Abstract
In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild-type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA-induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA.
Highlights
Than 100-fold resistant to the differentiating andcy- Comparable PMA-induced alterations inmRNA levels tostatic activities of phorbol 12-myristate 13-acetate were not observed in PMA-resistant cells, even under (PMA), the protein kinase C phenotype was found to conditions of significantactivationandsubsequent be nearly identical to thaotf wild-type HL-60 cells
Taken measurable decrease (30%)in thespecific activities of together, these data suggest that activation and downcrude preparationsof PR-17 cell protein kinaseC was regulation of the isozymes of protein kinaseC may not observed whenthe enzyme was measured with histonerepresent absolute determinants of the PMA-induced as the phosphate acceptor substrate, but other aspects differentiation of HL-60 cells, but that specific alterof the protein kinase C phenotype were equiv-by PMA
In wildtype HL-60 cells, the intracellular concentrations of mRNA for each of the isozymes were increased 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expressionof HL-60 cell tibilities to down-regulation by proteases) [3,4,5,6,7,8]
Summary
The loss of proliferative capacity in maturing cells, in that in the PMA-resistant subline, PR-17. Thepossible stabili- enzyme could be defective or deficient One such targetcould zation of p isozyme mRNA in PMA-treated HL-60 cells was be the AP-1 or fos-jun transcription factocromplexes, which examined to determineif this could account for the observed are known to modulate the expression of genes under the increases in steady-state mRNA levels. These results revealed that and tumor necrosis factor was not impaired in the PR-17cell the levels of the @ isozyme protein recovered to control levels line (data not shown) This result suggested that the lack of within 1 h in wild-type cells which had accumulated mRNA differentiation response in PR-17cells treated with PMA was for the @ isozyme. Given the isozyme-specific increase in protein kinase C p
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