatt et al, 1994). Therefore, an understanding of the functions of p53 may help elucidate key steps in carcinogenesis. The cellular effects of p53 are mediated either by protein-protein interaction or by binding to DNA regulatory elements. In the first case, p53 interacts with factors of the replication, transcription and repair machinery (reviewed by:

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Section: Discussionmentioning

confidence: 91%

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene

Gorgoulis

Zacharatos²,

Manolis³

et al. 1998

Br J Cancer

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“…This would add further support for`gain of function' characteristics of such mutations. Conversely, it will be interesting to examine whether DNA contact mutations in squamous epithelial cells lose the capacity to transactivate apoptosis promoting genes such as bax (Ory et al, 1994;Friedlander et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

TP53 DNA contact mutations are selectively associated with allelic loss and have a strong clinical impact in head and neck cancer

et al. 1998

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Recent studies have suggested that di erent mutation types within the core domain of the tumour suppressor protein p53, i.e. DNA contact mutations and structural mutations, confer di erent biological properties. We have analysed in 86 head and neck squamous cell carcinomas (HNSCC), whether these p53 mutation types have a di erential clinical impact. Thirty-seven missense mutations were identi®ed. Thirteen of these (36%) were DNA contact mutations, occurring in the L3 loop, in the H2 loop sheet helix motif, in the S10 b strand and in Zinc binding residues. Microsatellite marker analysis revealed a selective association between these mutations and the loss of wild-type alleles (100% LOH vs 50% LOH in tumours with structural mutations; P=0.0034, Fisher's exact, 2-tailed). In comparison to structural mutations or to the absence of mutations in the core domain, DNA contact mutations were associated with higher tumour stages (84.6% vs 62%), a higher incidence of lymph node metastasis (91.7% vs 56%; P=0.014, Fisher's exact, 2-tailed), a shortened recurrence-free survival (8.1 months vs 23.7 months, P=0.047, log rank test) and overall survival (11 months vs 29.2 months; P=0.003, log rank test). The latter was also the case when only stage IV tumours were analysed (P=0.0055, log rank test). These data indicate that in HNSCC, TP53 DNA contact mutations confer a strong selection pressure to eliminate wild-type alleles, and that they result in an accelerated tumour progression and reduced therapeutic responsiveness.

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“…mAbs directed to the p53 protein have been invaluable tools for both clinical and basic research. In particular, they have been able to distinguish several biological conformations of the protein (Gannon et al, 1990;Legros et al, 1994b;Ory et al, 1994). The recent report that mAbs directed to the carboxy-terminus of Hp53 can activate sequence speci®c DNA binding of Hp53 emphasizes their usefulness (Abarzua et al, 1995;Hupp et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…The expression of p53 was controlled by a CMV promoter. The vectors CMV27A (Xp53 cDNA) and CMVTrp 248 (Hp53 mutant at codon 248) have already been described (Ory et al, 1994;Soussi et al, 1989). The plasmid ptk-luc was obtained from D Beach (Okamoto and Beach, 1994).…”

Section: Plasmidsmentioning

confidence: 99%

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Regulation of the specific DNA binding activity of Xenopus laevis p53: evidence for conserved regulation through the carboxy-terminus of the protein

et al. 1998

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Recombinant human p53 isolated either from E. coli or from insect cells is poorly active for binding to DNA but it can be dramatically stimulated by phosphorylation, antibody binding to the carboxy-terminal negative regulatory domain, short peptides derived from this negative regulatory domain or short single strands of DNA. We report here that Xenopus p53 has a very similar behavior. Using a new set of monoclonal antibodies directed either to the amino-or the carboxyterminus of Xenopus p53, we demonstrate that the frog protein can be activated by speci®c carboxy-terminus monoclonal antibodies in order to bind to human p53 DNA response element. In addition, we report that such activation of both humans and frogs protein can also be achieved by small peptides derived from the carboxyterminus of both p53. Although, the sequence of this region is not conserved in the various p53 species, the presence of conserved basic residues indicates that such activation is charge-dependent. This is con®rmed by the ®nding that small poly-lysine peptides can activate both human and Xenopus p53. In vivo expression of Xenopus p53 indicates that this protein is able to transactivate a wide variety of human p53 response elements as long as the experiments are performed at 328C since activity at 378C, a temperature well above the natural temperature of Xenopus, is lost. Finally, we demonstrate that human mdm2 is able to down regulate the transcriptional activity of Xenopus p53.

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Analysis of the most representative tumour-derived p53 mutants reveals that changes in protein conformation are not correlated with loss of transactivation or inhibition of cell proliferation.

Cited by 217 publications

References 51 publications

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene

Effects of p53 mutants derived from lung carcinomas on the p53-responsive element (p53RE) of the MDM2 gene

TP53 DNA contact mutations are selectively associated with allelic loss and have a strong clinical impact in head and neck cancer

Regulation of the specific DNA binding activity of Xenopus laevis p53: evidence for conserved regulation through the carboxy-terminus of the protein

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