RESUMEN
PURPOSE: Li-Fraumeni syndrome (LFS) is a rare genetic disease with strong predispositions to multiple early-onset neoplasms, mostly sarcomas, breast cancers, brain tumors and adrenocortical carcinomas (LFS core cancers). In most LFS families the germline mutations of TP53 tumor suppressor gene were found. Lung cancer does not belong to the core cancers of LFS, however its higher incidence is observed in families with TP53 mutations. Our aim was to search for TP53 mutations in female lung cancer patients whose clinico-demographic characteristics suggested a probable genetic predisposition to the disease. MATERIALS AND METHODS: The coding region of TP53 from blood DNA was sequenced using Sanger method. The functioning of detected mutation was tested by luciferase reporter assay. RESULTS: We found a nucleotide substitution c.569C>G, p.Pro190Arg, which was not described in the TP53 germline mutation database (http://p53.iarc.fr/TP53GermlineMutations.aspx). The mutation destroys the ability of p53 to transactivate BAX promoter and significantly reduces transactivation potential of p53 toward the promoter of MDM2 gen. CONCLUSION: We identified novel germline mutation of TP53.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Neoplasias Pulmonares/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias de la Mama/patología , Femenino , Genotipo , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Linaje , Pronóstico , Regiones Promotoras GenéticasRESUMEN
The activation of the p53 pathway by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a molecule that mimics metabolic stress, is attenuated by rapamycin, an inhibitor of mTOR kinase, immunosuppressant, and cancer drug. Rapamycin also extends lifespan in experimental animals. Because AICAR is a relatively weak activator of p53, we investigated whether stimulation of p53 by the strong activator actinomycin D is also sensitive to the inhibitory effect of rapamycin. In A549 lung cancer cells, activation of p53 by actinomycin D was associated with phosphorylation of p53 on Ser46. Rapamycin inhibited the accumulation of phospho-Ser46 p53, attenuated upregulation of some p53 target genes, and altered cell-cycle progression. Moreover, in cells exposed to actinomycin D, rapamycin attenuated the accumulation of PML, a protein that in some conditions stimulates Ser46 phosphorylation. However, Ser46 phosphorylation was not diminished in PML-knockdown cells, suggesting that in our system PML does not play a major role in stimulating p53 phosphorylation on Ser46. Knockdown of p53 diminished the upregulation of PML by stress-inducing agents, consistent with the idea that PML is a p53-regulated gene. Our data suggest that the attenuation of p53 phosphorylation on Ser46 may play a significant role in the biological activity of anti-aging rapamycin.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Dactinomicina/farmacología , Neoplasias Pulmonares/metabolismo , Sirolimus/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Envejecimiento/efectos de los fármacos , Envejecimiento/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Proteína de la Leucemia Promielocítica , Serina , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Resveratrol is a natural compound that has been intensely studied due to its role in cancer prevention and potential as an anti-cancer therapy. Its effects include induction of apoptosis and senescence-like growth inhibition. Here, we report that two cancer cell lines (U-2 OS and A549) differ significantly in their molecular responses to resveratrol. Specifically, in U-2 OS cells, the activation of the p53 pathway is attenuated when compared to the activation in A549 cells. This attenuation is accompanied by a point mutation (458: CGAâTGA) in the PPM1D gene and overexpression of the encoded protein, which is a negative regulator of p53. Experimentally induced knockdown of PPM1D in U-2 OS cells resulted in slightly increased activation of the p53 pathway, most clearly visible as stronger phosphorylation of p53 Ser37. When treated with nutlin-3a, a non-genotoxic activator of p53, U-2 OS and A549 cells both responded with substantial activation of the p53 pathway. Nutlin-3a improved the clonogenic survival of both cell lines treated with resveratrol. This improvement was associated with lower activation of DNA-damage signaling (phosphorylation of ATM, CHK2, and histone H2AX) and higher accumulation of cells in the G1 phase of the cell cycle. Thus, the hyperactivation of p53 by nutlin-3a helps to preserve the replicative potential of cells exposed to resveratrol.
Asunto(s)
Anticarcinógenos/farmacología , Proliferación Celular/efectos de los fármacos , Imidazoles/farmacología , Fosfoproteínas Fosfatasas/genética , Piperazinas/farmacología , Estilbenos/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Datos de Secuencia Molecular , Mutación Puntual/genética , Proteína Fosfatasa 2C , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Reacción en Cadena en Tiempo Real de la Polimerasa , Resveratrol , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Proteína p53 Supresora de Tumor/agonistasRESUMEN
Polymorphisms in DNA repair genes may modulate not only an individual DNA repair capacity, DNA damage levels and cancer risk but also clinical outcome after DNA damage-inducing anticancer therapy. In this study, we analyzed the association between the XPA -4G>A, XPD Asp312Asn, hOGG1 Ser326Cys, XRCC1 Arg399Gln, XRCC2 -4234G>C, XRCC3 -4541A>G and Thr241Met polymorphisms and prognosis in 250 inoperable non-small cell lung cancer (NSCLC) patients treated with radiotherapy and platinum-based chemotherapy. In univariate model, the XPA-4A and XRCC1 399Gln alleles alone and in combination influenced survival only in stage III group. In multivariate analysis, the XPA-4 GA/AA was associated with poor survival (HR 1.55, p = 0.011 overall and HR 1.72, p = 0.008 in stage III). In chemoradiotherapy group, the XPA-4A carriers were at increased risk of death and progression (HR 1.73, p = 0.013 and HR 1.65, p = 0.016, respectively), especially in stage III (p = 0.008). Moreover, individuals with ≥ 2 XPA/XRCC1 adverse alleles showed a higher risk of death (HR 1.46, p = 0.036 overall; HR 1.85, p = 0.004 in stage III and HR 1.71, p = 0.022 in chemoradiotherapy group) and progression (HR 1.75, p = 0.011 overall and HR 1.93, p = 0.005 in stage III). The XPA-4 GA/AA genotype individually and together with the XRCC1 399Gln was an independent unfavorable prognostic factor in our study. Thus, our findings indicate a prognostic potential of the XPA-4G>A in unresected NSCLC treated with radiotherapy and chemoradiotherapy. The results require validation in an independent population.