A truncated human xeroderma pigmentosum complementation group A protein expressed from an adenovirus sensitizes human...

Individuals with the genetic disease xeroderma pigmentosum (XP) have impaired 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 sensitivity to certain cancer therapeutics, a concept that perhaps can be applied toward developing more effective cancer treatments. We have made a replication-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 pigmentosum 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 truncated XPA protein would work as a decoy and impair NER and, thus, sensitize cells to UV and drugs that produce bulky DNA lesions. Because the truncated XPA protein is "tagged" with the Flag epitope, an anti-Flag antibody can 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 days postinfection. Moreover, in a pull-down assay using anti-Flag antibody, we show that ERCC1 is present in the FlagXPA complex but not in a complex isolated from cells infected with a control virus. Most importantly, cells infected with AdCMV-FlagXPA(59-114) are significantly more sensitive than control cells to UV-induced damage as determined by host-cell reactivation of UV-irradiated AdLacZ adenovirus and in a cytotoxicity assay that appears to be the result of aberrant processing of 6-4 photoproducts. Infected cells were also more sensitive to treatment with cisplatin, an important cancer drug. These results suggest that NER, and the XPA protein in particular, can be a direct target for sensitizing tumor cells to UV and cisplatin and perhaps also certain other clinically important drugs.