Restoration of UV sensitivity in UV‐resistant HeLa cells by antisense‐mediated depletion of damaged DNA‐binding protein 2 (DDB2)

Abstract

Damaged DNA-binding activity comprises two major protein components, DDB1 and DDB2, which are implicated in the repair of ultraviolet (UV) radiation-induced DNA damage. The possible role of DDB2 as a determinant of cellular sensitivity to UV was investigated. The abundance of DDB2 in UVresistant HeLa cell lines was increased compared with that in the parental UV-sensitive cells. Stable transfection of the resistant cells with DDB2 antisense cDNA resulted in marked depletion of DDB2 protein and restored cellular sensitivity to UV-induced apoptosis. Whereas the extent of UV-induced activation of apoptosis executioners, including DNA fragmentation factor, and caspase-3 were reduced in the UV-resistant cells compared with those apparent in the sensitive cells, depletion of DDB2 from the resistant cells restored the normal activation patterns for these proteins. In contrast, overexpressing DDB2 in DDB2-depleted cells with recombinant adenovirus, which carries ddb2 cDNA, markedly inhibited the extent of UV-induced activation of DNA fragmentation factor, and caspase-3. Interestingly, a mutated form of DDB2, which is defective in interacting with DDB1 and binding to UV-damaged DNA, also markedly inhibited the activation of apoptosis executioners. These results indicate that DDB2 is a modulator of UV-induced apoptosis, and that UV resistance can be overcome by inhibition of DDB2. The findings also suggest that modulation of UV-induced apoptosis by DDB2 may be independent of DNA repair. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Potential attenuation of p38 signaling by DDB2 as a factor in acquired TNF resistance

“…Cultured cell lines resistant to cisplatin frequently cross-resist to other types of DNA damaging agents such as UV. 4,18 This cross-resistance phenotype may be explained by enhanced DNA repair as observed in the HeLa cells. 4,13,19 In this sense, cellular sensitivity to the treatment depends heavily on the ability of cells to sense and respond to DNA damage.…”

“…4,18 This cross-resistance phenotype may be explained by enhanced DNA repair as observed in the HeLa cells. 4,13,19 In this sense, cellular sensitivity to the treatment depends heavily on the ability of cells to sense and respond to DNA damage. For example, overexpression of DNA repair protein such as DDB2 may result in enhanced DNA repair.…”

“…HR3 cells, originally selected with cisplatin, 13 were also resistant to TNF-a, a non-DNA damaging agent. HR18 cells, established by inhibiting DDB2 with antisense cDNA in HR3 cells, 4 became sensitive to TNF-a (Fig. 1a, lower panel), but not cisplatin (Fig.…”

“…Despite improving genomic DNA repair, DDB2 did not protect V79 cells from UV cytotoxicity 3 but exerted a protective effect in HeLa cells. 4 Therefore, protection of cells from DNA damaging agents may depend on cell type. Recently, this investigation found protective DDB2 in apoptosis of UV-treated cells, 4,5 suggesting an alternative explanation for the contradictory observations.…”

“…4 Therefore, protection of cells from DNA damaging agents may depend on cell type. Recently, this investigation found protective DDB2 in apoptosis of UV-treated cells, 4,5 suggesting an alternative explanation for the contradictory observations.…”