Salivary cortisol and cardiovascular activity during stress in oppositional-defiant disorder boys and normal controls

The erythroleukemic cell line K562 can undergo further differentiation in erythroid or megakaryocytic lineage depending on the nature of the stimulus. Phorbol ester (PMA) stimulates megakaryocytic development whereas hemin promotes erythroid differentiation of these cells. We have examined the effect of PMA and hemin on the expression of the Kell blood group and CD10 antigens, two related proteins that belong to a family of membrane-bound neutral metalloendopeptidases. We show here that differentiation of K562 cells by PMA in the megakaryocytic lineage results in abolishment of Kell mRNA accumulation and protein expression and, in parallel, the induction of CD10 mRNA accumulation, protein expression, and enzymatic activity. Conversely, differentiation of these cells by hemin in the erythroid lineage is accompanied by an up-regulation of Kell mRNA and protein expression, with no changes in CD10 mRNA and protein expression. Thus, CD10 and Kell can be regarded as specific markers of the differentiation of K562 cells in the megakaryocytic and erythroid lineages, respectively.     

Expression of erythroid-specific genes in acute megakaryoblastic leukaemia and transient myeloproliferative disorder in Down's syndrome

Acute megakaryoblastic leukaemia (M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. To clarify properties of the blast cells in M7 and TMD cases, we examined erythroid markers expression in blasts from six cases with M7 and seven cases with TMD in this study. Erythroid-specific mRNAs encoding gamma-globin and erythroid delta-aminolevulinate synthase were found to be expressed in blasts from most of these cases, indicating that majorities of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. We also found that mRNAs encoding GATA-1 and GATA-2 are expressed in all these cases. These results suggest that M7 blasts and TMD blasts correspond to the erythroid/megakaryocytic bipotential progenitor cells.     

Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells

The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.