RESUMEN
DNA polymerase beta (pol beta) is an essential enzyme that has been shown to localize as discrete foci to the synaptonemal complex during meiosis in the mouse. To identify proteins that associate with pol beta during meiosis, we employed the yeast two-hybrid screen. Here we show that a multiple PDZ domain-containing protein, the glutamate receptor interacting protein 1 (GRIP1), interacts specifically with pol beta. The PDZ domain-containing proteins, including GRIP1, act as scaffolds to promote rapid and localized biochemical events that require the interaction of multiple proteins. GRIP1 localizes to discrete foci on meiotic bivalents of both spermatocyte and oocyte nuclei, and colocalizes with pol beta. Together, these findings provide evidence that GRIP1 interacts with pol beta during meiosis. Our findings are consistent with the possibility that GRIP1 acts as a scaffold to promote interaction between proteins that function during meiosis.
Asunto(s)
ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Meiosis , Factores de Transcripción/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Modelos Genéticos , Coactivador 2 del Receptor Nuclear , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Testículo/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The multiple genetic hit model of cancer predicts that normal individuals should have stable populations of cancer-prone, but noncancerous, mutant cells awaiting further genetic hits. We report that whole-mount preparations of human skin contain clonal patches of p53-mutated keratinocytes, arising from the dermal-epidermal junction and from hair follicles. These clones, 60-3000 cells in size, are present at frequencies exceeding 40 cells per cm2 and together involve as much as 4% of the epidermis. In sun-exposed skin, clones are both more frequent and larger than in sun-shielded skin. We conclude that, in addition to being a tumorigenic mutagen, sunlight acts as a tumor promoter by favoring the clonal expansion of p53-mutated cells. These combined actions of sunlight result in normal individuals carrying a substantial burden of keratinocytes predisposed to cancer.
Asunto(s)
Genes p53 , Queratinocitos/metabolismo , Mutación , Piel/metabolismo , Adulto , Anciano , Células Cultivadas , Cartilla de ADN , Células Epidérmicas , Epidermis/metabolismo , Cabello/citología , Cabello/metabolismo , Humanos , Queratinocitos/citología , Microscopía Confocal , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Piel/citología , Luz SolarRESUMEN
Sunlight is a carcinogen to which everyone is exposed. Epidemiology indicates that most carcinogenic sunlight exposure takes place several decades before the tumor arises. Some of the early events have been identified by searching for genes having ultraviolet (UV)-specific mutations. Over 90% of squamous cell carcinomas and more than 50% of basal cell carcinomas from New England patients contain UV-like mutations in the p53 tumor suppressor gene. From the mutation pattern, it can be concluded that the carcinogenic DNA lesions were pyrimidine-cytosine photoproducts caused by the UVB portion of sunlight. Particular codons of the p53 gene are most susceptible, apparently because of slower DNA repair at specific sites. Sunlight is sufficiently mutagenic often to mutate both p53 alleles. These mutations are also found in the precancer for squamous cell carcinoma, actinic keratosis, implying an early role. The function of p53 in normal skin is indicated by the observation that inactivating p53 in mouse skin reduces the appearance of sunburn cells, apoptotic keratinocytes generated by UV overexposure. Skin thus appears to possess a p53-dependent "cellular proofreading" response to DNA damage in which precancerous cells self-destruct. If this response is reduced in a single cell by a prior p53 mutation, sunburn can thereafter select for clonal expansion of the p53-mutated cell into an actinic keratosis. Sunlight appears to act twice: as tumor initiator and as tumor promoter.
Asunto(s)
Apoptosis , Genes p53/fisiología , Neoplasias Cutáneas/etiología , Quemadura Solar/complicaciones , Luz Solar/efectos adversos , Animales , Humanos , Ratones , Mutación , Lesiones Precancerosas/etiología , Rayos Ultravioleta/efectos adversosRESUMEN
Squamous cell carcinoma of the skin (SCC) can progress by stages: sun-damaged epidermis, with individual disordered keratinocytes; actinic keratosis (AK), spontaneously regressing keratinized patches having aberrant cell differentiation and proliferation; carcinoma in situ; SCC and metastasis. To understand how sunlight acts as a carcinogen, we determined the stage at which sunlight mutates the p53 tumour-suppressor gene and identified a function for p53 in skin. The p53 mutations induced by ultraviolet radiation and found in > 90% of human SCCs were present in AKs. Inactivating p53 in mouse skin reduced the appearance of sunburn cells, apoptotic keratinocytes generated by overexposure to ultraviolet. Skin thus appears to possess a p53-dependent 'guardian-of-the-tissue' response to DNA damage which aborts precancerous cells. If this response is reduced in a single cell by a prior p53 mutation, sunburn can select for clonal expansion of the p53-mutated cell into the AK. Sunlight can act twice: as tumour initiator and tumour promoter.