RESUMEN
We investigated whether Rad51 overexpression plays a role in soft tissue sarcoma (STS) chemoresistance as well as the regulatory mechanisms underlying its expression. The studies reported here show that Rad51 protein is overexpressed in a large panel of human STS specimens. Human STS cell lines showed increased Rad51 protein expression, as was also observed in nude rat STS xenografts. STS cells treated with doxorubicin exhibited up-regulation of Rad51 protein while arrested in the S-G(2) phase of the cell cycle. Treatment with anti-Rad51 small interfering RNA decreased Rad51 protein expression and increased chemosensitivity to doxorubicin. Because we previously showed that reintroduction of wild-type p53 (wtp53) into STS cells harboring a p53 mutation led to increased doxorubicin chemosensitivity, we hypothesized that p53 participates in regulating Rad51 expression in STS. Reintroduction of wtp53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression. Using luciferase reporter assays, we showed that reconstitution of wtp53 function decreased Rad51 promoter activity. Deletion constructs identified a specific Rad51 promoter region containing a p53-responsive element but no p53 consensus binding site. Electrophoretic mobility shift assays verified activator protein 2 (AP2) binding to this region and increased AP2 binding to the promoter in the presence of wtp53. Mutating this AP2 binding site eliminated the wtp53 repressive effect. Furthermore, AP2 knockdown resulted in increased Rad51 expression. In light of the importance of Rad51 in modulating STS chemoresistance, these findings point to a potential novel strategy for molecular-based treatments that may be of relevance to patients burdened by STS.
Asunto(s)
Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Recombinasa Rad51/fisiología , Sarcoma/tratamiento farmacológico , Neoplasias de los Tejidos Blandos/tratamiento farmacológico , Factor de Transcripción AP-2/fisiología , Proteína p53 Supresora de Tumor/fisiología , Ciclo Celular , Línea Celular Tumoral , Genes Reporteros , Humanos , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/metabolismo , Elementos de RespuestaRESUMEN
Human soft tissue sarcoma (STS) is a highly lethal malignancy in which control of metastasis determines survival. Little is known about the molecular determinants of STS dissemination. Here, we show that human STS express high levels of matrix metalloproteinase-9 (MMP-9) and that MMP-9 expression levels correlate with sequence analysis-defined p53 mutational status. Reintroduction of wild-type p53 (wtp53) into mutant p53 STS cell lines decreased MMP-9 mRNA and protein levels, decreased zymography-assessed MMP-9 proteolytic activity, and decreased tumor cell invasiveness. Reintroduction of wtp53 into STS xenografts decreased tumor growth and MMP-9 protein expression. Luciferase reporter studies showed that reintroduction of wtp53 into mutant p53 STS cells decreased MMP-9 promoter activity. Deletion constructs of the MMP-9 promoter identified a region containing a p53-responsive element that lacked a p53 consensus binding site but did contain a nuclear factor-kappaB (NF-kappaB) site. Mutating this NF-kappaB binding site eliminated the wtp53-repressive effect. Electrophoretic mobility shift assays confirmed decreased NF-kappaB binding in STS cells in the presence of wtp53. Our findings suggest a role for MMP-9 in STS progression and expand the role of p53 in molecular control of STS growth and metastasis. Therapeutic interventions in human STS targeting MMP-9 activity directly or via reintroduction of wtp53 merit further investigation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 9 de la Matriz/genética , FN-kappa B/antagonistas & inhibidores , Sarcoma/genética , Sarcoma/secundario , Proteína p53 Supresora de Tumor/fisiología , Línea Celular Tumoral , Genes Reporteros , Humanos , Luciferasas/análisis , Luciferasas/genética , Metaloproteinasa 9 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/fisiología , FN-kappa B/metabolismo , Trasplante de Neoplasias , Regiones Promotoras Genéticas , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Sarcoma/enzimología , Eliminación de Secuencia , Activación Transcripcional , Proteína p53 Supresora de Tumor/genéticaRESUMEN
We have conducted an analysis of genetic alterations in spontaneous murine melanoma cell line B16F0 and its two metastatic clones, B16F1 and B16F10 and the carcinogen-induced murine melanoma cell lines CM519, CM3205, and K1735. We found that unlike human melanomas, the murine melanoma cell lines did not have activating mutations in the Braf oncogene at exon 11 or 15. However, there were distinct patterns of alterations in the ras, Ink4a/Arf, and p53 genes in the two melanoma groups. In the spontaneous B16 melanoma cell lines, expression of p16Ink4a and p19Arf tumor suppressor proteins was lost as a consequence of a large deletion spanning Ink4a/Arf exons 1alpha, 1beta, and 2. In contrast, the carcinogen-induced melanoma cell lines expressed p16Ink4a but had inactivating mutations in either p19Arf (K1735) or p53 (CM519 and CM3205). Inactivation of p19Arf or p53 in carcinogen-induced melanomas was accompanied by constitutive activation of mitogen-activated protein kinases (MAPKs) and/or mutation-associated activation of N-ras. These results indicate that genetic alterations in p16Ink4a/p19Arf, p53 and ras-MAPK pathways can cooperate in the development of murine melanoma.
Asunto(s)
Melanoma Experimental/genética , Melanoma/genética , Animales , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Genes ras , Immunoblotting , Inmunohistoquímica , Ratones , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas c-raf/genética , Transducción de Señal/genética , Proteína p14ARF Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/efectos de la radiación , Rayos UltravioletaRESUMEN
Upon DNA damage, phosphorylation and nuclear translocation of wild-type p53 tumor suppressor protein signals its functional activation. However, very little is known about phosphorylation and localization of mutant p53. We found that mutant p53 protein in UV-induced murine primary skin tumors and cultured cell lines was constitutively phosphorylated at serine 15 residue and localized in the cell's nuclei. To investigate the mechanism of constitutive phosphorylation of mutant p53, we tested the involvement of a wide range of protein kinases and found that ERK1/2 mitogen-activated protein kinase was physically associated with mutant p53 in the nucleus. Addition of active recombinant ERK2 kinase protein in vitro to immunoprecipitated mutant p53 resulted in increased phosphorylation at serine 15. Furthermore, ERK1/2 activity was higher in tumor cells than normal cells, suggesting that phosphorylation of mutant p53 at serine 15 depends on the level of ERK1/2 activation. Interestingly, accumulation of mutant p53 in tumor cells was paralleled by low levels of Murine Double Minute 2 protein (MDM2) expression. However, when MDM2 was overexpressed, the fraction of mutant p53 that was phosphorylated at serine 15 resisted degradation, whereas the level of total p53 decreased, suggesting that phosphorylation at serine 15 and downregulation of MDM2 protein may both contribute to stabilization of mutant p53 in tumor cells.