DDB2 decides cell fate following DNA damage

Abstract

The xeroderma pigmentosum complementation group E (XP-E) gene product damaged-DNA binding protein 2 (DDB2) plays important roles in nucleotide excision repair (NER). Previously, we showed that DDB2 participates in NER by regulating the level of p21(Waf1/Cip1). Here we show that the p21(Waf1/Cip1) -regulatory function of DDB2 plays a central role in defining the response (apoptosis or arrest) to DNA damage. The DDB2-deficient cells are resistant to apoptosis in response to a variety of DNA-damaging agents, despite activation of p53 and the pro-apoptotic genes. Instead, these cells undergo cell cycle arrest. Also, the DDB2-deficient cells are resistant to E2F1-induced apoptosis. The resistance to apoptosis of the DDB2-deficient cells is caused by an increased accumulation of p21(Waf1/Cip1) after DNA damage. We provide evidence that DDB2 targets p21(Waf1/Cip1) for proteolysis. The resistance to apoptosis in DDB2-deficient cells also involves Mdm2 in a manner that is distinct from the p53-regulatory activity of Mdm2. Our results provide evidence for a new regulatory loop involving the NER protein DDB2, Mdm2, and p21(Waf1/Cip1) that is critical in deciding cell fate (apoptosis or arrest) upon DNA damage.

Fig. 1.

DDB2^−/− MEFs exhibit cell cycle arrest and apoptosis defect upon DNA damage. (A) Wild-type or DDB2^−/− MEFs were treated with UV-C (50 J/m²) or cisplatin (30 μm) or aclarubicin (0.5 μM) for 24 hours. After the treatments, the cells were subjected to TUNEL assay using the ApopTag Red In Situ Apoptosis Detection Kit and procedure provided by the manufacturer (Chemicon International). Average percentages of the TUNEL-positive nuclei from 10 different fields from two independent experiments are plotted (B). (C) After treatment with the DNA-damaging drugs, cells were subjected to flow-cytometric analyses. An average of the cell cycle distribution (including the SubG1 cells) from three different sets is shown.

Fig. 2.

Expression of pro-apoptotic genes and accumulation of p53 and p21^Waf1/Cip1 in DDB2-deficient cells. Wild-type or DDB2^−/− MEFs were treated with cisplatin (30 μM) (A) or UV-C (50 J/m²) (B). Cells were harvested 2, 4, and 6 hours after treatment, followed by Western blot analysis for p21, Bax, PUMA, and α-Tubulin (as a loading control).

Fig. 3.

DDB2 targets p21^Waf1/Cip1 for proteolysis. (A) HeLa cells expressing DDB2-shRNA (shDDB2) or control (pSuper) were treated with cycloheximide (50 μg/ml). At the indicated time points, cells were harvested and the extracts (0.25 mg) were analyzed for the levels of p21 by Western blot. (B) HeLa cells expressing DDB2-shRNA were transfected with plasmids expressing p21 and DDB2 (two plates) or p21 alone (two plates). Twelve hours after transfection, the cells in the two plates were pooled and equally divided into five plates. Twenty-four hours after re-plating, cells were treated with cycloheximide and were harvested at the indicated time points. Extracts were assayed for the levels of p21. (C) HeLa–shDDB2 cells were infected for 16 hours with adenovirus expressing T7-epitope–tagged DDB2. Cells were also treated with or without UV-C. The extracts (2 mg) were subjected to immunoprecipitation with T7-epitope antibody. The immunoprecipitates were analyzed for the presence of p21 by Western blot. (D) Wild-type or DDB2-deficient cells were treated with MG132 for 4 hours, followed by harvesting, extraction, and Western blot analysis for p21. (E) HeLa cells (pSuper) or HeLa cells expressing DDB2-shRNA were transfected with a plasmid expressing His-tagged ubiquitin. The ubiquitinated proteins were isolated following a previously described procedure (11) and then subjected to Western blot assay with p21 antibody.

Fig. 4.

Deletion of p21^Waf1/Cip1 restores apoptosis in DDB2^−/− MEFs. (A) WT DDB2^−/− MEFs, p21^−/−MEFs, or DDB2^−/−p21^−/− MEFs were treated with UV-C, cisplatin, or aclarubicin or were infected with adenovirus-expressing E2F1 or virus-expressing lac Z. Twenty-four hours after the treatments, the cells were subjected to TUNEL assay. Averages of the TUNEL positive nuclei from 10 different fields were plotted. (B) The DDB2^−/−p21^−/− MEFs were subjected to flow-cytometric analysis after treatments with UV-C, cisplatin, or aclarubicin. Averages of the cell cycle distribution from three different sets are shown.

Fig. 5.

Mdm2 inhibits apoptosis in DDB2-deficient cells. (A) HeLa cells expressing DDB2-shRNA were transfected with control or Mdm2-siRNA or treated with nutlin-3 (48 hours). Extracts of the cells were assayed for Mdm2, p53, p21, and Cdk2. (B) DDB2-shRNA cells were treated with UV-C (50 J/m²), cisplatin (30 μM), or aclarubicin (0.5 μM), and after 18 hours the cells were subjected to TUNEL assay using the ApopTag Fluorescein In Situ Apoptosis Detection Kit. Averages of TUNEL-positive nuclei from 10 different fields were plotted. (C) HeLa cells expressing DDB2-shRNA were not treated or treated with nutlin-3 for 48 hours, followed by TUNEL assay using the ApopTag Fluorescein In Situ Apoptosis Detection Kit. Averages of TUNEL-positive nuclei from 10 different fields were plotted.

Fig. 6.

Stimulation of Cdk2-activity in DDB2-deficient cells by depletion of p21^Waf1/Cip1 or Mdm2. (A) Extracts from DDB2^−/− MEFs and DDB2^−/−p21^−/− MEFs were compared for Cdk2 activity after treatments with DNA-damaging agents. (B) DDB2-shRNA expressing HeLa cells were transfected with Mdm2-siRNA. The extracts were compared for Cdk2-activity (B). In both experiments, the Cdk2 activity was measured by assaying for histone-H1 phosphorylation (38). (C) Model for activation of apoptosis by DDB2.