Oncogenic WIP1 phosphatase attenuates the DNA damage response and sensitizes p53 mutant Jurkat cells to apoptosis

Abstract

Wild-type (wt) p53-induced phosphatase 1 (Wip1), encoded by the protein phosphatase, Mg²⁺/Mn²⁺ dependent 1D (PPM1D) gene, is a serine/threonine phosphatase induced upon genotoxic stress in a p53-dependent manner. Wip1/PPM1D is frequently overexpressed, amplified and mutated in human solid tumors harboring wt p53 and is thus currently recognized as an oncogene. Oncogenic Wip1 dampens cellular stress responses, such as cell cycle checkpoints, apoptosis and senescence, and consequently increases resistance to anticancer therapeutics. Targeting Wip1 has emerged as a therapeutic strategy for tumors harboring wt p53. However, little is known about the efficacy of Wip1-targeted therapies in tumors lacking p53. The present study aimed to investigate the potential role of oncogenic Wip1 in p53 mutant (mt) Jurkat cells. In the present study, it was demonstrated that p53 mt Jurkat cells exhibited PPM1D/Wip1 gene amplification and expressed relatively high levels of Wip1, as confirmed by gene copy number and RNA expression analysis. In addition, Jurkat cells underwent G2 cell cycle arrest, apoptotic cell death and senescence in response to etoposide and doxorubicin, although the phosphorylation levels of DNA damage response (DDR) elements, including ataxia-telangiectasia mutated, ataxia-telangiestasia and Rad3-related, checkpoint kinase (Chk)1 and Chk2 were significantly low. Accordingly, the targeting of Wip1 phosphatase by RNA interference increased the phosphorylation of DDR elements, but decreased the rate of apoptosis in response to etoposide or doxorubicin in Jurkat cells. The induction of senescence or cell cycle arrest was not affected by the knockdown of Wip1. The results suggest that increased Wip1 expression enhances the apoptotic sensitivity of Jurkat cells in response to chemotherapeutic agents by attenuating DDR signaling. The present study highlights the possible pro-apoptotic role of Wip1 in a p53 mt T-cell acute lymphoblastic leukemia cell line. The data suggest the careful consideration of future treatment strategies aiming to manipulate or target Wip1 in human cancers lacking p53.

Keywords: Jurkat; apoptosis; doxorubicin; etoposide; p53; p53-induced phosphatase 1; senescence.

Figure 1.

Jurkat cells exhibit PPM1D/Wip1 gene amplification and increased expression of Wip1. (A) Genomic DNA was isolated from BJ human diploid fibrobasts, Jurkat T-cell acute lymphoblastic leukemia cells and MCF-7 breast cancer cells and used for subsequent PPM1D gene copy number analysis by qPCR. RNAse P was used as the reference gene. (B) BJ, Jurkat and MCF7 cells were used for total RNA isolation. Relative Wip1 mRNA levels were analyzed by reverse transcription-qPCR. Data shown are the means ± SD of three independent experiments. The statistical significance of differences in data was analyzed using one-way ANOVA followed by Bonferroni multiple comparisons tests. *P≤0.001 vs. BJ. PPM1D, protein phosphatase, Mg²⁺/Mn²⁺ dependent 1D; Wip1, p53-induced phosphatase 1; qPCR, quantitative PCR.

Figure 2.

Eto and Doxo induce G2 cell cycle arrest and cell death. Jurkat cells treated with DMSO, 1 µg/ml Doxo or 5 µg/ml Eto for 24 h or 72 were subjected to (A) WST-1 assay and (B) cell cycle analysis. The percentage of Jurkat cells in the G0/G1, S and G2/M phases was calculated using a Muse Cell Analyzer. Data shown are the means ± SD of three independent experiments. The statistical significance of differences in the data was analyzed using one-way ANOVA followed by Bonferroni multiple comparison tests. *P≤0.001 vs. C; ^#P≤0.001 vs. Eto. C, DMSO control; Doxo, doxorubicin; Eto, etoposide.

Figure 3.

DNA damage response signaling is impaired in Jurkat cells, but the induction of apoptosis is retained. Jurkat cells treated with DMSO, 1 µg/ml Doxo or 5 µg/ml Eto for 24 h or 72 h were analyzed for (A) apoptosis using Annexin V/7AAD and (B) caspase-3/7 activity using a Muse Cell Analyzer, and (C) subjected to the western blot analysis of Wip1, p-ATM, T-ATM, ATR, p-Chk1, T-Chk1, p-Chk2, T-Chk2, gH2AX and H2AX. GAPDH was used as the loading control. Data shown are the means ± SD of three independent experiments. The statistical significance of differences in the data was analyzed using one-way ANOVA followed with Bonferroni multiple comparison tests. *P≤0.001 vs. C; ^#P≤0.001 vs. Eto. C, DMSO control; Doxo, doxorubicin; Eto, etoposide; Wip1, p53-induced phosphatase 1; p-, phospho-; T-, total; ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiestasia and Rad3-related; Chk, checkpoint kinase; H2AX, H2A histone family member X; gH2AX, p-H2AX (S139).

Figure 4.

Induction of senescence is unaffected by increased Wip1 in Jurkat cells. Jurkat cells were treated with DMSO, 0.2 µg/ml Doxo or 1 µg/ml Eto for 72 h and the induction of senescence was assayed by measuring (A) cell viability by WST-1 assay, (B) the cell cycle profile and (C) the amount of active H2AX [γH2AX; p-H2AX (S139)] and non-active (unphosphorylated) H2AX. (D) Staining of cells for senescence-associated β-galactosidase activity. Scale bar, 50 mm. (E) Western blot analysis of Wip1, p-ATM, T-ATM, ATR, p-Chk1, T-Chk1, p-Chk2 and T-Chk2. GAPDH was used as the loading control. Data shown are the means ± SD of three independent experiments. The statistical significance of differences in the data was analyzed by one-way ANOVA followed by Bonferroni multiple comparison tests. *P≤0.001 vs. C. C, DMSO control; Doxo, doxorubicin; Eto, etoposide; H2AX, H2A histone family member X; Wip1, p53-induced phosphatase 1; p-, phospho-; T-, total; ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiestasia and Rad3-related; Chk, checkpoint kinase.

Figure 5.

Knockdown of Wip1 restores DNA damage response signaling but decreases apoptosis. Jurkat cells were transfected with siWip1 or siC siRNA. (A) The cells were subjected to western blot analysis of Wip1, p-ATM, T-ATM, p-ATR, T-ATR, p-Chk1, T-Chk1, p-Chk2 and T-Chk2. GAPDH was used as the loading control. siRNA. The transfected cells were treated with DMSO, 1 µg/ml Doxo or 5 µg/ml Eto for 72 h and subjected to (B) cell cycle analysis (C) Annexin/7AAD staining for apoptosis analysis and (D) caspase-3/7 activity assay. Data are expressed the as means ± SD of three independent experiments. The statistical significance of differences in the data was analyzed using one-way ANOVA followed by Bonferroni multiple comparison tests. *P≤0.001 as indicated. Wip1, p53-induced phosphatase 1; siWip1, siRNA targeting Wip1; siC, inverted siRNA control that does not target any gene; siRNA, small interfering RNA; C, DMSO control; Doxo, doxorubicin; Eto, etoposide; p-, phospho-; T-, total; ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiestasia and Rad3-related; Chk, checkpoint kinase.

Figure 6.

Knockdown of Wip1 does not affect cell cycle or senescence. (A) Jurkat cells were transfected with siWip1 or siC siRNA and treated with DMSO, 0.2 µg/ml Doxo or 1 µg/ml Eto for 72 h and induction of senescence was assayed by measuring the cell cycle profile. (B) Quantitative γH2AX assay and (C) senescence-associated β-galactosidase activity staining and quantification were also performed. Scale bar, 20 mm. Data are shown as the means ± SD of three independent experiments. The statistical significance of differences in the data were analyzed using one-way ANOVA followed by Bonferroni multiple comparison tests. *P≤0.001 as indicated. Wip1, p53-induced phosphatase 1; siWip1, siRNA targeting Wip1; siC, inverted siRNA control that does not target any gene; siRNA, small interfering RNA; C, DMSO control; Doxo, doxorubicin; Eto, etoposide.