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

Sun NK, Sun CL, Lin CH, Pai LM, Chao CC - J. Biomed. Sci. (2010)

Bottom Line: Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP.On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger.Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Chang Gung University, Gueishan, Taoyuan 333, Taiwan.

ABSTRACT

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.

Conclusion: 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.

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Upregulation of cFLIP promoter activity by forced expression of DDB2. HEK293 cells were co-transfected with cFLIP reporter together with either control vector (pcDNA3) or DDB2-expressing vector (pcDNA3-DDB2). After 24 hrs, the luciferase activity was measured. The plotted values represent means ± S.D. from three independent transfections. The schematic presentation of full-length cFLIP promoter (-920/+43) and its deletion mutants are indicated below. Putative cis-elements are also indicated at positions relative to the transcription initiation site (+1). The construct number at the top indicates the length of the tested promoter region upstream of the putative transcription initiation site (designated by the bent arrow at +1). Luciferase activity is shown relative to the full-length cFLIP promoter (-920/+43). Significant difference to the control for each promoter is indicated.
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Figure 4: Upregulation of cFLIP promoter activity by forced expression of DDB2. HEK293 cells were co-transfected with cFLIP reporter together with either control vector (pcDNA3) or DDB2-expressing vector (pcDNA3-DDB2). After 24 hrs, the luciferase activity was measured. The plotted values represent means ± S.D. from three independent transfections. The schematic presentation of full-length cFLIP promoter (-920/+43) and its deletion mutants are indicated below. Putative cis-elements are also indicated at positions relative to the transcription initiation site (+1). The construct number at the top indicates the length of the tested promoter region upstream of the putative transcription initiation site (designated by the bent arrow at +1). Luciferase activity is shown relative to the full-length cFLIP promoter (-920/+43). Significant difference to the control for each promoter is indicated.

Mentions: To examine whether DDB2 may upregulate cFLIP gene by activating its transcriptional, a cFLIP promoter (-920/+43, setting the transcription initiation site as +1), which had been fused to a luciferase cDNA as a reporter gene, was co-transfected with a plasmid expressing DDB2 (pcDNA3-DDB2) in HEK293 cells. The cFLIP promoter contains several potential cis-acting elements for transactivators, including E2F (Fig. 4, bottom panel. A series of deletion mutants were constructed as indicated (Fig. 4, bottom panel). Transient expression analysis in HEK293 cells indicated the presence of cFLIP core promoters located 920 bp upstream of the putative transcription initiation sites. Deletion of these elements reduced basal promoter activity (Fig. 4, top panel, open bars). The core promoters contain multiple active E2F sites, followed by a site at -488 to -258, which represents a critical determinant of negative regulation for this promoter activity. In addition, Sp1 and AP1 sites located at -158 to -67 may be essential transcription elements. Overexpression of DDB2 induced nearly a two-fold increase of FLIP promoter activity in HEK293 cells (Fig. 4, top panel). Notably, all the 5'-deletion mutants displayed similar promoter activities as the full-length promoters (Fig. 4, -920/+43). The promoter activity was undetected in the 3'-deletion mutants (-920/-487 and -920/-258) where sequences spanning the transcriptional initiation site were deleted. These results suggest that DDB2 slightly enhances cFLIP promoter activity, and that the trans-activation effect may involve multiple transcription factors.


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

Sun NK, Sun CL, Lin CH, Pai LM, Chao CC - J. Biomed. Sci. (2010)

Upregulation of cFLIP promoter activity by forced expression of DDB2. HEK293 cells were co-transfected with cFLIP reporter together with either control vector (pcDNA3) or DDB2-expressing vector (pcDNA3-DDB2). After 24 hrs, the luciferase activity was measured. The plotted values represent means ± S.D. from three independent transfections. The schematic presentation of full-length cFLIP promoter (-920/+43) and its deletion mutants are indicated below. Putative cis-elements are also indicated at positions relative to the transcription initiation site (+1). The construct number at the top indicates the length of the tested promoter region upstream of the putative transcription initiation site (designated by the bent arrow at +1). Luciferase activity is shown relative to the full-length cFLIP promoter (-920/+43). Significant difference to the control for each promoter is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864207&req=5

Figure 4: Upregulation of cFLIP promoter activity by forced expression of DDB2. HEK293 cells were co-transfected with cFLIP reporter together with either control vector (pcDNA3) or DDB2-expressing vector (pcDNA3-DDB2). After 24 hrs, the luciferase activity was measured. The plotted values represent means ± S.D. from three independent transfections. The schematic presentation of full-length cFLIP promoter (-920/+43) and its deletion mutants are indicated below. Putative cis-elements are also indicated at positions relative to the transcription initiation site (+1). The construct number at the top indicates the length of the tested promoter region upstream of the putative transcription initiation site (designated by the bent arrow at +1). Luciferase activity is shown relative to the full-length cFLIP promoter (-920/+43). Significant difference to the control for each promoter is indicated.
Mentions: To examine whether DDB2 may upregulate cFLIP gene by activating its transcriptional, a cFLIP promoter (-920/+43, setting the transcription initiation site as +1), which had been fused to a luciferase cDNA as a reporter gene, was co-transfected with a plasmid expressing DDB2 (pcDNA3-DDB2) in HEK293 cells. The cFLIP promoter contains several potential cis-acting elements for transactivators, including E2F (Fig. 4, bottom panel. A series of deletion mutants were constructed as indicated (Fig. 4, bottom panel). Transient expression analysis in HEK293 cells indicated the presence of cFLIP core promoters located 920 bp upstream of the putative transcription initiation sites. Deletion of these elements reduced basal promoter activity (Fig. 4, top panel, open bars). The core promoters contain multiple active E2F sites, followed by a site at -488 to -258, which represents a critical determinant of negative regulation for this promoter activity. In addition, Sp1 and AP1 sites located at -158 to -67 may be essential transcription elements. Overexpression of DDB2 induced nearly a two-fold increase of FLIP promoter activity in HEK293 cells (Fig. 4, top panel). Notably, all the 5'-deletion mutants displayed similar promoter activities as the full-length promoters (Fig. 4, -920/+43). The promoter activity was undetected in the 3'-deletion mutants (-920/-487 and -920/-258) where sequences spanning the transcriptional initiation site were deleted. These results suggest that DDB2 slightly enhances cFLIP promoter activity, and that the trans-activation effect may involve multiple transcription factors.

Bottom Line: Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP.On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger.Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Chang Gung University, Gueishan, Taoyuan 333, Taiwan.

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus