Limits...
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.

Show MeSH

Related in: MedlinePlus

Protection against UV-induced cytotoxicity by forced expression of DDB2. (A, B, C) Sensitivity to UV is presented for stable cell lines indicated or for (D, E, F) HR18 cells which express DDB2. The protein levels of DDB2, DDB1, and β-actin are shown in the insets. The plotted values represent means ± S.D. of experiments performed in triplicates. IC50 values were also indicated in A and D.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2864207&req=5

Figure 1: Protection against UV-induced cytotoxicity by forced expression of DDB2. (A, B, C) Sensitivity to UV is presented for stable cell lines indicated or for (D, E, F) HR18 cells which express DDB2. The protein levels of DDB2, DDB1, and β-actin are shown in the insets. The plotted values represent means ± S.D. of experiments performed in triplicates. IC50 values were also indicated in A and D.

Mentions: 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]. By western blot analysis, we observed that the amount of DDB2 protein in HR3 cells was around two times that seen in control HeLa cells (Fig. 1A). On the other hand, DDB2 in HR18 cells was lower than in control HeLa cells (Fig. 1A). When the viability of these cells upon UV irradiation was monitored, we noted that HR3 cells were more resistant to UV than HR18 or control HeLa cells (Fig. 1A). The level of DDB1 in HR3 and HR18 was similar to control cells (Fig. 1A), an observation which suggested that resistance to UV in these cells may be associated mostly with DDB2. Next, we measured the level of apoptosis in the UV-irradiated cells by assessing nuclear morphology (Fig. 1B) or by flow cytometry (Fig. 1C). We confirmed that HR3 cells were more resistant to UV than HR18 or control HeLa cells in both assays (Fig. 1B and 1C). Overexpression of DDB2 in HR18 cells was shown to increase resistance to UV in these cells compared to overexpression of β-Gal or to control HR18 cells (Fig. 1D). In addition, HR18 cells overexpressing DDB2 showed lower apoptosis in response to UV compared to control cells (Fig. 1E and 1F). These results indicate that resistance to UV is associated with an increased level of DDB2.


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)

Protection against UV-induced cytotoxicity by forced expression of DDB2. (A, B, C) Sensitivity to UV is presented for stable cell lines indicated or for (D, E, F) HR18 cells which express DDB2. The protein levels of DDB2, DDB1, and β-actin are shown in the insets. The plotted values represent means ± S.D. of experiments performed in triplicates. IC50 values were also indicated in A and D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Protection against UV-induced cytotoxicity by forced expression of DDB2. (A, B, C) Sensitivity to UV is presented for stable cell lines indicated or for (D, E, F) HR18 cells which express DDB2. The protein levels of DDB2, DDB1, and β-actin are shown in the insets. The plotted values represent means ± S.D. of experiments performed in triplicates. IC50 values were also indicated in A and D.
Mentions: 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]. By western blot analysis, we observed that the amount of DDB2 protein in HR3 cells was around two times that seen in control HeLa cells (Fig. 1A). On the other hand, DDB2 in HR18 cells was lower than in control HeLa cells (Fig. 1A). When the viability of these cells upon UV irradiation was monitored, we noted that HR3 cells were more resistant to UV than HR18 or control HeLa cells (Fig. 1A). The level of DDB1 in HR3 and HR18 was similar to control cells (Fig. 1A), an observation which suggested that resistance to UV in these cells may be associated mostly with DDB2. Next, we measured the level of apoptosis in the UV-irradiated cells by assessing nuclear morphology (Fig. 1B) or by flow cytometry (Fig. 1C). We confirmed that HR3 cells were more resistant to UV than HR18 or control HeLa cells in both assays (Fig. 1B and 1C). Overexpression of DDB2 in HR18 cells was shown to increase resistance to UV in these cells compared to overexpression of β-Gal or to control HR18 cells (Fig. 1D). In addition, HR18 cells overexpressing DDB2 showed lower apoptosis in response to UV compared to control cells (Fig. 1E and 1F). These results indicate that resistance to UV is associated with an increased level of DDB2.

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