RETRACTED: Identification of phosphorylated p38 as a novel DAPK-interacting partner during TNFalpha-induced apoptosis in colorectal tumor cells

Abstract

Death-associated protein kinase (DAPK) is a serine/threonine kinase that contributes to pro-apoptotic signaling on cytokine exposure. The role of DAPK in macrophage-associated tumor cell death is currently unknown. Recently, we suggested a new function for DAPK in the induction of apoptosis during the interaction between colorectal tumor cells and tumor-associated macrophages. Using a cell-culture model with conditioned supernatants of differentiated/activated macrophages (U937) and human HCT116 colorectal tumor cells, we replicated DAPK-associated tumor cell death; this model likely reflects the in vivo tumor setting. In this study, we show that tumor necrosis factor-alpha exposure under conditions of macrophage activation induced DAPK-dependent apoptosis in the colorectal tumor cell line HCT116. Simultaneously, early phosphorylation of p38 mitogen-activated protein kinase (phospho-p38) was observed. We identified the phospho-p38 mitogen-activated protein kinase as a novel interacting protein of DAPK in tumor necrosis factor-alpha-induced apoptosis. The general relevance of this interaction was verified in two colorectal cell lines without functional p53 (ie, HCT116 p53(-/-) and HT29 mutant) and in human colon cancer and ulcerative colitis tissues. Supernatants of freshly isolated human macrophages were also able to induce DAPK and phospho-p38. Our findings highlight the mechanisms that underlie DAPK regulation in tumor cell death evoked by immune cells.

Figure 1

Macrophage-induced cell death in HCT116 cells is accompanied by DAPK up-regulation. A: Annexin-V measurements of HCT116 cells (ctrl) and HCT116 cells subjected to diffM supernatant (sup.) or actM supernatant. B: HCT116 subjected to diffM supernatant or actM supernatant for 48 hours were analyzed by caspase 3/7 activity assay. C: Lysates prepared from diffM supernatant- and actM supernatant-exposed HCT116 cells were analyzed by anti-DAPK or anti-β-actin Western blotting. Untreated HCT116 cells served as control. D: Lysates of HCT116 cells treated with supernatants from human actM isolated from PBMCs were analyzed by anti-DAPK, anti-Caspase3, and anti-β-actin Western blotting. Untreated HCT116 served as control.

Figure 2

TNF-α and IFN-γ are significantly released by macrophage cell line U937 on activation. A and B: U937 cells were stimulated with PMA or stimulated and activated with PMA and LPS, and subsequently, the supernatants were analyzed by TNF-α-ELISA (A) and IFN-γ-ELISA (B). *#P < 0.001.

Figure 3

TNF-α is critical for macrophage-induced cell death in HCT116 cells, and this apoptosis is mediated by DAPK. A: Annexin-V measurements of control HCT116 cells (ctrl) and HCT116 cells subjected to TNF-α. B: Control HCT116 cells (ctrl) and HCT116 cells subjected to 0.665 ng/ml TNF-α for 48 hours were analyzed by caspase 3/7 activity assay. C: Lysates of HCT116 cells subjected to TNF-α were analyzed by anti-DAPK, anti-DAPK-Ser308, or anti-β-actin Western blotting. Untreated HCT116 cells (ctrl) served as a control. D: Lysates of HCT116 cells subjected to TNF-α for 24 hours were analyzed by immunprecipitation (IP) and by in vitro kinase assay with p-RB-S6P as the substrate. DAPK-immunoprecipitate of untreated HCT116 cells (ctrl) and IgG-immunoprecipitate subjected to 0.665 ng/ml TNFα served as controls. E: Lysates of HCT116 cells knocked down for DAPK and subsequently subjected to TNFα for 48 hours were analyzed by anti-DAPK or anti-β-actin Western blotting. HCT116 cells subjected to transfection reagent (TR), DAPK-siRNA, and TNF-α-treatment served as controls. F: HCT116 cells transfected with DAPK-siRNA, stimulated with TNF-α were analyzed by Annexin-V assay. Untreated HCT116 cells (ctrl), transfected HCT116 cells (DAPK-siRNA), and TNFα-exposed HCT116 cells (TNFα 48 hours) and the transfection reagent (TR) served as controls.

Figure 4

p-p38 is a direct interacting partner of DAPK during TNFα-induced apoptosis. A: Lysates of HCT116 cells subjected to TNFα were analyzed by Western blotting using anti-p38, anti-p-p38, anti-ERK1/ERK2, anti-p-ERK1/ERK2, anti-JNK, anti-p-JNK, or anti-β-actin. Untreated HCT116 cells (ctrl) served as a control. B: HCT116 cells (ctrl or subjected to TNFα) were assayed for p38 MAPK activity; the control was adjusted to one. C: Lysates of HCT116 cells treated with supernatants from human actM isolated from PBMCs were analyzed by anti p-p38 and anti-β-actin Western blotting. Untreated HCT116 served as a control. D: HCT116 cells subjected to TNFα were lysed, and DAPK or p-p38 proteins were immunoprecipitated (IP) using anti-DAPK or anti-p-p38. Precipitates were analyzed by Western blotting for the presence of p-p38 and DAPK. E: TNFα-induced co-localization of DAPK and p-p38 after 6 hours was determined by fluorescence immunolabeling analysis using anti-DAPK, anti-p-p38, and DAPI.

Figure 5

TNFα-induced and DAPK-mediated apoptosis is triggered by p-p38. A: Lysates of HCT116 cells knocked down for p38 and subsequently subjected to TNFα for 48 hours were analyzed by Western blotting using anti-p38, anti-p-p38, or anti-β-actin and were then assayed for p38 MAPK activity; the control was adjusted to one. Untreated HCT116 cells, HCT116 cells stimulated with transfection reagent (TR), p38-siRNA, or TNFα served as controls. B: DAPK-immunoprecipitate (IP) of HCT116 cells transfected with p38-siRNA before TNFα-exposure was assayed for in vitro kinase activity: DAPK-IP of untreated HCT116 cells, of HCT116 cells transfected with p38-siRNA, of HCT116 cells transfected with p38-siRNA and stimulated with TNFα. IgG-IP subjected to TNFα served as a control. C: HCT116 cells transfected with p38-siRNA, subjected TNFα, were analyzed by Annexin-V measurements. Untreated HCT116 cells (ctrl), transfected HCT116 cells (p38-siRNA), and TNFα-subjected HCT116 cells (TNFα 48 hours) served as controls. D: HCT116 cells treated with 1 μmol/L p38 inhibitor SB203580 and/or TNFα were analyzed by Annexin-V measurements. Untreated HCT116 cells (ctrl), inhibitor-treated HCT116 cells (SB203580), and TNFα-subjected HCT116 cells (TNFα 48 hours) served as controls. E: Lysates of HCT116 cells subjected to p38 inhibitor SB203580 and/or TNFα for 6 hours were analyzed by Western blotting using anti-DAPK, anti-p38, anti-p-p38, or anti-β-actin.

Figure 6

TNFα induced DAPK-mediated apoptosis and DAPK/p38 co-localization in HCT116 p53 deficient cells. A: Annexin-V measurements of control HCT116 p53^−/− cells (ctrl) and HCT116 p53^−/− cells subjected to TNFα. B: Lysates of HCT116 p53^−/− cells subjected to TNFα were analyzed by Western blotting using anti-DAPK, anti-pDAPK³⁰⁸, anti-p38, anti-p-p38, anti- anti-Caspase3, or anti-β-actin. Untreated HCT116 p53^−/− cells (ctrl) served as control. C: Lysates of HCT116 p53^−/− cells knocked down for DAPK and subsequently subjected to TNFα for 48 hours were analyzed by anti-DAPK or anti-β-actin Western blotting. HCT116 p53^−/− cells subjected to transfection reagent (TR), DAPK-siRNA, and TNFα-treatment served as controls. D: HCT116 p53^−/− cells transfected with DAPK-siRNA, stimulated with TNFα were analyzed by Annexin-V assay. Untreated HCT116 p53^−/− cells (ctrl), transfected HCT116 p53^−/− cells (DAPK-siRNA), and TNFα-exposed HCT116 p53^−/− cells (TNFα 48 hours) and the transfection reagent (TR) served as controls. E: TNFα-induced co-localization of DAPK and p-p38 in HCT116 p53^−/− cells after 6 hours was determined by fluorescence immunolabeling analysis using anti-DAPK, anti-p-p38, and DAPI.

Figure 7

TNFα induced DAPK-mediated apoptosis and DAPK/p38 co-localization in HT29 p53 mutant cells. A: Annexin-V measurements of control HT29 cells (ctrl) and HT29 cells subjected to TNFα. B: Lysates of HT29 cells subjected to TNFα were analyzed by Western blotting using anti-DAPK, anti-pDAPK³⁰⁸, anti-p38, anti-p-p38, anti- anti-Caspase3, or anti-β-actin. Untreated HT29 cells (ctrl) served as control. C: Lysates of HT29 cells knocked down for DAPK and subsequently subjected to TNFα for 48 hours were analyzed by anti-DAPK or anti-β-actin Western blotting. HT29 cells subjected to transfection reagent (TR), DAPK-siRNA, and TNFα-treatment served as controls. D: HT29 cells transfected with DAPK-siRNA, stimulated with TNFα were analyzed by Annexin-V assay. Untreated HT29 cells (ctrl), transfected HT29 cells (DAPK-siRNA), and TNFα-exposed HT29 cells (TNFα 48 hours) and the transfection reagent (TR) served as controls. E: TNFα-induced co-localization of DAPK and p-p38 in HT29 cells after 6 hours was determined by fluorescence immunolabeling analysis using anti-DAPK, anti-p-p38, and DAPI.

Figure 8

DAPK co-localizes with p-p38 in human colorectal carcinoma. Immunohistochemical analysis of DAPK and p-p38 in normal colonic mucosa and colorectal carcinoma tissue with and without DAPK expression was detected by anti-DAPK and anti-p-p38. un, umethylated tumors; meth, methylated tumor (Microscope: Zeiss Axioscope 50; camera: Nikon coolpix 990; magnification: ×200, ×400). Arrows show single tumor cells expressing p-p38. Statistical analysis of differences in immunohistochemical DAPK and p-p38 expression (Student’s t-test).

Figure 9

DAPK co-localizes with p-p38 in ulcerative colitis. Immunohistochemical analysis of DAPK and p-p38 in different stages of acute and chronic inflammation and remission stage in ulcerative colitis was performed using the anti-DAPK and anti-p-p38. (Microscope: Zeiss Axioscope 50; camera: Nikon coolpix 990; magnification: ×200, ×400). Statistical analysis of differences in immunohistochemical DAPK and p-p38 expression (Student′s t-test).