p38 mitogen-activated protein kinase promotes cell survival in response to DNA damage but is not required for the G(2) DNA damage checkpoint in human cancer cells

Abstract

p38 mitogen-activated protein kinase (MAPK) is rapidly activated by stresses and is believed to play an important role in the stress response. While Chk1 is known to mediate G(2) DNA damage checkpoint control, p38 was also reported to have an essential function in this checkpoint control. Here, we have investigated further the roles of p38 and Chk1 in the G(2) DNA damage checkpoint in cancer cells. We find that although p38 activation is strongly induced by DNA damage, its activity is not required for the G(2) DNA damage checkpoint. In contrast, Chk1 kinase is responsible for the execution of G(2) DNA damage checkpoint control in p53-deficient cells. The inhibition of p38 activity has no effect on Chk1 activation and gamma-H2AX expression. Global gene expression profiling of cancer cells in response to tumor necrosis factor alpha (TNF-alpha) revealed that p38 plays a strong prosurvival role through the coordinated downregulation of proapoptotic genes and upregulation of prosurvival genes. We show that the inhibition of p38 activity during G(2) DNA damage checkpoint arrest triggers apoptosis in a p53-independent manner with a concurrent decrease in the level of Bcl2 family proteins. Our results suggest that although p38 MAPK is not required for the G(2) DNA damage checkpoint function, it plays an important prosurvival role during the G(2) DNA damage checkpoint response through the upregulation of the Bcl2 family proteins.

FIG. 1.

DNA damage induces G₂ arrest and activates p38 MAPK and Chk1 pathways. (A) Cell cycle profiles of control HeLa cells or HeLa cells treated with 160 nM adriamycin, 500 J/m² UV irradiation, or 0.01% MMS for 24 h. (B and C) Western blot analysis of the activation of p38α and phosphorylation of its substrate MK2 in unsynchronized HeLa cells treated with increasing doses of UV irradiation, with 10 μg/ml anisomycin treatment as a positive control (B) and with 0.01% MMS for various time intervals for up to 10 h (C). (D) Synchronized HeLa cells treated with 160 nM adriamycin 5 h after release from a second thymidine block. Cell cycle progression and p38 activation were analyzed by flow cytometry and Western blotting at the time points indicated after release. (E to G) DNA damage in synchronized cells. HeLa cells were synchronized at G₁ by serum starvation (E), at G₁/S by double-thymidine block (F), and at G₂ with the CDK1 inhibitor RO-3306 (G) and were then released into 0.01% MMS-containing fresh medium. The activation of p38 and Chk1 kinases was followed by Western blotting at the time intervals indicated.

FIG. 2.

Inhibition of Chk1 but not p38 abrogates the G₂ DNA damage checkpoint in HeLa cells. (A) Mitotic index of unsynchronized HeLa cells that were pretreated for 20 h with adriamycin prior to the addition of 320 nM p38 inhibitor (p38i) or 1.25μM Chk1 inhibitor (Chk1i) and 150 nM nocodazole. Mitotic indexes were quantified by using the high-content/high-throughput Acumen Explorer for cells expressing phospho-histone H3 at the time points indicated after inhibitor treatment. (B) Western blot analysis of double-thymidine-synchronized HeLa cells treated with various kinase inhibitors in the presence of 160 nM adriamycin and 150 nM nocodazole 20 h after release from the thymidine block. Lane 1, double-thymidine-treated cells; lane 2, untreated HeLa cells; lanes 3 to 9, synchronized cells treated with adriamycin; lane 3, adriamycin alone; lane 4, adriamycin plus 320 nM p38i; lane 5, adriamycin plus 2 μM MK2 inhibitor (MK2i); lane 6, adriamycin plus 1.25 μM Chk1 inhibitor; lane 7, adriamycin plus 6 mM caffeine; lane 8, adriamycin plus 1.25 μM Chk1 inhibitor and 320 nM p38i; lane 9, adriamycin plus 6 mM caffeine and 320 nM p38i. (C) Western blot analysis of the relationship between p38 activation/inactivation and the DNA damage response. Thymidine-synchronized HeLa cells were released into 0.01% MMS, and cells were analyzed at the times indicated. (D) Mitotic index of unsynchronized HeLa cells treated with 500 J/m² UV-B irradiation for 20 h prior to the addition of 320 nM p38i or 1.25 μM Chk1 inhibitor in the presence of 150 nM nocodazole (Noc). (E) Western blot of HeLa cells treated with 1,000 J/m² UV-B and the indicated doses of p38i, MK2 inhibitor, or Chk1 inhibitor 24 h after kinase inhibitor treatment.

FIG. 3.

Inhibition of Chk1 but not p38 by small-molecule kinase inhibitors or RNAi abrogates the UV-induced G₂ DNA damage checkpoint. (A) Western blot analysis of the response of cells after the inactivation of Chk1, p38, or MK2 by two specific and validated siRNA oligonucleotides directed against each gene to adriamycin. (B) Mitotic index plot of HeLa cells with p38 or Chk1 knockdown and treated with 160 nM adriamycin or 500 J/m² UV for 24 h. (C) p53-dependent abrogation of the G₂ DNA damage checkpoint by Chk1 inhibition. A549, U2OS, and Calu-6 cancer cells were treated with 160 nM adriamycin for 24 h before the addition of kinase inhibitors (320 nM p38i or 1.25 μM Chk1 inhibitor). Mitotic indexes were determined at 3 and 24 h after the addition of kinase inhibitors. (D and E) MK2 is not required for G₂ DNA damage checkpoint control following UV-C irradiation. U2OS cells treated with green fluorescent protein (GFP) or MK2 (MAPKAP kinase 2) siRNA were irradiated with 20 J/m² of UV-C as described previously (31) and then placed into 50 ng/ml nocodazole-containing medium for an additional 16 h. Target protein knockdown, DNA content, and phospho-histone H3 of cells were analyzed by Western blotting (D) and flow cytometry (E).

FIG. 4.

Nongenotoxic activation of p38 with anisomycin does not impede entry into mitosis in HeLa cells. (A) Mitotic index (phospho-histone H3) of HeLa cells synchronized with the CDK1 inhibitor RO-3306 in the presence or absence of 2 μg/ml anisomycin. (B) Western blot for the p38 activation of HeLa cells synchronized with the CDK1 inhibitor RO-3306 by 2 μg/ml anisomycin.

FIG. 5.

Inactivation of p38 inhibits immediate-early response and antiapoptotic pathways in TNF-α-treated Calu-6 cells. (A) Cluster analysis of genes differentially expressed 1 h after TNF-α treatment and effect of 320 nM p38i LY479754 on these expression changes. Gene expression values are represented by colors, with red and green indicating high and low expressions, respectively. (B) Box plots of TNF-α response of immediate-early response genes and members of apoptosis pathway component genes at the 1-h time point in the presence or absence of p38 kinase inhibitor. (C) Western blots of p38 activation, apoptosis, and cell death/survival pathway protein expression in Calu-6 cells treated with TNF-α in the presence of 320 nM p38i LY479754. (D) Apoptotic index (percentage of cells expressing cleaved PARP) of Calu-6 cells treated with 25 ng/ml TNF-α and various concentrations of p38i using an Acumen Explorer high-content imaging assay. CTRL, control with no treatment.

FIG. 6.

Inhibition of p38 MAPK sensitizes cells to adriamycin and induces cell death. (A) Apoptosis index (percentage of cells expressing cleaved caspase 3) of HeLa cells treated with increasing doses of the p38i LY479754 in the presence of 160 nM adriamycin for 48 h. (B) Western blot for apoptosis of HeLa cells treated with siRNA targeting p38α, MK2, or Chk1 in the presence or absence of 160 nM adriamycin for 48 h. NS-si, nonspecific/scramble siRNA. (C) Western blot analysis of synchronized A549 cells by serum starvation for 48 h after treatment with 1 μM p38i LY479754 in the presence of DNA damage induced by 160 nM adriamycin for 48 h. (D) Western blot of A549 cells treated with 0.01% MMS and 1 μM p38i LY479754 for 48 h. (E) Apoptosis induced by p38 inhibition in the presence of DNA damage is associated with G₂ arrest. The apoptosis index and cell cycle state were determined for A549 cells treated with 160 nM adriamycin and 1 μM p38i LY479754 using Acumen Explorer.

FIG. 7.

New model for a novel role of p38 MAPK in the DNA damage response in cancer cells. DNA damage elicits the activation of cell cycle checkpoint, DNA repair, and cell survival signaling pathways. In response to DNA damage, the Chk1-mediated G₂ DNA damage checkpoint is activated to prevent entry into mitosis with damaged DNA, whereas the p38-mediated cell survival pathway is activated to keep cells alive. Together, these two pathways afford cell the time to repair DNA damage and ultimately to allow recovery from DNA damage.