Acetylcholinesterase expression in early human meqakaryoblasts is supressed under phorbol ester and thrombopoietin induced differentiation

The acetylcholine hydrolyzing enzyme acetylcholinesterase (AChE) is expressed in murine megakaryocytes (MK). Despite its important role in murine hematopoiesis (Soreq, PNAS 91:7907,1994, Lev-Lehman, Gene Therapy 1:127,1994), AChE was never detected In human MK by conventional methods. Using RT-PCR we detected in DAMI cells two alternatively spliced AChE mRNAs, 3' terminated with exon 6 or 5, encoding the common brain and muscle and the erythrccyte GPI-anchored form of AChE respectively. Semi-quantitative kinetics of RT-PCR amplification revealed about 100 AChE RNA molecules per ceH. High resolution in situ hybridization revealed AChE mRNA transcripts surrounding the nudear lobes in small and targe MK. Cytochemical staining, demonstrated nudear association of catalyticalry active AChE. Substrate hydrolysis assays revealed enzyme activity in DAMI cells and in the CHRF4288 and MEG01 megakaryoblast eel lines. Both phorbol ester (PMA) and Hu-rthrombopoietin (TPO) induced a dear reduction in enzyme activity, primarily in the larger celte, as detected by computerized image analysis of cytochemically stained cells. The PMA induced suppression reflected a dramatic suppression of AChE mRNA levels after 24 hours (>100 fold. p=<0.0001). Reductions in nudear associated enzyme activities by 35% with PMA and 20% with TPO were observed after 48 h. Moreover the residual nuclear associated enzyme which remained in the PMA treated cells was considerably more stable than that of proliferating control or TPO treated cells. Normal human bone marrow MK, detected with GPIIb/llla antibodies, also displayed dear nuclear associated AChE activity which was highly prominent in the small 2N MK and almost not detectable in the large mature MK. The AChE activity lost during proliferation arrest and differentiation indicates that AChE may be involved in early human megakaryocytopoiesis and can provide a unique marker for early proliferating MK progenitors.