Abstract
The human erythroleukemia (K562) cell line undergoes megakaryocytic differentiation and cessation of proliferation when treated with phorbol myristate acetate (PMA). To investigate the role of individual protein kinase C (PKC) isotypes in these events, we have assessed PKC isotype expression during leukemic proliferation and PMA-induced differentiation. Immunoblot analysis using isotype-specific antibodies demonstrates that proliferating K562 cells express the alpha, beta II, and zeta PKC isotypes. PMA-induced differentiation and cytostasis lead to a decrease in beta II PKC and increases in alpha and zeta PKC levels. The role of the alpha and beta II PKC isotypes was further assessed in cells overexpressing these isotypes. K562 cells overexpressing human alpha PKC grew more slowly and were more sensitive to the cytostatic effects of PMA than control cells, whereas cells overexpressing beta II PKC were less sensitive to PMA. PMA-induced cytostasis is reversed upon removal of PMA. Resumption of proliferation is accompanied by reexpression of beta II PKC to near control levels, whereas alpha and zeta PKC levels remain elevated for several days after removal of PMA. Proliferation of PMA-withdrawn cells can be partially inhibited by antisense beta II PKC oligodeoxyribonucleotide. Growth inhibition is dose-dependent, specific for beta II PKC-directed antisense oligonucleotide, and associated with significant inhibition of beta II PKC levels indicating that beta II PKC is essential for K562 cell proliferation. Sodium butyrate, which unlike PMA induces megakaryocytic differentiation without cytostasis, causes increases in both alpha and beta II PKC levels. These data demonstrate that beta II PKC is required for K562 cell proliferation, whereas alpha PKC is involved in megakaryocytic differentiation.
Highlights
From the $Departmentof Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 and §Sphinx PharmaceuticalZnc., Durham, NorthCarolina 27717
Immunoblot analysis using isotype-specific antibodies demonstrates that proliferating K562cells express thea,[811], single celltype (6).These observationssuggest that each Proteinkinase C (PKC) isotype participates in distinct signal transduction pathways within the cell
Resumption of proliferation is accompanied by reexpression of &I PKC to near control levels, whereas a and b PKC levels remain elevated for decrease in decrease in kemic cells cellular PKC protein levels with a corresponding kinase activity (9,lO)
Summary
PMA and harvested after 0,4,8,122,4, and 48 h. Cells overexpressingPKC showed no appreciable change in growth rate (a doubling time of about 24 h), but these cellsshowed relativeresistance to PMAinduced cytostasis (half-maximal response at 3 nM). Changes in PMA sensitivity are consistent with thperoposed roles of a and 011 PKC suggested by the isotype expression studies of cells undergoing PMA-induceddifferentiation PMA withdrawalbefore gradually returning to their normal, lower levels (data not shown) These results indicate that the level of PKCexpressioncorrelates positivelywith the proliferation rate of K562 cells, whereas a and { PKC levels show little or no correlation to this parameter. PMA-induced cytostasis and rapidreexpression upon return, to logarithmic growth phase after PMAwithdrawal suggest a direct role for BIrPKC in theproliferative capacity of K562
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