Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

Patinkin, Deborah; Seidman, Shlomo; Eckstein, Fritz; Benseler, Fritz; Zakut, Haim; Soreq, Hermona

doi:10.1128/mcb.10.11.6046

Cited by 45 publications

(15 citation statements)

References 23 publications

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“…6 and Table 1). To overcome the variabilities in sample size and cell-type composition, arbitrary ratios between CHED mRNA and actin mRNA levels for reference were independently calculated for each culture, based on direct PCR amplification of cDNA reversetranscribed from cell cultures mRNA and subsequent densitometry.…”

Section: Qislygkxiv Syvsedr Sra---------------------czd Qikkm*-------mentioning

confidence: 98%

“…Phosphorothioate antisense oligodeoxynucleotides (15) were prepared and HPLC-purified as described (6). Phosphate oligodeoxynucleotides were synthesized in an Applied Biosystems 380B DNA synthesizer and were gel-purified.…”

Section: Libraries Several Cdna Libraries All Prepared In the Agt1omentioning

confidence: 99%

“…Hence, after their uptake into the cultured cells (6,26), these agents could be expected to hybridize with their complementary mRNA sequences through base pairing, inducing nucleolytic degradation of these mRNAs (26) and disrupting the production of their protein products.…”

Section: Qislygkxiv Syvsedr Sra---------------------czd Qikkm*-------mentioning

confidence: 99%

“…In developing megakaryocytes, the progenitors of blood platelets, both phases involve nuclear multiplications (4), implicating cell division control (CDC) genes in these processes. Involvement of cholinergic signaling in hematopoiesis was independently inferred from observations that the cholinesterase genes amplify in leukemias (5) and that antisense oligodeoxynucleotides complementary to butyrylcholinesterase (BCHE) mRNA (AS-BCHE) block megakaryocyte maturation (6). Moreover, acetylcholine stimulates inositol phospholipid hydrolysis in cholinoceptive cells (7,8), which induces the phosphorylation of cdc2 kinases, altering their biochemical properties and enhancing cell proliferation (9)(10)(11).…”

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confidence: 99%

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Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

Lapidot-Lifson

Patinkin

Prody

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Qislygkxiv Syvsedr Sra---------------------czd Qikkm*-------mentioning

confidence: 98%

Section: Libraries Several Cdna Libraries All Prepared In the Agt1omentioning

confidence: 99%

Section: Qislygkxiv Syvsedr Sra---------------------czd Qikkm*-------mentioning

confidence: 99%

mentioning

confidence: 99%

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Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

Lapidot-Lifson

Patinkin

Prody

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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“…To examine the basis of these phenomena, we employed primary murine bone marrow cultures as an ex vivo system for modulating hematopoietic proliferation and development in nontransformed cells. In such cultures interleukin 3 (IL-3) induces expansion of a fraction of the existing pluripotent stem cells into multipotent progenitors, which can differentiate into megakaryocyte colony-forming units (CFU-MK) composed of granulocytes, megakaryocytes, and macrophages (11,12); addition of erythropoietin and transferrin to IL-3 and longer incubation times induce CFU-GEMM colonies, which contain granulocytes, erythroid cells, megakaryocytes, and macrophages (13). This implies that colony counts reflect expansion and survival ofprogenitors that have given rise to progeny, whereas cell numbers reflect proliferation rates (9), and differential cell compositions demonstrate which cell lineages developed and which were programmed to die (9).…”

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confidence: 99%

Antisense oligonucleotide inhibition of acetylcholinesterase gene expression induces progenitor cell expansion and suppresses hematopoietic apoptosis ex vivo.

Soreq

Patinkin²,

Lev-Lehman³

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells

Cellai¹,

Matucci²,

Vannucchi

et al. 1999

J. Cell. Physiol.

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Binding experiments with the specific muscarinic ligand [3H]N-methylscopolamine (3H-NMS) have shown the presence of constitutive muscarinic acetylcholine receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1-M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K(D)) for 3H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B+) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B+ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells.

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Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

Cited by 45 publications

References 23 publications

Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

Antisense oligonucleotide inhibition of acetylcholinesterase gene expression induces progenitor cell expansion and suppresses hematopoietic apoptosis ex vivo.

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells

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