Bimodal regulation of p21(waf1) protein as function of DNA damage levels

Abstract

Human p21(Waf1) protein is well known for being transcriptionally induced by p53 and activating the cell cycle checkpoint arrest in response to DNA breaks. Here we report that p21(Waf1) protein undergoes a bimodal regulation, being upregulated in response to low doses of DNA damage but rapidly and transiently degraded in response to high doses of DNA lesions. Responsible for this degradation is the checkpoint kinase Chk1, which phosphorylates p21(Waf1) on T145 and S146 residues and induces its proteasome-dependent proteolysis. The initial p21(Waf1) degradation is then counteracted by the ATM-Chk2 pathway, which promotes the p53-dependent accumulation of p21(Waf1) at any dose of damage. We also found that p21(Waf1) ablation favors the activation of an apoptotic program to eliminate otherwise irreparable cells. These findings support a model in which in human cells a balance between ATM-Chk2-p53 and the ATR-Chk1 pathways modulates p21(Waf1) protein levels in relation to cytostatic and cytotoxic doses of DNA damage.

Keywords: BLM, bleomycin; DDR, DNA damage response; DSBs, double strand breaks; FCS, fetal calf serum; IF, immunofluorescence; apoptosis, cell survival, DNA damage response, double strand breaks.

Figure 1

(See previous page). p21^Waf1 downregulation in U2OS cells following treatment with DNA damaging agents. (A) Western blot analysis of p21^Waf1, p53 and cleaved PARP in U2OS cells exposed to increasing doses of bleomycin (BLM), neocarzionostatin (NCS) and etoposide (Eto). The histograms for p21^Waf1 and p53 proteins were obtained from densitometric analysis of the bands, normalized against actin, from 3 independent western blot experiments. (B) p21^Waf1 protein detected by immunofluorescence in cells exposed for 3 hrs to genotoxic agents. The intensity of single cell signal was assessed by ImageJ software analysis on microscope images. Cells with p21^Waf1 signal intensity lower than 10% of the average signal detectable in the positive control (undamaged cells), were considered as p21^Waf1 negative. (C) EdU and p21^Waf1 double staining. To mark DNA replication, EdU was added to cell cultures 15 minutes before BLM treatment. p21^Waf1 protein levels were evaluated as described in (B), in untreated and treated cells (120 μM BLM, 3 hrs) and in EdU-positive (cells that transit in S-phase during the time of the experiment) and negative populations.

Figure 2.

p21^Waf1 regulation in normal and cancer cell lines. (A) Human normal lymphoblastoid cells (LCL), normal fibroblast h-TERT immortalized (BJ-hTERT), ovarian carcinoma (IGROV-1), colon carcinoma (HCT116), neuroblastoma (SH-SY5Y), breast cancer (T47D), cervix adenocarcinoma (HeLa) and osteosarcoma (Saos-2) cell lines were tested by immunoblot for p21^Waf1 and p53 before and after 3 hrs of treatment with increasing concentrations of BLM. HeLa and Saos-2 are negative for p53, T47D cells have a stabilized, but not inducible form of p53. (B) p53 wt or p53 KO HCT116 cells were analyzed by protein gel blot for p21^Waf1 and p53 before and after treatment for up to 24 hrs with 240 μM BLM. Relative quantification of band intensities, obtained by densitometric analyses, normalized to actin for loading, is shown.

Figure 3.

p21^Waf1 downregulation is due to protein degradation. (A) p21^Waf1 transcript and protein levels following BLM or Eto treatments were analyzed on the same U2OS samples by semi-quantitative RT-PCR and western blot. Relative quantification of band intensities was carried out considering the untreated sample as 1. The experiment shown is representative of 3 independent experiments. (B) U2OS were pre-treated for 30 min with 10 μM MG132 or 10 μg/ml cycloheximide (CHX) before addition of 120 μM BLM for 3 hrs. Total lysates were analyzed by protein gel blotting for p21^Waf1. Relative quantification of band intensities, obtained by densitometric analyses normalized to actin loading is shown. (C) p21^Waf1 protein half life was assessed in presence of CHX alone or CHX and 120 μM BLM. The data plotted in the graph were obtained by western blot and densitometric analysis of the bands. Untreated samples were considered as 1. (D) Immunofluorescence analysis of p21^Waf1 protein localization in U2OS cells pretreated for 30 min with 10 μM MG132 and treated for 3 hrs with 120 μM BLM. Nuclei were visualized by DAPI staining. Data in the graph were obtained analyzing the microscope images as in Fig. 1B.

Figure 4.

Chk1-dependent degradation of p21^Waf1 by bleomycin. (A) U2OS cells were pre-treated for 1 hr with inhibitors of ATM (KU55933, KU, 10mM) or Chk2 (VRX0466617, VRX, 10 μM), prior to treatment with BLM for 3 hrs. Cell lysates were analyzed by protein gel blot. pS966-Smc1 was used as a reporter of the ATM/ATR kinase activity. Relative quantification of band intensities, obtained by densitometric analyses normalized to actin loading sample, is shown. The samples with no inhibitors and no BLM were considered as 1. (B) U2OS were transfected with either mock or HA-Chk2 vector and 48 hrs later treated or not with 120 μM BLM for 3 hrs. (C) U2OS cells were pre-treated for 1 hr with 300 nM of the Chk1 inhibitor UCN-01, then treated for 3 hrs with BLM and analyzed by western blot. (D) U2OS cells were pre-treated with UCN-01 (300 nM, 1 hr) or PF-477736 (400 nM, 1 hr) and then exposed to 120 μM BLM for the indicated times. Total Chk1 and the pS296 fraction, p21^Waf1, p53 and actin were evaluated by protein gel blotting. (E) p21^Waf1 mRNA levels in presence of 120 μM BLM and PF-477736 (PF). Relative quantification of band intensities, obtained by densitometric analyses normalized to actin mRNA is shown. (F) Edu and p21^Waf1 double staining in presence of PF-477736. p21^Waf1 positivity was evaluated in EdU-positive and negative cells as described in Fig. 1B. PF-477736 was added 1 hr before BLM treatment, EdU 15 min before BLM. The image is representative of 3 independent experiments. (G) The ATR inhibitor VE-821 (1 μM) was added 1 hr before 120 μM BLM. Total lysates were obtained at the indicated time point after BLM addition. Total Chk1 and the pS345 fraction, p21^Waf1 and actin were evaluated by western blotting. Relative quantification of band intensities, obtained by densitometric analyses normalized to actin loading is shown. Untreated samples were considered as 1. (H) Time course analysis of p21^Waf1 degradation and Chk1 autophosphorylation in U2OS cells exposed to 120 μM BLM. (I) Pre-treatment with PF-477736 (PF, 400 nM, 1 hr) of T47D, HeLA and LCL cells partially or completely represses p21^Waf1 downregulation 3 hrs following 120 μM BLM.

Figure 5.

Chk1 phosphorylates in vitro p21^Waf1 at sites involved in p21^Waf1 degradation. (A) In vitro kinase assays performed with recombinant GST-Chk1 (upper) or GST-Chk2 (lower) kinases and GST-p21^Waf1 or GST-Cdc25C fragment as substrates. Low amounts of kinases were added to increase the specificity of the assay. Left panel: autoradiography of incorporated ³²P; right panel: Coomassie staining. (B) The non radioactive in vitro kinase assay containing GST-Chk1 and GST-p21^Waf1 proteins was immunoblotted with an anti-RXXpS/T specific antibody or an anti-pS146- p21^Waf1 antibody. Normalization was done by Coomassie staining. (C) U2OS cells were transfected with wt or T145A/S146A p21^Waf1 double mutant. Tests were conducted 72 hrs after transfection, when the expression levels of the exogenous proteins were reduced to less than fold2- of the endogenous p21^Waf1 and to avoid possible effects of the overexpression on cell cycle progression. Cells were pre-treated or not with 400 nM PF-477736 for 1 hr and then exposed to 120 μM BLM for 3 hrs. Whole cell extracts were analyzed by protein gel blot. The densitometric analyses of the experiment, normalized to actin and to mock value, are shown.

Figure 6.

p21^Waf1 degradation impacts on apoptosis. (A) To assess G1 to S transition, LCL replicating cells were labeled with EdU and evaluated before and 3 and 6 hrs after the indicated DNA damaging treatments. The fraction of EdU positive cells before drugs addition or IR exposure was subtracted from each time point. (B) The G2 to M transition was assessed in U2OS cells pre-treated with 100 ng/ml nocodazole before exposure to genotoxic agents to trap cells in M phase to correctly enumerate cells entering in M phase avoiding mitosis exit. (C) U2OS cells were transfected with siRNA to knock down p21^Waf1 (sip21) or a control sequence (sictrl). 48 hrs after transfection, cells were treated for 24 hrs with the genotoxic agents, stained with propidium iodide and the subdiploid apoptotic fraction quantitated by DNA flow cytofluorimetry. (D) The same cells were also lysed and tested for PARP cleavage by immunoblotting. The densitometric analysis of the cleaved form of PARP is shown after normalization on the 6 μM BLM sample. (E) U2OS cells transiently transfected with wild type (OP WT) or mutated (OP S-T/A) forms of p21^Waf1, were exposed (72 hrs after transfection to reduce the amount of exogenous p21^Waf1 protein and limit the impact of overexpression on cell cycle progression) for 24 hrs to BLM. The subdiploid apoptotic fraction quantified by DNA flow cytofluorimetry was evaluated as above. (F) The same cells were also lysed and tested for PARP cleavage by immunoblotting. The densitometric analyses of the cleaved form of PARP, normalized with vinculin and relative to the wt sample, are shown.