RESUMO
We have studied the mechanism of mutant p53-mediated oncogenesis using several tumor-derived mutants. Using a colony formation assay, we found that the majority of the mutants increased the number of colonies formed compared to the vector. Expression of tumor-derived p53 mutants increases the rate of cell growth, suggesting that the p53 mutants have 'gain of function' properties. We have studied the gene expression profile of cells expressing tumor-derived p53-D281G to identify genes transactivated by mutant p53. We report the transactivation of two genes, asparagine synthetase and human telomerase reverse transcriptase. Quantitative real-time PCR confirms this upregulation. Transient transfection promoter assays verify that tumor-derived p53 mutants transactivate these promoters significantly. An electrophoretic mobility shift assay shows that tumor-derived p53-mutants cannot bind to the wild-type p53 consensus sequence. The results presented here provide some evidence of a possible mechanism for mutant p53-mediated transactivation.
Assuntos
Transformação Celular Neoplásica/genética , Genes p53 , Neoplasias/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/fisiologia , Substituição de Aminoácidos , Aspartato-Amônia Ligase/biossíntese , Aspartato-Amônia Ligase/genética , Divisão Celular , Linhagem Celular Tumoral , Sequência Consenso , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/fisiologia , Relação Estrutura-Atividade , Telomerase/biossíntese , Telomerase/genética , Proteína Tumoral p73 , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/química , Proteínas Supressoras de TumorRESUMO
Tumor-derived p53 mutants activate transcription from promoters of various growth-related genes. We tested whether this transactivation function of the mutant protein is sufficient to induce tumorigenesis ('gain of function'). Tumor-derived mutant p53-281G transactivates the promoters of human epidermal growth factor receptor (EGFR) and human multiple drug resistance gene (MDR-1). To determine whether the C-terminal domain functions only as an oligomerization domain in mutant p53-mediated transactivation, we have replaced the tetramerization domain of p53 by a heterologous tetramerization domain; although this mutant protein formed tetramers in solution, it failed to transactivate significantly. Therefore, for successful mutant p53-mediated transactivation, sequences near the C-terminus of mutant p53 are required to perform functions in addition to tetramerization. We also demonstrate that co-expression of a deletion mutant of p53 (p53 del 1-293), which retains the p53 oligomerization domain, inhibits this transactivation. p53 del 1-293 co-immunoprecipitates with p53-281G suggesting that hetero-oligomers of p53-281G and p53 del 1-293 are defective in transactivation. We also show that a cell line stably transfected with p53-281G expresses higher levels of endogenous NF-kappaB and proliferating cell nuclear antigen (PCNA) compared to that transfected with vector alone. On co-expression, p53 del 1-293 lowered the levels of NF-kappaB and PCNA in p53-281G-expressing cells. However, on co-expression, p53 del 1-293 did not inhibit the tumorigenicity and colony forming ability of p53-281G expressing cells. Our earlier work showed that a deletion of the C-terminal sequences of p53-281G overlapping the oligomerization domain obliterates 'gain of function'. Taken together, the above information suggests that the C-terminal sequences have some critical role in 'gain of function' in addition to transactivation.
