ABSTRACT
The nucleotide excision repair (NER) is one of the major human DNA repair pathways. Defects in one of the proteins that act in this system result in three distinct autosomal recessive syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). TFIIH is a nine-protein complex essential for NER activity, initiation of RNA polymerase II transcription and with a possible role in cell cycle regulation. XPD is part of the TFIIH complex and has a helicase function, unwinding the DNA in the 5' --> 3' direction. Mutations in the XPD gene are found in XP, TTD and XP/CS patients, the latter exhibiting both XP and CS symptoms. Correction of DNA repair defects of these cells by transducing the complementing wild-type gene is one potential strategy for helping these patients. Over the last years, adenovirus vectors have been largely used in gene delivering because of their efficient transduction, high titer, and stability. In this work, we present the construction of a recombinant adenovirus carrying the XPD gene, which is coexpressed with the EGFP reporter gene by an IRES sequence, making it easier to follow cell infection. Infection by this recombinant adenovirus grants full correction of SV40-transformed and primary skin fibroblasts obtained from XP-D, TTD and XP/CS patients.
Subject(s)
Adenoviridae/genetics , DNA Helicases/genetics , DNA Repair , DNA-Binding Proteins/genetics , Recombination, Genetic , Transcription Factors/genetics , Blotting, Western , Cell Line , Cell Line, Transformed , Cell Line, Tumor , Cell Separation , Cell Survival , Cells, Cultured , Cockayne Syndrome/genetics , Cockayne Syndrome/therapy , DNA/metabolism , Dose-Response Relationship, Radiation , Ectodermal Dysplasia/genetics , Ectodermal Dysplasia/therapy , Fibroblasts/metabolism , Flow Cytometry , Genetic Complementation Test , Humans , Kinetics , Male , Microscopy, Fluorescence , Models, Genetic , Mutation , Phenotype , RNA Polymerase II/metabolism , Sensitivity and Specificity , Skin/metabolism , Transcription Factor TFIIH , Transcription Factors, TFII/genetics , Ultraviolet Rays , Xeroderma Pigmentosum/genetics , Xeroderma Pigmentosum/therapy , Xeroderma Pigmentosum Group D ProteinABSTRACT
Nucleotide excision repair (NER) is one of the most versatile DNA repair mechanisms, ensuring the proper functioning and trustworthy transmission of genetic information in all living cells. The phenotypic consequences caused by NER defects in humans are autosomal recessive diseases such as xeroderma pigmentosum (XP). This syndrome is the most sun-sensitive disorder leading to a high frequency of skin cancer. The majority of patients with XP carry mutations in the XPA or XPC genes that encode proteins involved in recognition of DNA damage induced by UV light at the beginning of the NER process. Cells cultured from XPA and XPC patients are hypersensitive to UV light, as a result of malfunctioning DNA repair. So far there is no effective long-term treatment for these patients. Skin cancer prevention can only be achieved by strict avoidance of sunlight exposure or by the use of sunscreen agents. We have constructed recombinant adenoviruses carrying the XPA and XPC genes that were used to infect XP-A and XP-C immortalized and primary fibroblast cell lines. UV survival curves and unscheduled DNA synthesis confirmed complete phenotypic reversion in XP DNA repair deficient cells with no trace of cytotoxicity. Moreover, transgene expression is stable for at least 60 days after infection. This efficient adenovirus gene delivery approach may be an important tool to better understand XP deficiency and the causes of DNA damage induced skin cancer.
