Damaged DNA-binding protein 2 DDB2 protects against UV irradiation in human cells and DrosophilaReportar como inadecuado




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Journal of Biomedical Science

, 17:27

First Online: 17 April 2010Received: 05 February 2010Accepted: 17 April 2010DOI: 10.1186-1423-0127-17-27

Cite this article as: Sun, NK., Sun, CL., Lin, CH. et al. J Biomed Sci 2010 17: 27. doi:10.1186-1423-0127-17-27

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.

AbbreviationsASOantisense oligonucleotides

CDDPcisplatin

cFLIPFLICE inhibitory proteins

DDB2UV-DNA damage binding protein 2

DFFDNA fragmentation factor

β-Galβ-Galactosidase

GAPDHglyceraldehyde 3-phosphate dehydrogenase

GFPgreen fluorescence protein

MTT3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide

PARPPoly-ADP ribose polymerase

PCRpolymerase chain reaction

SDS-PAGEsodium dodecyl sulfate-polyacrylamide gel electrophoresis

UVultraviolet radiation. XP-A: xeroderma pigmentosum group A.

Electronic supplementary materialThe online version of this article doi:10.1186-1423-0127-17-27 contains supplementary material, which is available to authorized users.

Nian-Kang Sun, Chun-Ling Sun contributed equally to this work.

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Autor: Nian-Kang Sun - Chun-Ling Sun - Chia-Hua Lin - Li-Mai Pai - Chuck CK Chao

Fuente: https://link.springer.com/







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