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Protein phosphatase 4 regulates apoptosis in leukemic and primary human T-cells.

Mourtada-Maarabouni M, Williams GT - Leuk. Res. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Science and Technology in Medicine and School of Life Sciences, Huxley Building, Keele University, Keele ST5 5BG, UK. bia19@biol.keele.ac.uk

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.

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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).
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fig4: 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).

Mentions: To confirm that PP4c expression plays a role in apoptosis and cell proliferation in untransformed lymphoid cells, we manipulated the endogenous levels of PP4c expression in human peripheral blood lymphocytes stimulated with the mitogen phytohaemagglutinin (PHA) [50]. PHA-stimulated T-lymphocytes were transiently transfected with pcDNA3.1-PP4c or with pcDNA3.1. Expression of PP4c was assessed by qRT-PCR 24 h post-transfection and was found to be increased by 4-fold (results not shown). Transfected cells were then plated for 4 days in the presence of PHA and their proliferation rate was monitored every 24 h, based on viable cell counting. Apoptosis was assessed after 24 and 48 h. PP4c-transfected cells showed a 1.5- to 2-fold increase in apoptosis relative to cells transfected with vector only (Fig. 4a), indicating that over-expression of PP4c by itself enhances apoptosis in normal human T-lymphocytes. Cells transfected with pcDNA3.1-PP4c showed a 3-fold decrease in the number of viable cells at every time point, compared to cells transfected with vector only (Fig. 4b), showing the inhibitory effect of PP4c on cell proliferation in primary human T-lymphocytes.


Protein phosphatase 4 regulates apoptosis in leukemic and primary human T-cells.

Mourtada-Maarabouni M, Williams GT - Leuk. Res. (2009)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2734887&req=5

fig4: 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).
Mentions: To confirm that PP4c expression plays a role in apoptosis and cell proliferation in untransformed lymphoid cells, we manipulated the endogenous levels of PP4c expression in human peripheral blood lymphocytes stimulated with the mitogen phytohaemagglutinin (PHA) [50]. PHA-stimulated T-lymphocytes were transiently transfected with pcDNA3.1-PP4c or with pcDNA3.1. Expression of PP4c was assessed by qRT-PCR 24 h post-transfection and was found to be increased by 4-fold (results not shown). Transfected cells were then plated for 4 days in the presence of PHA and their proliferation rate was monitored every 24 h, based on viable cell counting. Apoptosis was assessed after 24 and 48 h. PP4c-transfected cells showed a 1.5- to 2-fold increase in apoptosis relative to cells transfected with vector only (Fig. 4a), indicating that over-expression of PP4c by itself enhances apoptosis in normal human T-lymphocytes. Cells transfected with pcDNA3.1-PP4c showed a 3-fold decrease in the number of viable cells at every time point, compared to cells transfected with vector only (Fig. 4b), showing the inhibitory effect of PP4c on cell proliferation in primary human T-lymphocytes.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Science and Technology in Medicine and School of Life Sciences, Huxley Building, Keele University, Keele ST5 5BG, UK. bia19@biol.keele.ac.uk

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.

Show MeSH
Related in: MedlinePlus