The human monocytic cell line U937 was used as a model system to investigate the effects of glucocorticoids on monocytic differentiation. Upon incubation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (5 x 10(-9) M) for 48 to 72 h, the immature U937 cells ceased to proliferate and became morphologically and functionally macrophage-like. Preincubation of the cells with glucocorticoids (dexamethasone and prednisolone, 10(-7) and 10(-6) M) but not progesterone (10(-6) M) had marked effects: The cells remained in suspension and developed very little cell-cell interaction. This correlated with decreased expression of the surface molecules ICAM-1 and CD18 as determined by fluorescence-activated cell sorter analysis. The TPA-induced ability of the cells to release lysozyme or to generate reactive oxygen radicals (determined as reduction of nitroblue tetrazolium) was markedly reduced. The induction of cyclooxygenase activity and thus the ability to release prostanoids was almost completely abolished. Inhibition of prostanoid synthesis was also observed when the glucocorticoids were administered 24 or 48 h after TPA. The primary step of TPA induction, the activation and translocation of protein kinase C, however, was not affected by glucocorticoids as determined by activity measurements and Western blot analysis. There was no change in the subsequent TPA-induced induction of c-fos. The down-regulation of the differentiation-related oncogenes c-myc and c-myb was the same in cells treated with TPA in the presence or absence of glucocorticoids. Furthermore, no significant effect of glucocorticoids on the TPA-induced growth arrest was observed. Glucocorticoids thus interfere with TPA-induced functions, which are typical for activated macrophages; however, they do not impair the differentiation process and concomitant growth inhibition.
Conventional Synthesis of a Selective Peptide Substrate for Measurements of Protein Kinase C
Protein kinase C (PKC), a family of serin/threonin kinases, plays a key role in signal transduction. We have prepared the PKC‐selective peptide substrate H‐Phe‐Lys‐Lys‐Ser‐Phe‐Lys‐Leu‐NH2 (7) by classical solution synthesis. 7 allows PKC‐measurements in crude extracts or permeabilized cells. The protection of the N‐terminal amino acid and the side chains by Boc resp. tert‐butyl groups enables a one‐step liberation of the desired heptapeptide amide 7.
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