No abstract

To examine the role of acetylcholinesterase (EC 3.1.1.7) in hematopoietic cell proliferation and differentiation, we administered a 15-mer phosphorothioate oligonucleotide, antisense to the corresponding ACHE gene (AS-ACHE), to primary mouse bone marrow cultures. Within 2 hr of AS-ACHE addition to the culture, ACHE mRNA levels dropped by approximately 90%, as compared with those in cells treated with the "sense" oligomer, S-ACHE. Four days after AS-ACHE treatment, ACHE mRNA increased to levels 10-fold higher than in S-ACHE cultures or in fresh bone marrow. At this later time point, differential PCR display revealed significant differences between cellular mRNA transcripts in bone marrow and those in AS-ACHE- or S-ACHE-treated cultures. These oligonucleotide-triggered effects underlay considerable alterations at the cellular level: AS-ACHE but not S-ACHE increased cell counts, reflecting enhanced proliferation. In the presence of erythropoietin it also enhanced colony counts, reflecting expansion of progenitors. AS-ACHE further suppressed apoptosis-related fragmentation of cellular DNA in the progeny cells, and it diverted hematopoiesis toward production of primitive blasts and macrophages in a dose-dependent manner promoted by erythropoietin. These findings suggest that the hematopoietic role of acetylcholinesterase, anticipated to be inverse to the observed antisense effects, is to reduce proliferation of the multipotent stem cells committed to erythropoiesis and megakaryocytopoiesis and macrophage production and to promote apoptosis in their progeny. Moreover, these findings may explain the tumorigenic association of perturbations in ACHE gene expression with leukemia.

Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines ofevidence suggested correlation between cholinergic snaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically alignd amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these fmding strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and cholinesterases in this differentiation process.

No abstract

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.