Efficient repair of cyclobutane pyrimidine dimers at mutational hot spots is restored in complemented Xeroderma pigmentosum group C and trichothiodystrophy/xeroderma pigmentosum group D cells

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare heritable diseases. Patients suffering from XP and 50% of TTD afflicted individuals are photosensitive and have a high susceptibility to develop skin tumors. One solution to alleviating symptoms of these diseases is to express the deficient cDNAs in patient cells as a form of gene therapy. XPC and TTD/XPD cell lines were complemented using retroviral transfer. Expressed wild-type XPC or XPD cDNAs in these cells restored the survival to UVC radiation to wild-type levels in the respective complementation groups. Although complemented XP cell lines have been studied for years, data on cyclobutane pyrimidine dimer (CPD) repair in these cells at different levels are sparse. We demonstrate that CPD repair is faster in the complemented lines at the global, gene, strand specific, and nucleotide specific levels than in the original lines. In both XPC and TTD/XPD complemented lines, CPD repair on the non-transcribed strand is faster than that for the MRC5SV line. However, global repair in the complemented cell lines and MRC5SV is still slower than in normal human fibroblasts. Despite the slower global repair rate, in the complemented XPC and TTD/XPD cells, almost all of the CPDs at "hotspots" for mutation in the P53 tumor database are repaired as rapidly as in normal human fibroblasts. Such evaluation of repair at nucleotide resolution in complemented nucleotide excision repair deficient cells presents a crucial way to determine the efficient re-establishment of function needed for successful gene therapy, even when full repair capacity is not restored.     

A comparison of the response of unstimulated and stimulated T-lymphocytes and fibroblasts from normal, xeroderma pigmentosum and trichothiodystrophy donors to the lethal action of UV-C.

Unstimulated T-lymphocytes from normal donors are significantly more sensitive to the lethal effects of UV-C than either stimulated T-lymphocytes or fibroblasts as judged by colony-forming ability. Data from other studies suggest that excision repair is more effective in stimulated than unstimulated T-lymphocytes leading to the prediction that these differences in survival should be minimal in cells established from excision defective donors. The prediction was met with XP6BR, a donor of unknown complementation group. For 3 XP's from complementation group D, however, enhanced survival in stimulated T-cells was observed. With cells from an excision-defective TTD who was included in complementation group D of XP both fibroblasts and unstimulated T-lymphocytes were hypersensitive. For a second excision defective TTD patient who was excluded from complementation group D, the unstimulated T-lymphocytes were more resistant than those of normal donors although the fibroblasts were hypersensitive. These results suggest that the in vitro response of stimulated T-lymphocytes or fibroblasts may not reflect the in vivo response of cells, as measured by the response of unstimulated T-lymphocytes.