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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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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.
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fig1: 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.

Mentions: The EST clone PP4c (accession # BG913014) was cloned directionally in pcDNA3.1 and transfected into Jurkat and CEM-C7 cells to generate stable clones selected by growth in G418. Since very similar results were obtained from both cell lines, only CEM-C7 results are shown (the Jurkat cell data are provided as supplementary data online). Transfection of the PP4c expression construct into both cell lines led to the growth of 8- to 12-fold fewer colonies than vector-only transfected cells (Fig. 1a), reflecting the inhibitory effects of PP4c over-expression on cell growth and colony-forming ability. Three empty vector-containing clones and six PP4c-transfected clones from each cell line were further characterized. The degree of over-expression of PP4c was determined, firstly, by qRT-PCR (a 6–8-fold over-expression was observed (data not shown), and, secondly, by Western blotting (Fig. 1b). The effect of PP4c over-expression on CEM-C7 and Jurkat cell growth rate and apoptosis was examined. A significant difference in the rate of proliferation between cells transfected with PP4c and cells transfected with vector only was consistently observed. Fig. 1c shows that PP4c-transfected cells proliferated at a significantly lower rate than control pcDNA3.1-transfected cells. The observed regulatory effects of PP4c were not due to clonal idiosyncrasies, since the results were also confirmed using polyclonal populations of CEM-C7 and Jurkat cells transfected with PP4c or vector only (results not shown).


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

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

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.
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fig1: 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.
Mentions: The EST clone PP4c (accession # BG913014) was cloned directionally in pcDNA3.1 and transfected into Jurkat and CEM-C7 cells to generate stable clones selected by growth in G418. Since very similar results were obtained from both cell lines, only CEM-C7 results are shown (the Jurkat cell data are provided as supplementary data online). Transfection of the PP4c expression construct into both cell lines led to the growth of 8- to 12-fold fewer colonies than vector-only transfected cells (Fig. 1a), reflecting the inhibitory effects of PP4c over-expression on cell growth and colony-forming ability. Three empty vector-containing clones and six PP4c-transfected clones from each cell line were further characterized. The degree of over-expression of PP4c was determined, firstly, by qRT-PCR (a 6–8-fold over-expression was observed (data not shown), and, secondly, by Western blotting (Fig. 1b). The effect of PP4c over-expression on CEM-C7 and Jurkat cell growth rate and apoptosis was examined. A significant difference in the rate of proliferation between cells transfected with PP4c and cells transfected with vector only was consistently observed. Fig. 1c shows that PP4c-transfected cells proliferated at a significantly lower rate than control pcDNA3.1-transfected cells. The observed regulatory effects of PP4c were not due to clonal idiosyncrasies, since the results were also confirmed using polyclonal populations of CEM-C7 and Jurkat cells transfected with PP4c or vector only (results not shown).

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