An assay to detect DNA-damaging agents that induce nucleotide excision-repairable DNA lesions in living human cells

摘要

Biochemical risk assessment studies of chemicals that induce DNA lesions are important, because lesions in genomic DNA frequently result in cancer, neurodegeneration, and aging in humans. Many classes of DNA lesions induced by chemical agents are eliminated via DNA repair mechanisms, such as nucleotide excision repair (NER) and base excision repair (BER), for the maintenance of genomic integrity. Individuals with NER-defective xeroderma pigmentosum (XP), in which bulky DNA lesions are not efficiently removed, are cancer-prone and suffer neurodegeneration. For research into cancer and neurological diseases, therefore, it might be important to identify DNA damage from agents that induce NER-repairable bulky DNA lesions. However, simple and quick assays to detect such damaging agents have not been developed using human cells. Here, we report a simple, non-isotopic assay for determining DNA damaging agents that induce NER-repairable DNA lesions by visualizing gene expression from treated fluorescent protein vectors in a mammalian cell system. This assay is based on a comparison of fluorescent protein expression in NER-proficient and NER-deficient cells. When we tested UV-irradiated fluorescent protein vectors, the fluorescent protein was observed in NER-proficient cells, but not in NER-deficient cells. Similar results were obtained for vectors treated with the anticancer drug, cisplatin. In contrast, when treated with the DNA alkylating agent methyl methanesulfonate, believed to cause BER-repairable damage, no difference in gene expression between NER-proficient and NER-deficient cells was observed. These results suggest that our assay can specifically detect DNA-damaging agents that induce NER-repairable DNA lesions, and could be used to analyze chemicals with the potential to cause cancer and neurological diseases. With further validation, the assay might be also applicable to XP diagnosis.