A truncated human xeroderma pigmentosum complementation group a protein expressed from an adenovirus sensitizes human tumor cells to ultraviolet light and cisplatin

Individuals with the genetic disease xeroderma pigmentosum (XP) have impair ed nucleotide excision repair (NER), Group A XP cells are defective in the XPA protein essential for NER and serve, together with other NER proteins, as a nucleation factor for the demarcation of bulky DNA damage, Because XPA cells are extremely sensitive to UV and drugs that cause bulky DNA damage, the XPA protein is an attractive target for manipulating cellular sensitiv ity to certain cancer therapeutics, a concept that perhaps can be applied t oward developing more effective cancer treatments. We have made a replicati on-defective adenovirus, AdCMV-FlagXPA(59-114), that expresses a truncated form of XPA encompassing amino acids (59-114) sufficient for binding to the excision repair cross-complementing protein 1 (ERCC1)/xeroderma pigmentosu m complementation group F (XPF) nuclease essential for making an incision 5 ' of the damage. On the basis of previous work, it was expected that this t runcated XPA protein would work as a decoy and impair NER and, thus, sensit ize cells to UV and drugs that produce bulky DNA lesions. Because the trunc ated XPA protein is "tagged" with the Flag epitope, an anti-Flag antibody c an be used to detect protein expression and to isolate proteins associated with the XPA complex. We show that relatively large quantities of truncated XPA protein are present in infected human lung carcinoma A549 cells 2-4 da ys postinfection, Moreover, in a pull-down assay using anti-Flag antibody, me show that ERCC1 is present in the FlagXPA complex but not in a complex i solated from cells infected with a control virus. Most importantly, cells i nfected with AdCMV-FlagXPA(59-114), are significantly more sensitive than c ontrol cells to UV-induced damage as determined by host-cell reactivation o f UV-irradiated AdLacZ adenovirus and in a cytotoxicity assay that appears to be the result of aberrant processing of 6-4 photoproducts. Infected cell s were also more sensitive to treatment with cisplatin, an important cancer drug, These results suggest that NER, and the XPA protein in particular, c an be a direct target for sensitizing tumor cells to UV and cisplatin and p erhaps also certain other clinically important drugs.