Summary
1. To investigate the possibility that cholinesterase inhibitors may cause adverse hematopoietic effects, we employed antisense oligodeoxynucleotides selectively inhibiting butyrylcholinesterase gene expression (AS-BCHE). Complementary sense (S) oligonucleotides served as controls.
2. In primary bone marrow cell cultures grown with interleukin 3 (IL-3), AS-BCHE but not S-BCHE reduced growth of megakaryocyte colony-forming units (CFU-MK) in a dose-dependent manner at the micromolar range.
3. In cultures grown with IL-3, transferrin, and erythropoietin (Epo), cell counts increased up to twofold, yet colony counts (CFU-GEMM) remained unchanged under AS-BCHE treatment.
4. Electrophoretic measurements of DNA ladder as an apoptotic index revealed that the above oligonucleotide effects were not due to nonspecific induction of programmed cell death.
5. Differential cell counts demonstrated increased myeloidogenesis and reduced levels of early megakaryocytes in CFU-GEMM under AS-BCHE, suggesting requirement of the BuChE protein for megakaryopoiesis.
6.In vivo injection of AS-BCHE reduced BCHE mRNA levels in both young and mature megakaryocytes for as long as 20 days, as shown byin situ hybridization.
7.Ex vivo growth of primary bone marrow cells revealed a twofold reduction in CFU-MK colonies grown from the AS-BCHE- but not the S-BCHE-injected mice, 15 days posttreatment.
8. These findings demonstrate that deficient butyrylcholinesterase expression, and hence interference with this enzyme's activity through treatment with or exposure to cholinesterase inhibitors, may cause hematopoietic differences in treated patients.
References
Anderson, K. M., Levin, J., Jajeh, A., Seed, T., and Harris, J. E. (1993). Induction of apoptosis in blood cells from a patient with acute myelogenous leukemia by SC41661A, a selective inhibitor of 5-lipoxygenase.Prostaglandins Leukot. Essent. Fatty Acids 4323–326.
Brown, L. M., Blair, A., Gibson, R., Everett, G. D. Cantor, K. P., Schuman, L. M., Burmeister, L. F., Van Lier, S. F., and Dick, F. (1990). Pesticide exposures and other agricultural risk factors for leukemia among men in Iowa and Minnesota.Cancer Res. 506585–6591.
Campbell, J. L., Abraham, C. R., Mashiah, E., Kemper, P., Inglis, J. D., Oldstone, M. B. A., and Mucke, L. (1993). Neurologic disease induced in transgenic mice by cerebral overexpression of interleukin 6.Proc. Natl. Acad. Sci. USA 9010061–10065.
Eckstein, F. (1985). Nucleoside phosphorothioates.Annu. Rev. Biochem. 54367–402.
Ehrlich, G., Ginzberg, D., Loewenstein, Y., Glick, D., Kerem, B., Ben-Ari, S., Zakut, H., and Soreq, H. (1994a). Population diversity and distinct haplotype frequencies associated with ACHE and BCHE genes of Israeli Jews from Trans-Caucasian Georgia and from Europe.Genomics 22288–295.
Ehrlich, G., Patinkin, D., Ginzberg, D., Zakut, H., Eckstein, F., and Soreq, H. (1994b). Use of partially phosphorothioated “antisense” oligodeoxynucleotides for sequence-dependent modulation of hematopoiesis.Antisense Res. Dev. 4173–183.
Escary, J. L., Perreau, J., Dumenil, D., Ezine, S., and Brulet, P. (1993). Leukemia inhibitory factor is necessary for maintenance of haematopoietic stem cells and thymocyte stimulation.Nature 363361–364.
Gewirtz, A. M. (1993). Potential therapeutic applications of antisense oligodeoxynucleotides in the treatment of chronic myelogenous leukemia.Leuk. Lymphona 1131–137.
Gnatt, A., Loewenstein, Y., Yaron, A., Schwarz, M., and Soreq, H. (1994). Site-directed mutagenesis of active site residues reveals plasticity in substrate and inhibitor interactions of human butyrylcholinesterase.J. Neurochem. 62749–755.
Kelly, L. L., Koury, M. J., Bondurant, M., Koury, S. T., Sawyer, T., and Wickrema, A. (1993). Survival or death of individual proerythroblasts results from differeint erythropoietin sensitivities: A mechanism for controlled rates of erythrocyte production;Blood 822340–2352.
Koury, M. J., and Bondurant, M. C. (1990). Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells.Science 248378–381.
Lapidot-Lifson, Y., Prody, C. A., Ginzberg, D., Meytes, D., Zakut, H., and Soreq, H. (1989). Co-amplification of human acetylcholinesterase and butyrylcholinesterase genes in blood cells: Correlation with various leukemias and abnormal megakaryocytopoiesis.Proc. Natl. Acad. Sci. USA 864715–4719.
Lapidot-Lifson, Y., Patinkin, D., Prody, C. A., Ehrlich, G., Seidman, S., Ben-Aziz, R., Benseler, F., Eckstein, F., Zakut, H., and Soreq, H. (1992). Cloning and antisense oligodeoxynucleotide inhibition of a human cdc2 homologue required in hematopoiesis.Proc. Natl. Acad. Sci. USA 89579–583.
Lev-Lehman, E., Hornreich, G., Ginzberg, D., Gnatt, A., Meshorer, A., Eckstein, F., Soreq, H., and Zakut, H. (1994). Antisense inhibition of acetylcholinesterase gene expression causes transient hematopoietic alterationsin vivo.Gene Ther. 1127–135.
Loewenstein, Y., Denarie, M., Zakut, H., and Soreq, H. (1993). Molecular dissection of cholinesterase domains responsible for carbamate toxicity.Chem. Biol. Interact. 87209–216.
Lord, K. A., Abdollahi, A., Hoffman-Liebermann, B., and Liebermann, D. A. (1993). Protooncogenes of the fos/jun family of transcription factors are positive regulators of myeloid differentiation.Mol. Cell Biol. 13841–851.
Metcalf, D. (1992). Hemopoietic regulators.Trends Biochem. Sci. 17286–289.
Methia, N., Louache, F., Vainchenker, W., and Wendling, F. (1993). Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis.Blood 821395–1401.
Neville, L. F., Gnatt, A., Loewenstein, Y., and Soreq, H. (1992). Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE.EMBO J. 111641–1649.
Okumura, N., Tsuji, K., and Nakahata, T. (1992). Changes in cell surface antigen expressions during proliferation and differentiation of human erythroid progenitors.Blood 80642–650.
Patinkin, D., Seidman, S., Eckstein, F., Benseler, F., Zakut, H., and Soreq, H. (1990). Manipulations of cholinesterase gene expression modulate Murine Megakaryocytopoiesisin vitro.Mol. Cell. Biol. 106046–6050.
Pech, N., Hermine, O., and Golswasser, E. (1993). Further study of internal autocrine regulation of multipotent hematopoietic cells.Blood 821502–1506.
Prody, C. A., Zevin-Sonkin, D., Gnatt, A., Goldberg, O., and Soreq, H. (1987). Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues.Proc. Natl. Acad. Sci. USA 843555–3559.
Ratajczak, M. Z., and Gewirtz, A. M. (1994). Oligonucleotide-based therapies of human malignancies. InNucleic Acids and Molecular Biology, Vol. 8 (F. Eckstein and D. J. M. Lilley, Eds.), Springer-Verlag, Berlin and Heidelberg, pp. 298–326.
Shi, Y., Glynn, J. M., Guilbert, L. J., Cotter, T. G., Bissonette, R. P., and Green, D. R. (1992). Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas.Science 257212–214.
Stein, C. A., and Cheng, Y. C. (1993). Antisense oligonucleotides as therapeutic agents—Is the bullet really magical?Science 2611004–1012.
Soreq, H., and Zakut, H. (1993).Human Cholinesterases and Anticholinesterases, Academic Press, New York, pp. 1–314.
Soreq, H., Gnatt, A., Lowenstein, Y., and Neville, L. F. (1992). Excavations into the active site gorge of cholinesterases.Trends Biochem. Sci. 17353–358.
Soreq, H., Patinkin, D., Lev-Lehman, E., Grifman, M., Ginzberg, D., Eckstein, F., and Zakut, H. (1994). Antisense oligonucleotide inhibition of acetylcholinesterase gene expression induces progenitor cell expansion and suppresses hematopoietic apoptosisex vivo.Proc. Natl. Acad. Sci. USA 917907–7911.
Szczylik, C., Skorski, T., Ku, D. H., Nicolaides, N. C., Wen, S. C., Rudnicka, L., Bonati, A., Malaguarnera, L., and Calabretta, B. (1993). Regulation of proliferation and cytokine expression of bone marrow fibroblasts: Role of c-myb.J. Exp. Med. 178997–1005.
Watkins, P. B., Zimmerman, H. J., Knapp, M. J., Gracon, S. I., and Lewis, K. W. (1994). Hepatotoxic effects of tacrine administration in patients with Alzheimer's disease.JAMA 271992–998.
Winkler, M. A. (1994). Tacrine for Alzheimer's disease: Which patient, what does?JAMA 2721023–1024.
Zakut, H., Lapidot-Lifson, Y., Leibson, R., Ballin, A., and Soreq, H. (1992).In vivo gene amplification in non-cancerous cells: Cholinesterase genes and oncogenes amplify in thrombocytopenia associated with lupus erythematosus.Mutat. Res. 276275–284.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Patinkin, D., Lev-Lehman, E., Zakut, H. et al. Antisense inhibition of butyrylcholinesterase gene expression predicts adverse hematopoietic consequences to cholinesterase inhibitors. Cell Mol Neurobiol 14, 459–473 (1994). https://doi.org/10.1007/BF02088831
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02088831