Abstract
BackgroundWe observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process.MethodsWe explored the role of DDB2 in mediating UV resistance in both human cells and Drosophila. To do so, DDB2 was overexpressed by using a full-length open reading frame cDNA. Conversely, DDB2 and cFLIP were suppressed by using antisense oligonucleotides. Cell survival was measured using a colony forming assay. Apoptosis was monitored by examination of nuclear morphology, as well as by flow cytometry and Western blot analyses. A transcription reporter assay was also used to assess transcription of cFLIP.ResultsWe first observed that the cFLIP protein was upregulated in UV-resistant HeLa cells. In addition, the cFLIP protein could be induced by stable expression of DDB2 in these cells. Notably, the anti-apoptotic effect of DDB2 against UV irradiation was largely attenuated by knockdown of cFLIP with antisense oligonucleotides in HeLa cells. Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP. Interestingly, ectopic expression of human DDB2 in Drosophila dramatically inhibited UV-induced fly death compared to control GFP expression. On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger.ConclusionOur results show that DDB2 protects against UV stress in a cFLIP-dependent manner. In addition, the protective role of DDB2 against UV irradiation was found to be conserved in divergent living organisms such as human and Drosophila. In addition, UV irradiation may activate a cFLIP-regulated apoptotic pathway in certain cells.
Highlights
We observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of UV-DNA damage binding protein 2 (DDB2), a protein implicated in DNA repair
Resistance to UV in cisplatin-resistant HeLa cells is associated with increased levels of DDB2 We first assessed the level of DDB2 protein in the cisplatin-resistant HeLa cells HR3 and HR18
While HR3 cells were obtained by treating HeLa cells with repeated cycles of cisplatin, HR18 were derived from HR3 cells following expression of antisense cDNA to knockdown DDB2 [22]
Summary
We observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. We found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by deathreceptor signaling. We investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is involved in this process. Apoptosis plays an important role during the development and the lifespan of multicellular organisms by eliminating various cells via processes mediated mainly by caspase enzymes [1,2]. The Fas signaling pathway is a complex set of events that can be regulated by both cellular and viral proteins, including cellular-FLICE inhibitory proteins (cFLIP) [9]. The importance of cFLIP in humans was shown by the finding that dysregulation of cFLIP expression is observed in numerous autoimmune diseases and cancers [11]
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