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Possible involvement of protein kinase C-epsilon in phorbol ester-induced growth inhibition of human lymphoblastic
R Mihalik1, G Farkas, L Kopper
1Department of Immunology, National Institute of Haematology, Blood Transfusion and Immunology, Budapest Hungary.
Abstract:
Sustained activation of members of the protein kinase C (PKC) family is known to influence the growth and differentiation of various cell types, however, the specific roles for individual isoforms mediating these cellular events have yet to be elucidated. Activation of PKC by phorbol esters leads to growth inhibition in certain cell lines. The HT58 human B lymphoblastic cell may serve as a cellular model system to investigate the participation of individual isoforms in the initial events of growth arrest induced by phorbol ester. Determination of cell cycle and investigation of apoptosis were performed by flow cytometric measurements. Phorbol ester-induced translocation and down-regulation of the conventional alpha, beta and the novel epsilon isoforms of PKC were demonstrated by Western blot analysis. At lower concentrations (o.5 ng/ml) phorbol myristate acetate (PMA) stimulated a G1 arrest with retention of viability in the human HT58 B lymphoblastic cell. The protein kinase inhibitor staurosporine at a concentration of 25 nM did not significantly alter HT58 cell viability. However, staurosporine (25 nM) induced apoptosis in cells preincubated for 4 hr with 0.5-1.0 ng/ml PMA. The translocation of PKC-epsilon was observed within 39 min exposure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regulation and and altered phosphorylation state of PKC-epsilon was seen. In contrast, the conventional alpha and beta isoforms were practically uneffected by this PMA treatment. At higher PMA concentrations (50 ng/ml) the alpha and beta isoforms showed a significant down-regulation. The preferential alterations in PKC-epsilon observed under the conditions required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca(2+)-independent epsilon isoform in mediating the initial events of the phorbol ester stimulated cellular responses.
Insights
Protein kinase C (PKC) epsilon isoform mediates phorbol ester-induced growth arrest and apoptosis in HT58 B lymphoblastic cells. This study reveals PKC-epsilon
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Protein kinase C (PKC) activation influences cell growth and differentiation.
- Specific roles of individual PKC isoforms in these processes remain unclear.
- Phorbol esters can induce growth inhibition in certain cell lines.
Purpose of the Study:
- Investigate the role of individual PKC isoforms in phorbol ester-induced growth arrest.
- Utilize the HT58 human B lymphoblastic cell line as a model system.
- Elucidate the specific PKC isoforms involved in early cellular responses to phorbol esters.
Main Methods:
- Flow cytometry for cell cycle and apoptosis analysis.
- Western blot analysis for PKC isoform translocation and down-regulation.
- Treatment with phorbol myristate acetate (PMA) and staurosporine.
Main Results:
- PMA at low concentrations induced G1 arrest and maintained HT58 cell viability.
- PKC-epsilon translocation occurred rapidly, followed by down-regulation and altered phosphorylation.
- Conventional PKC alpha and beta isoforms were unaffected at low PMA concentrations but down-regulated at high concentrations.
- Staurosporine induced apoptosis in PMA-pretreated cells, suggesting a role for PKC in survival pathways.
Conclusions:
- PKC-epsilon isoform plays a preferential role in mediating initial phorbol ester-stimulated cellular responses.
- The Ca(2+)-independent nature of PKC-epsilon may be significant in these events.
- Findings suggest distinct functions for different PKC isoforms in cellular regulation.