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Interactions between PP4 and PEA-15 in the regulation of cell proliferation and apoptosis of breast cancer cellsBackground The serine/threonine protein phosphatase 4 (PP4) is recognised to regulate a variety of cellular functions. Our previous work has shown that the catalytic subunit of PP4 (PP4c) promotes cell death and inhibits proliferation in breast cancer cells, suggestive of a role of PP4c as tumour suppressor gene. Phosphoprotein enriched in astrocytes 15 (PEA-15), a member of the death effector domain protein family known to control cell survival, is reported to be regulated by PP4c. The aims of this study were to investigate the involvement of PEA-15 in mediating the effects of PP4c on breast cancer cells. Method PEA-15 phosphorylation was examined by western blot analysis on proteins extracted from MCF7 and MDA-MB-231 cells over-expressing PP4 and PP4 knock down cells. To investigate the role of PEA-15 in mediating the effects of PP4c, MCF7 and MDA-MB-231 were transfected with control (-) siRNA or with three different PEA-15 specific siRNAs. 48 h post-transfection, control cells (transfected with negative control siRNA) and cells transfected with PEA-15 siRNAs were transiently transfected with pcDNA3.1-PP4c expression construct or pcDNA3.1. Cell viability and apoptosis level were assessed post transfection. Results In MCF7 and MDA-MB-231 cells, the phosphorylation state of PEA-15 increased when PP4c expression was suppressed and decreased when PP4c was over-expressed. Over-expression of PP4c in cells transfected with (-) siRNA caused 50% reduction in viability compared to cells transfected with empty vector. Cells transfected with PEA-15 siRNAs showed a decrease in viable cell number and long term survival. However, over-expression of PP4c in these cells did not have any additional effect on the decrease in cell viability. Conclusion These observations suggest that the induction of apoptosis by over-expression of PP4c is mediated, at least in part, by the dephosphorylation of PEA-15. The interactions between PEA-15 and PP4c may therefore be critical in breast cancer tumorigenesis.
Regulation of the cell cycle and cell death by protein phosphatase 4 in breast cancer cell linesBackground At the molecular level, cell death is often regulated by the level of phosphorylation of particular proteins, i.e. by the balance of between opposing kinase and phosphatase activities on those proteins. Protein phosphatase 4 (PP4) is a PP2A-related serine/threonine phosphatase. PP2A has already been implicated in the control of cell proliferation, cell cycle and tumorigenesis. Using a functional expression cloning strategy, we have previously identified the catalytic subunit of PP4 (PP4c) as an important gene influencing the regulation of both apoptosis and cell proliferation in human leukaemic cell lines and in normal lymphocytes. The aims of this study were to examine the effects of PP4c overexpression and silencing on the cell death and survival of breast cancer cell lines. Method MCF7 and MDA-MB-231 cells were transfected with pcDNA3.1 encoding PP4c (pcDNA3-PP4c) or siRNAs to different PP4c sequences. Cells transfected with scrambled siRNA or empty vector were considered as controls. Culture viability, apoptosis and cell cycle were assessed post transfection. Results In MCF7 and metastatic MDA-MB-231 cells, PP4c over-expression exerted an inhibitory effect on cell proliferation, enhanced spontaneous apoptosis and decreased their colony forming ability. Conversely, siRNA mediated silencing of PP4 enhanced the proliferation and survival of MCF7 and MDA-MB-231 cells, affected cell cycle kinetics by enhancing the proportion of cells in S and G2/M phases, increased the colony forming ability and stimulated the anchorage independent growth. Conclusion PP4c promotes cell death and inhibits proliferation in breast cells, suggestive of a role of PP4c as tumour suppressor gene. Down regulation of PP4c expression increases cell survival, proliferation and anchorage independent growth of breast cancer cells, indicating a potential link between the PP4c expression levels, tumorigenesis and metastasis.