RESUMEN
XPC is an important DNA damage recognition protein involved in DNA nucleotide excision repair. We have studied the role of the XPC protein in cisplatin treatment-mediated cell cycle regulation. Through the comparison of microarray data obtained from human normal fibroblasts and two individual XPC-defective cell lines, 486 genes were identified as XPC-responsive genes in the cisplatin treatment (with a minimal 1.5-fold change) and 297 of these genes were further mapped to biological pathways and gene ontologies. The cell cycle and cell proliferation-related genes were the most affected genes by the XPC defect in the cisplatin treatment. Many other cellular function genes were also affected by the XPC defect in the treatment. Western blot hybridization results revealed that the XPC defect reduced the p53 responses to the cisplatin treatment. The ability to activate caspase-3 was also attenuated in the XPC cells with the treatment. These results suggest that the XPC protein plays a critical role in initiating the cisplatin DNA damaging treatment-mediated signal transduction process, resulting in activation of the p53 pathway and cell cycle arrest that allow DNA repair and apoptosis to take place. These results reveal an important role of the XPC protein in the cancer prevention.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Cisplatino/farmacología , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Apoptosis/genética , Caspasa 3 , Caspasas/metabolismo , Ciclo Celular/genética , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Reparación del ADN/genética , Activación Enzimática/efectos de los fármacos , Perfilación de la Expresión Génica , Genes cdc , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismo , Xerodermia PigmentosaRESUMEN
Many commonly used drugs, such as psoralen and cisplatin, can generate a very unique type of DNA damage, namely ICL (interstrand cross-link). An ICL can severely block DNA replication and transcription and cause programmed cell death. The molecular mechanism of repairing the ICL damage has not been well established. We have studied the role of XPF (xeroderma pigmentosum group F) protein in psoralen-induced ICL-mediated DNA repair and mutagenesis. The results obtained from our mutagenesis studies revealed a very similar mutation frequency in both human normal fibroblast cells and XPF cells. The mutation spectra generated in both cells, however, were very different: most of the mutations generated in the normal fibroblast cells were T167-->A transversions, whereas most of the mutations generated in the XPF cells were T167-->G transversions. When a wild-type XPF gene cDNA was stably transfected into the XPF cells, the T167-->A mutations were increased and the T167-->G mutations were decreased. We also determined the DNA repair capability of the XPF cells using both the host-cell reactivation and the in vitro DNA repair assays. The results obtained from the host-cell reactivation experiments revealed an effective reactivation of a luciferase reporter gene from the psoralen-damaged plasmid in the XPF cells. The results obtained from the in vitro DNA repair experiments demonstrated that the XPF nuclear extract is normal in introducing dual incisions during the nucleotide excision repair process. These results suggest that the XPF protein has important roles in the psoralen ICL-mediated DNA repair and mutagenesis.
Asunto(s)
Reactivos de Enlaces Cruzados/farmacología , Daño del ADN , Reparación del ADN/fisiología , ADN Recombinante/efectos de los fármacos , Proteínas de Unión al ADN/fisiología , Mutagénesis , Oligonucleótidos/farmacología , Trioxsaleno/farmacología , Xerodermia Pigmentosa/genética , Secuencia de Bases , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Células Cultivadas/efectos de la radiación , Aductos de ADN/metabolismo , Reparación del ADN/genética , ADN Complementario/genética , ADN Recombinante/genética , ADN Recombinante/efectos de la radiación , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Genes Reporteros , Genes Supresores , Genes Sintéticos/efectos de los fármacos , Genes Sintéticos/efectos de la radiación , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fotoquímica , Plásmidos/efectos de los fármacos , Plásmidos/efectos de la radiación , ARN de Transferencia/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Transfección , Trioxsaleno/análogos & derivados , Rayos Ultravioleta , Xerodermia Pigmentosa/metabolismo , Xerodermia Pigmentosa/patologíaRESUMEN
DNA damage recognition plays an important role in DNA repair and mutagenesis. Failure to recognize DNA damage may lead to DNA replication without damage repair as well as mutation accumulation. Mutations can lead to many disease conditions. XPC is a DNA damage recognition protein that binds to damaged DNA templates at a very early stage during the DNA repair process. We have studied the role of the XPC protein in DNA cross-link reagents, psoralen and cisplatin, mediated DNA repair and mutagenesis. When psoralen and cisplatin-damaged plasmid DNA was transfected into xeroderma pigmentosum group C (XPC) cells, which were defective in the XPC gene, very distinct mutation frequency and spectrum was observed: a decreased mutation frequency for psoralen-damaged plasmid and an increased mutation frequency for cisplatin-damaged plasmid; in contrast, most mutations generated by psoralen in XPC cells were T-to-G transversions and most mutations generated by cisplatin in XPC cells were large deletions. We also determined the DNA repair ability of XPC cells by both host cell reactivation (HCR) assay and in vitro DNA repair assay. The HCR results showed greatly reduced host cell reactivation of a luciferase reporter for both psoralen and cisplatin-damaged plasmid DNA in XPC cells. The in vitro DNA repair results revealed a defective repair capacity for both psoralen and cisplatin-damaged plasmid DNA in nuclear extract prepared from XPC cells. However, this defective DNA repair activity was partially restored when a functional XPC protein was supplemented into the XPC nuclear extract prior to the reaction. These results suggest that the XPC protein DNA damage recognition function plays a crucial role in DNA repair initiation and mutation avoidance and XPC defects may lead to increased mutations and high risk for disease progression.