RESUMO
BACKGROUND: UV radiation can produce mutations in skin cells and correlates strongly with the onset of actinic keratoses and basal and squamous cell carcinomas. Xeroderma pigmentosum (XP) is a heritable disease characterized by an extreme sensitivity of skin to UV radiation. Recently, studies in cultured cells as well as in XP patients have demonstrated that the recombinant T4 endonuclease V UV-specific endonuclease could enhance repair of UV-induced photoproducts. OBJECTIVE: We aimed to obtain a stable UV-specific DNA recombinant endonuclease, pharmacologically active in mammalian cells so as to be used in treatment and prophylaxis of sun damage. METHODS: The UV-specific DNA endonuclease gene obtained from Micrococcus luteus, was fused to a leader peptide and expressed (alphaUveA), refolded and purified. A construction under the control of an eukaryotic promoter was used to transfect XP fibroblasts deficient in DNA damage repair. Transformed cells were UV irradiated and cell survival was assessed. RESULTS: alphaUveA was obtained as a highly active UV-specific repair enzyme stable for at least 2 years. XP fibroblasts transfected with alphaUveA gene increased the resistance to UV radiation and, in consequence, cell survival. CONCLUSION: alphaUveA is stable and pharmacologically active in human cells. The topical administration of this long-term stable new active principle could help diminish the risks of skin cancer after sun exposure.
Assuntos
Reparo do DNA , Linhagem Celular , Sobrevivência Celular , DNA/metabolismo , Dano ao DNA , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Relação Dose-Resposta à Radiação , Eletroforese em Gel de Ágar , Fibroblastos/metabolismo , Vetores Genéticos , Humanos , Luz , Micrococcus luteus/enzimologia , Neoplasias/metabolismo , Peptídeos/química , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Queimadura Solar , Temperatura , Fatores de Tempo , Transfecção , Raios Ultravioleta , Proteínas Virais/metabolismo , Xeroderma Pigmentoso/metabolismoRESUMO
The block in differentiation from pro-B to pre-B cells results in a selective defect in the humoral immune response characteristic of human X-linked agammaglobulinemia (XLA). Mutations of Bruton tyrosine kinase (BTK) gene have been identified as the cause of XLA. Mutation detection is the most reliable method for making a definitive diagnosis, except when clinical and laboratory findings are distinctive and coupled with history of X-linked inheritance. To provide a definitive diagnosis to 40 families incorporated in the Argentinian Primary Immunodeficiencies Registry we analysed the BTK gene by SSCP analysis as screening method for XLA, followed by direct sequencing. The molecular defect was localized in 45 patients from 34 unrelated families. From the 34 independent mutations identified, 16 were previously undescribed, 31 were unique mutations, 22 were exonic single nucleotide changes (16 missense and 6 nonsense) and four intronic mutations. Because five families had clinical, immunological and inheritance data sufficient for a definitive diagnosis, our study allowed 37 patients from 29 families previously categorized probable/ possible XLA, have now definitive diagnosis leading to appropriate genetic counseling.