Modulation of the Megakaryoblastic Dami Cell Line Differentiation by Phosphodiesterase Inhibitors and Imidazo[1,2‐a]pyrazine Derivatives

Abstract

Phosphodiesterase inhibitors have been shown to modulate cell differentiation. We have previously shown that a series of imidazo[ 1,2-a]pyrazine derivatives displayed inhibitory effects on phosphodiesterase isoenzymes types 111, IV and V isolated from Dami cells and on Dami cell growth. In the present study we have investigated the effect of these derivatives on the expression of two differentiation markers, glycoproteins Ib and IIb/IIIa of the human megakaryoblastic leukaemic Dami cell line in comparison to those elicited by 3-isobutyl-1-methylxanthine and selective phosphodiesterase inhibitors of types I (8-methoxymethyl-l-methyl-3-(2-methylpropyl) xanthine), 111 (Mikinone), IV (RO-201724) and V (Zaprinast). Imidazo[l,2-a]pyrazine derivatives, 3-isobutyl-1 -methylxanthine and selective phosphodiesterase inhibitors, except 8-methoxymethyl-l-methyl-3-(2-methylpropyl) xanthine, decreased glycoprotein Ib expression. SCA40, SCA41, SCA44 and 3-isobutyl-1-methylxanthine but not the other compounds affected the expression of glycoprotein IIb/IIIa in a positive manner. The effects of imidazo [ 1,2-u]pyrazine derivatives on glycoprotein expression appeared to be related to their phosphodiesterase inhibitory potency.Cyclic nucleotides are known to be involved in cell differentiation ( Materials and MethodsMaterials. Dami cells were obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). Horse serum was purchased from Techgen International (Les Ulis, France), and cell culture medium and reagents were from Gibco BRL (Eragny, France).Monoclonal antibodies P2 anti-glycoprotein IIb/IIIa (CD4la) and SZ2 anti-glycoprotein Ib (CD42b) were obtained from Immunotech (Marseilles, France) and the FITC-labeled goat rabbit anti-mouse immunoglobuline f(ab'), from Dako SA (Versailles, France). Milrinone (1,6-dihydro-2-mCthyl-6-0~0-(3,4'-bipyridine)-5-carbonitrile), RO-201724 amino)imidazo[l,2-a]pyrazine-2-carbonitrile), SCA44 (6-bromo-8-(cyclopen tylamino)imidazo[ 1,2-a]pyrazine-2-carboni trile) were synthesised in our laboratory as previously described (Bonnet et al. 1992). Other reagents were from Sigma (St. Quentin Fallavier, France).Cell culture conditions. Cells were grown in RPMI 1640 medium supplemented with 10% heat inactivated horse serum, 2 mM gluta-