Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Nov;33(11):1539-51.
doi: 10.1016/j.leukres.2009.05.013. Epub 2009 Jun 18.

Protein Phosphatase 4 Regulates Apoptosis in Leukemic and Primary Human T-cells

Affiliations
Free PMC article

Protein Phosphatase 4 Regulates Apoptosis in Leukemic and Primary Human T-cells

Mirna Mourtada-Maarabouni et al. Leuk Res. .
Free PMC article

Abstract

The control of T-cell survival is of overwhelming importance for preventing leukemia and lymphoma. The present report demonstrates that the serine/threonine protein phosphatase PP4 regulates the survival of both leukemic T-cells and untransformed human peripheral blood T-cells, particularly after treatment with anti-leukemic drugs and other cytotoxic stimuli. PP4-induced apoptosis is mediated, at least in part, through de-phosphorylation of apoptosis regulator PEA-15, previously implicated in the control of leukemic cell survival. PP4 activity significantly affects the mutation rate in leukemic T-cells, indicating that PP4 dysfunction may be important in the development and progression of leukemia.

Figures

Fig. 1
Fig. 1
PP4c over-expression inhibits colony-forming ability, inhibits cell growth and increases apoptosis of CEM-C7 cells. CEM-C7 cells were transfected with either pcDNA3.1 or pcDNA3-PP4c. (a) 24 h post-transfection, cells were cloned in soft agar in the presence of G418 and the number of colonies was determined after 2–3 weeks. (b) Immunoblot of PP4c expression in CEM-C7 parental cells (lane 1), pcDNA3.1-transfected CEM-C7 cells (lane 2) and pcDNA.1-PP4c-transfected CEM-C7 cells (lanes 3 and 4). Each lane contains 50 μg of whole-cell lysate subjected to SDS-PAGE, followed by Western blot analysis with anti-PP4c antibody. Anti-β-actin antibody was used to reveal β-actin as a loading control. The resulting autoradiographs were analysed by densitometry. A representative autoradiograph is presented, and the bar graphs represent means ± S.E. from four independent experiments. Relative expression is the ratio of PP4c to β-actin. (c) Growth curve of CEM-C7, CEM-C7-pcDNA3.1-transcfected cells and CEM-C7-pcDNA3.1-PP4c-transfected cells over 96 h. Viable cell density was determined by nigrosin dye exclusion. Results are expressed as the means ± S.E., and are representative of data obtained from five separate experiments, *P < 0.01 compared with vector only and parental cells. (d) Cell cycle analysis of CEM-C7-pcDNA3.1-transfected cells and CEM-C7-pcDNA3.1-PP4c-transfected cells. DNA content was quantified by propidium iodide staining of fixed cells and fluorescence flow cytometry. Results are represented as the means ± S.E. (n = 5). Representative histograms are shown.
Fig. 2
Fig. 2
PP4c-specific siRNAs increase CEM-C7 proliferation and alter the cell cycle profile. CEM-C7 cells were transfected with control (−)siRNA or with PP4c-specific siRNA. (a) Expression of PP4c protein under the same conditions was determined by Western blotting and equivalent loading was demonstrated using anti-β-actin antibody. The resulting autoradiographs were analysed by densitometry. A representative autoradiograph is presented, and the bar graph shows means ± S.E. from five independent experiments. Relative expression is the ratio of PP4c to β-actin. (b) Viable cell number of (−)siRNA-, PP4s2- and PP4s1-siRNA-treated CEM-C7 cells over 96 h. *P < 0.01 compared with (−)siRNA. (c) Cell cycle profiles of (−)siRNA-transfected CEM-C7 cells and PP4s2 siRNA-transfected CEM-C7 cells. DNA content was quantified by propidium iodide staining of fixed cells and fluorescence flow cytometry. Results are represented as the means ± S.E. (n = 5). Representative histograms are shown.
Fig. 3
Fig. 3
PP4c knockdown inhibits apoptosis induced by Dexamethasone, anti-Fas antibody, TNFα, UV, cisplatin and butyrate in CEM-C7 cells. 72 h post-siRNA transfection, (−)siRNA-, PP4s2- and PP4s1-siRNA-treated CEM-C7 cells were exposed to 10 μM dexamethasone, 5 ng/ml anti-Fas antibody, 50 ng/ml TNFα, 40 J/m2 UV, 5 μg/ml cisplatin, 5 mM butyrate or 30 nM okadaic acid. (a) Apoptosis was quantified after 48 h using CaspaTag staining. (b) Colony-forming assays were carried out at 72 h. Data represent means ± S.E. from five independent experiments, *P < 0.01 compared with (−)siRNA-transfected cells.
Fig. 4
Fig. 4
Up-regulation and down-regulation of PP4c expression produce complementary effects on the survival and growth of human peripheral blood lymphocytes. (Graphs (a) and (b)) Peripheral blood lymphocytes were cultured in complete RPMI medium supplemented with 2.5 μg/ml PHA for 5 days and transiently transfected with either pcDNA3.1 or pcDNA3.1-PP4c. (a) Apoptosis was determined at 24 and 48 h time points using CaspaTag (means ± S.E. from five independent experiments). (b) Viable cell numbers were determined by vital dye staining, at the indicated time points (means ± S.E. from seven independent experiments). (Graphs (c) and (d)) Peripheral blood lymphocytes were cultured in complete RPMI medium supplemented with 2.5 μg/ml PHA for 5 days and transfected with (−)siRNA, PP4s1 or PP4s2. (c) Caspase activation, as a marker of apoptosis, was determined at 24 and 48 h (means ± S.E. from six independent experiments). (d) Viable cell number was determined by vital dye staining (means ± S.E. from six independent experiments).
Fig. 5
Fig. 5
Comparison between the effects of PP2cβ, PP4 phosphatase-dead mutant PP4-RL, and PP4c on viable cell number of human peripheral blood lymphocytes. PHA-stimulated lymphocytes were transfected with pcDNA3.1-PP4c or pcDNA3.1, pCMVSPORT6-PP2c or pCMVSPORT, or the phosphatase-dead PP4c mutant in the expression vector expression vector pCI-neo. Viable cell number was determined after 48 h. Results represent means ± S.E. and are representative of data obtained from five independent experiments, *P < 0.01 compared with vector only.
Fig. 6
Fig. 6
Effects of PP2cβ down-regulation on PHA-stimulated human peripheral blood lymphocyte cell viability. PHA-stimulated human peripheral blood lymphocytes were transfected with either (−)siRNA or one of the two specific PP2cβ-targeted siRNAs. (a) Viable cell number was determined after 48 and 72 h. Results represent means ± S.E. and are representative of data obtained from five independent experiments, *P < 0.01 compared with (−)siRNA. (b) and (c) Comparison between the effects of PP4c (b) and PP2cβ (c) down-regulation on growth inhibition of human peripheral blood lymphocytes induced by fetal calf serum withdrawal. Cell density was determined by nigrosin dye exclusion at different time points. Results are expressed as the means ± S.E., and are representative of data obtained from five separate experiments, *P < 0.01 compared with (−)siRNA transfected cells.
Fig. 7
Fig. 7
Modulation of PP4c expression affects colony-forming ability and mutation frequency at the HPRT locus in CEM-C7 and Jurkat cells. (a) CEM-C7 and Jurkat cells transfected with either pcDNA3.1, pcDNA3.1-PP4c, (−)siRNA, PP4s1 or PP4s2 siRNAs were exposed to 40 J/m2 UV, and colony-forming ability was determined 48 h after UV exposure. Results are expressed as means ± S.E. from five independent experiments, *P < 0.01 compared with vector-only and parental cells. (b) Mutation frequencies at the HPRT locus in CEM-C7 and Jurkat cells transfected with either pcDNA3.1, pcDNA3.1-PP4c, (−)siRNA, PP4s1 or PP4s2 were determined 10 days post-UV irradiation by cloning in soft agar in the presence or absence of 50 μM 6-thioguanine. Data represent means ± S.E. from five independent experiments. *P < 0.01 compared with vector only and (−)siRNA transfected cells.
Fig. 8
Fig. 8
PEA-15-specific siRNAs inhibit cell growth and increase apoptosis in CEM-C7 cells. CEM-C7 cells were transfected with control (−)siRNA, GAPDH siRNA or with three different PEA-15-specific siRNAs. (a) Expression of PEA-15 protein under the same conditions was determined by Western blotting and equivalent loading was demonstrated using anti-β-actin antibody. The resulting autoradiographs were analysed by densitometry. A representative autoradiograph is presented, and the bar graph (b) shows the means ± S.E. from five independent experiments. Relative expression is the ratio of PEA-15 to β-actin. (c) Viable cell count of CEM-C7 cells treated with GAPDH siRNA, (−)siRNA or PEA-15s1, PEA-15s2 or PEA-15s3 siRNA, after 48 h. Data represent means ± S.E. from five independent experiments, *P < 0.01 compared with (−)siRNA transfected cells. (d) Active caspase staining, as a marker of apoptosis, was determined using CaspaTag and fluorescence microscopy. Data shown are the means ± S.E from five separate experiments, *P < 0.01 compared with (−)siRNA-transfected cells. (e) Cell density was determined by nigrosin dye exclusion. (f) Effects of PP2cβ over-expression on PEA-15 siRNA-transfected cells. CEM-C7 cells were transfected with either control (−)siRNA or PEA-15-specific siRNA. 48 h post-transfection, control cells (transfected with negative control siRNA) and cells transfected with PEA-15 siRNAs were transiently transfected with pCMVSPORT6-PP2cβ or pCMVSPORT6. Viable cell number was determined after 24 h. Data represent means ± S.E. from five independent experiments. (a) P < 0.01 compared with (−)siRNA. (b) P < 0.01 compared to pcDNA3.1. (c) P < 0.01 compared to (−)siRNA. (d) P < 0.01 compared to pCMVSPORT6.

Similar articles

See all similar articles

Cited by 13 articles

See all "Cited by" articles

References

    1. Rathmell J.C., Thompson C.B. Pathways of apoptosis in lymphocyte development, homeostasis, and disease. Cell. 2002;109:S97–S107. - PubMed
    1. Green D.R., Evan G.I. A matter of life and death. Cancer Cell. 2002;1:19–30. - PubMed
    1. Hengartner M.O. The biochemistry of apoptosis. Nature. 2000;407:770–776. - PubMed
    1. Giovannetti A., Pierdominici M., Di Iorio A., Cianci R., Murdaca G., Puppo F. Apoptosis in the homeostasis of the immune system and in human immune mediated diseases. Curr Pharm Des. 2008;14:253–268. - PubMed
    1. Sun E.W., Shi Y.F. Apoptosis: the quiet death silences the immune system. Pharmacol Ther. 2001;92:135–145. - PubMed

Publication types

Substances

Feedback