Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila.
BACKGROUND
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. 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.


METHODS
We 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.


RESULTS
We 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.


CONCLUSIONS
Our 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.
Sun
Nian-Kang
NK
Department of Biochemistry and Molecular Biology, Chang Gung University, Gueishan, Taoyuan 333, Taiwan.
Sun
Chun-Ling
CL
Lin
Chia-Hua
CH
Pai
Li-Mai
LM
Chao
Chuck C K
CC
D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004330:Drosophila / Q000378:metabolism* / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D014162:Transfection; D014466:Ultraviolet Rays / Q000009:adverse effects*; D015854:Up-Regulation
C117669:DDB2 protein, human; D004268:DNA-Binding Proteins
J Biol Chem. 1997 Oct 10;272(41):25783-6
9325306
Mol Cell Biol. 2008 Jan;28(1):177-87
17967871
Cell. 1997 Nov 14;91(4):479-89
9390557
DNA Repair (Amst). 2003 Sep 18;2(9):1065-9
12967661
J Biol Chem. 1991 Nov 25;266(33):22493-500
1657999
Mol Cell Biol. 2002 May;22(10):3247-54
11971958
J Biol Chem. 2002 Jan 18;277(3):1637-40
11705987
Nucleic Acids Res. 2002 Apr 15;30(8):1859-67
11937641
Mol Cell. 2000 Apr;5(4):737-44
10882109
Proc Natl Acad Sci U S A. 1990 May;87(9):3324-7
2333286
J Pathol. 1984 Jan;142(1):67-77
6422024
Science. 1997 Feb 21;275(5303):1132-6
9027315
J Biol Chem. 2003 May 23;278(21):19245-56
12637505
PLoS One. 2008 Apr 23;3(4):e2002
18431487
Genes Dev. 1999 Feb 1;13(3):239-52
9990849
FEBS Lett. 2002 Feb 13;512(1-3):168-72
11852074
Int J Cancer. 2010 Aug 15;127(4):977-88
20013802
Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10690-5
19541625
DNA Repair (Amst). 2003 Jul 16;2(7):819-26
12826282
Cell Death Differ. 2006 Aug;13(8):1256-9
16710363
Nature. 2000 Oct 12;407(6805):770-6
11048727
Int J Biochem Cell Biol. 2010 Feb;42(2):210-3
19932761
FEBS Lett. 2001 Sep 14;505(2):206-12
11566177
Mol Cell Biol. 1998 Jul;18(7):4391-9
9632823
J Biol Chem. 2009 May 22;284(21):14165-76
19339246
Mol Pharmacol. 2005 Apr;67(4):1307-14
15644494
Genomics. 1995 Sep 1;29(1):62-9
8530102
Cell Death Differ. 2007 Jan;14(1):32-43
17082813
J Biol Chem. 1999 Jan 15;274(3):1541-8
9880531
Mutat Res. 2009 Apr 26;663(1-2):69-76
19428372
Mol Cell. 1999 Nov;4(5):771-81
10619024
Nature. 1997 Jul 10;388(6638):190-5
9217161
Cancer Res. 1997 Aug 1;57(15):3115-20
9242435
Oncogene. 2007 May 24;26(24):3572-81
17173070
Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2509-14
9482916
Chang Gung Med J. 2002 Nov;25(11):723-33
12553360
Cell. 1997 Feb 7;88(3):355-65
9039262
Science. 1998 Aug 28;281(5381):1305-8
9721089
J Biol Chem. 1993 Oct 5;268(28):21293-300
8407967
Biochem J. 2003 Nov 15;376(Pt 1):253-60
12911332
Mol Cell Biol. 1991 Apr;11(4):2075-80
2005898