RESUMEN
Deregulated AKT kinase activity due to PTEN deficiency in cancer cells contributes to oncogenesis by incompletely understood mechanisms. Here, we show that PTEN deletion in HCT116 and DLD1 colon carcinoma cells leads to suppression of CHK1 and CHK2 activation in response to irradiation, impaired G2 checkpoint proficiency and radiosensitization. These defects are associated with reduced expression of MRE11, RAD50 and NBS1, components of the apical MRE11/RAD50/NBS1 (MRN) DNA damage response complex. Consistent with reduced MRN complex function, PTEN-deficient cells fail to resect DNA double-strand breaks efficiently after irradiation and show greatly diminished proficiency for DNA repair via the error-free homologous recombination (HR) repair pathway. MRE11 is highly unstable in PTEN-deficient cells but stability can be significantly restored by inhibiting mTORC1 or p70S6 kinase (p70S6K), downstream kinases whose activities are stimulated by AKT, or by mutating a residue in MRE11 that we show is phosphorylated by p70S6K in vitro. In primary human fibroblasts, activated AKT suppresses MRN complex expression to escalate RAS-induced DNA damage and thereby reinforce oncogene-induced senescence. Taken together, our data demonstrate that deregulation of the PI3K-AKT/ mTORC1/ p70S6K pathways, an event frequently observed in cancer, exert profound effects on genome stability via MRE11 with potential implications for tumour initiation and therapy.
Asunto(s)
Inestabilidad Genómica/genética , Proteína Homóloga de MRE11/genética , Neoplasias/genética , Fosfohidrolasa PTEN/deficiencia , Reparación del ADN por Recombinación/genética , Daño del ADN/efectos de la radiación , Regulación hacia Abajo , Fibroblastos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Inestabilidad Genómica/efectos de la radiación , Células HCT116 , Humanos , Proteína Homóloga de MRE11/antagonistas & inhibidores , Proteína Homóloga de MRE11/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Neoplasias/radioterapia , Fosfohidrolasa PTEN/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinonas/farmacología , ARN Interferente Pequeño/metabolismo , Tolerancia a Radiación/genética , Reparación del ADN por Recombinación/efectos de la radiación , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/genética , Tionas/farmacología , Rayos X/efectos adversosRESUMEN
Forensic investigations involve several scientific branches among which biological analyses are much more frequently requested as a consequence of their importance and great versatility towards most of the traces found on the crime scene. Biological analyses are lead in subsequent steps: extraction, amplification and STR typing of the specimens collected on the crime scene. All of these techniques have been modified from original protocols according to the kind of sample to process. A critical point in our analysis is trying to amplify small amounts of DNA extracted from decomposed tissues or objects, small biological traces have been left on, with high fidelity and account. That's why we have decided to settle on an experimental procedure aimed to find the best DNA polymerase according to our purposes. We have tested different Taq polymerases on the same known DNA sample at several dilutions and have compared quality and amount of amplified DNA in order to appreciate the amplifying capability of each enzyme. These data have been analyzed as a function of the technical properties of each engineered Taq polymerase and results are shown in details.