PKR, a p53 target gene, plays a crucial role in the tumor-suppressor function of p53

Abstract

Type I IFN-induced expression of dsRNA-activated protein kinase (PKR) during viral infection is a well-established antiviral mechanism. However, little is known about the expression of PKR in the context of p53 and about PKR involvement in p53-mediated tumor suppression. Here, we report that PKR is a p53 target gene and plays an important role in the tumor-suppressor function of p53. Activation of p53 by genotoxic stress induces a significant level of PKR expression by acting on the newly identified cis-acting element (ISRE), which is separated from the IFN-stimulated responsive element on the PKR promoter, resulting in translational inhibition and cell apoptosis. The genotoxin-mediated inhibition of translation is associated with the p53/PKR/elF2a (eukaryotic initiation factor-2alpha) pathway. To some extent, p53 activation induced by DNA damage facilitates cell apoptosis by activating PKR. PKR-knockdown human colon cancer cells grew rapidly in nude mice and proved resistant to anti-cancer drugs. These data indicate that p53-mediated tumor suppression can be attributed at least in part to the biological functions of PKR induced by p53 in genotoxic conditions.

Fig. 1.

p53 induces PKR expression. (A) Expression of endogenous PKR and other p53 target genes in HCT116 (p53^+/+ or p53^−/−) cells was assessed by Western blot analysis. (B) mRNA levels of PKR and other p53 target genes in the p53-transfected (3 μg pcDNA3-p53) or IFNα-treated (1,000U/ml for 24 h) HCT116 p53^−/− cells and in untreated cells (UT) were examined by real-time quantitative RT-PCR. Results are expressed as means ± SEM (n = 3). (C) Western blot analysis of PKR and other p53 target genes in p53-transfected (1 or 3 μg of pcDNA3-p53) or IFNα-treated HCT116 p53^−/− cells. (D) Immunocytochemistry of p53-transfected (1 μg pcDNA3-p53) or IFNα-treated (1,000U/ml for 24 h) HCT116 p53^−/− cells after staining with anti-PKR antibody and anti-rabbit IgG-FITC (green) and anti-p53 antibody (DO-1) and anti-mouse IgG-Rhodamine (red), together with each isotype control IgG, as described in Materials and Methods. (E) PKR expression in p53 wild-type, mutant, and null cells under conditions of DNA damage (0.5 μM doxorubicin [Dox], 5 μM etoposide [Ets], and 1 mM hydroxyurea [HU]) for 12 h or 6 h after 20 J/m² UV) and in untreated cells (UT). (F) PKR expression in RKO cells treated with IFN-α or doxorubicin (Dox) in the presence or absence of 20 μM pifithrin alpha (PFTα), a p53-specific inhibitor. (G) Western blot analysis of PKR and p53 in HCT116 p53^+/+ and p53^−/− cells treated with IFNα for 12 h. (H) HCT116 cells (p53^+/+, p53^−/−, p53^+/+/sh-con, or sh-PKR) infected for 1 h with VSV (multiplicity of infection = 1) were cultured in presence or absence of doxorubicin (Dox, 1 μM). After 12 h, VSV in the culture supernatant was titrated by the 50% tissue culture infectious dose (TCID₅₀) method. PKR-knockdown and p53-knockout was evaluated (Right). *, P < 0.01 and **, P < 0.02, as compared with indicated control, respectively.

Fig. 2.

p53 acts directly on the PKR promoter. (A) Promoter deletion assay using luciferase reporters (Ppkr-Luc). Luciferase activities were assessed in p53-transfected HCT116 (p53^−/−) cells. Results are reported as mean ± SEM (n = 3). (B) DNase I footprinting with ³²P-labeled wild-type (wt) and mutant (mt, sequence shown in Fig. S4) PKR promoter fragments (-160/-29) and p53 protein (BaculoV-p53). Consensus p53RE sequence is aligned with the identified Ppkr-p53RE sequence (Lower). (C) EMSA using nuclear extracts (Nuc ext) obtained from doxorubicin-treated HCT116 p53^+/+ cells (Dox), ³²P-labeled PKR_81/-29/p21 promoter fragments, and α-p53 antibody (DO-1). Nuclear extracts were assessed with anti-histone 2A (H2A) antibody and DO-1 (Lower). UT, untreated; NS, nonspecific. (D) Luciferase reporter assay with wild-type (wt) and mutant (mt) PKR promoters (Ppkr) used in EMSA in panel C and in Fig. S4B. Luciferase assay was performed 48 h after transfection of HCT116 p53^+/+ cells with wild-type or mutant PKR promoter (Ppkr-81/-29)-attached luciferase reporters together with p53-expressing plasmid (1 μg pCDNA-p53). Data shown are from 3 independent experiments and are expressed as means ± SEM. (E) ChIP assay with chromatins obtained from p53-transfected HCT116 (p53^−/−) cells or from doxorubicin-treated (0.5 μM for 12 h) p53^+/+ cells (Dox), and DO-1 (α-p53) or control mouse IgG (mIgG).

Fig. 3.

p53 activates PKR promoter independently of ISRE. (A) Activity of the PKR promoters harboring mutant ISRE (mISRE) was assessed by luciferase assays in untreated (UT), p53-transfected (3 μg pcDNA3-p53) or IFNα-treated (1,000U/ml for 24 h) HCT116 p53^−/− cells. Results are expressed as means ± SEM (n = 3). (B) Additive effects of p53 and IFN-α on the activity of the PKR promoter, accessed in p53-transfected HCT116 (p53^−/−) cells (Left), or in genotoxin-treated (0.5 μM doxorubicin [Dx], 1 mM hydroxyurea [HU] for 12 h) HCT116 (p53^+/+) cells in the presence (+) or absence (−) of IFN-α (Right). HU, hydroxyurea; UT, untreated Results are reported as means ± SEM (n = 3). (C, D) Additive effects of p53 and type 1 IFN on the expression of PKR at protein (C) and mRNA (D) levels in HCT116 (p53^+/+ or p53^−/−) cells were assessed by Western blotting and real-time RT-PCR, respectively. Results are reported as means ± SEM (n = 3). UT, untreated.

Fig. 4.

PKR plays a role in the p53-mediated inhibition of translation and apoptosis. (A) Genotoxin-treated (5 μM etoposide [Ets] and 1 mM hydroxyurea [HU] for 12 h) RKO cells were examined by immunocytochemistry with phospho-PKR and phospho-p53 antibodies and each isotype control (control data not shown). (B) Phospho-PKR and phospho-eIF2α were assessed in HCT116 (p53^+/+ or p53^−/−) cells following genotoxin-treatment (0–1 μM doxorubicin [Dox], 0–5 μM etoposide [Ets], and 0–2 mM hydroxyurea [HU] for 12 h). (C, D) A metabolic labeling assay performed with p53^+/+, p53^−/−, PKR^+/+, and PKR^KD isogenic HCT116 cells and p53^+/+, p53^−/−, PKR^+/+, and PKR^−/− MEF cells after genotoxin treatment with increasing concentrations for 12 h (Top). Relative band intensities are represented by histograms (Middle). PKR-knockdown, p53-knockout, and PKR knockout were confirmed (Bottom). Dox, doxorubicin; Ets, etoposide. (E) A metabolic labeling assay was performed with normal and eIF2α constitutively active cells (eIF2α^CA by permanent expression of eIF2α/S51/A) after doxorubicin (Dox) treatment with increasing concentrations for 12 h. Expression of cellular (c-eIF2α) and mutant eIF2α (Myc-S51A) was confirmed. (F) Cell apoptosis was assessed by flow cytometry in untreated (UT) PKR^KD (sh-PKR) HCT116 (p53^+/+ and p53^−/−) cells and after genotoxin treatment. Early-stage apoptosis was assessed by annexin V staining of genotoxin-treated (0.5 μM doxorubicin [Dox] and 5 μM etoposide [Ets] for 36 h) cells (Left). Later-stage apoptosis was assessed by measuring subG1 cells after propidium iodide (PI) staining of genotoxin-treated (0.5 μM doxorubicin [Dox] and 5 μM etoposide [Ets] for 48 h) cells, as described in Materials and Methods. (G, H) PKR-associated pro-apoptotic molecules and cleaved PARP were assessed in PKR^KD (sh-PKR) isogenic HCT116 (p53^+/+ and p53^−/−) cells after genotoxin treatment (0.5 μM doxorubicin [Dox] and 5 μM etoposide [Ets]) for 12 h. (I) Phospho-eIF2α, ATF4, and cleaved PARP were determined in normal and eIF2α^CA cells after genotoxin treatment with increasing concentrations for 12 h. Dox, doxorubicin.

Fig. 5.

PKR contributes to the tumor-suppressor function of p53. (A) Normal (sh-con) and PKR^KD (sh-PKR) HCT116 cells were treated or not treated with 0.2 μM doxorubicin (Dox) for 12 h. Each sample was subjected to cell-cycle analysis by flow cytometry with CellQuest software (Adobe) after propidium iodide staining. (B) PKR-normal (sh-con) and PKR^KD (sh-PKR) HCT116 (p53^+/+ or p53^−/−) cells were cultured in the presence or absence of 50 nM doxorubicin (Dox) or 1 μM etoposide (Ets), respectively, and the number of cells was recorded every day for 4 days (shown in Fig. S9C). Growth inhibition ratios [(1 − number of cells after drug treatment/number of cells without drug treatment) × 100] were calculated with the data obtained on day 4 in Fig. S9C. *, P < 0.01 and **, P < 0.05, as compared with the group of cells harboring control sh-RNA, respectively. Results are reported as means ± SEM (n = 5). (C) Nude mice were inoculated s.c. in the dorsal area (10⁷ cells/injection, 4 mice/sample) with sh-con (left dorsal) and sh-PKR (right dorsal) HCT116 (p53^+/+ or p53^−/−) cells. Three days later, mice were treated i.p. once with doxorubicin (Dox) (2 mg kg⁻¹) or etoposide (25 mg kg⁻¹), and tumor growth was monitored for 18 days. Results are reported as means ± SEM (n = 4). (D) Inhibition ratios of tumor growth in tumor-bearing mice by doxorubicin (Dox) or etoposide (Ets) treatment were recalculated with the data on day 18 and are represented as means ± SEM (n = 4). *, P < 0.01 and **, P < 0.02, respectively, as compared with the mouse group bearing tumors expressing control sh-RNA. (E) Untreated (UT) and genotoxin-treated tumor-bearing mice were imaged on day 15 (Top). Yellow arrows and white arrows indicate the tumors established by inoculating sh-con (left dorsal) and sh-PKR (right dorsal) HCT116 (p53^+/+ or p53^−/−) cells, respectively. The expression of p53 and PKR was examined from each tumor (Bottom). (F) p53-induced PKR expression and associated tumor-suppression mechanisms are described together with other p53 target genes under conditions of DNA damage. Our findings described in this article are boxed.