Abstract
The demethylating agents 5-azacytidine and 5-aza-2′-deoxycytidine (DAC) have been shown to induce differentiation and inhibit growth of leukemic myeloid cells at low concentrations. However, the effect of DAC in changing the differentiation and proliferation behavior of normal human myeloid progenitors has rarely been investigated. Therefore, we established an in vitro model of normal hematopoietic differentiation, using CD34+ cells from mobilized peripheral blood, to study proliferation and colony formation, expression of several myeloid maturation markers and of the inhibitor of cyclin-dependent kinases p15/INK4b. Upon DAC treatment, cell growth was significantly decreased in a dose-dependent manner, without an increase in cytotoxicity. DAC treatment also resulted in a substantial increase of lysozyme-positive cells, which could be enhanced by G-CSF, a modest increase of myeloperoxidase+ and CD15+ cells, as well as an increase of colony-forming cells (CFU-GM) compared to control cells. p15/INK4b protein expression was strongly upregulated upon myeloid maturation, and additional DAC treatment did not change p15 expression or the methylation status of the p15 promoter at the noncytotoxic concentrations used. Taken together, these data indicate a role of DAC in changing myeloid progenitor cell expansion and differentiation. This model appears suitable also for global analyses of multiple differentially methylated genes.
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References
Lübbert M, Brugger W, Mertelsmann R, Kanz L . Developmental regulation of myeloid gene expression and demethylation during ex vivo culture of peripheral blood progenitor cells. Blood 1996; 87: 447–455.
Lübbert M, Henschler R, Kreutz M, Andreesen R, Mertelsmann R, Herrmann F . The human lysozyme gene undergoes stepwise demethylation during phagocyte maturation. Leukemia 1997; 11: 990–997.
Felgner J, Kreipe H, Heidorn K, Jaquet K, Zschunke F, Radzun HJ et al. Increased methylation of the c-fms protooncogene in acute myelomonocytic leukemias. Pathobiology 1991; 59: 293–298.
Felgner J, Heidorn K, Korbacher D, Frahm SO, Parwaresch R . Cell lineage specificity in G-CSF receptor gene methylation. Leukemia 1999; 13: 530–534.
Lübbert M, Miller CW, Koeffler HP . Changes of DNA methylation and chromatin structure in the human myeloperoxidase gene during myeloid differentiation. Blood 1991; 78: 345–356.
Lehmann U, Brakensiek K, Kreipe H . Role of epigenetic changes in hematological malignancies. Ann Hematol 2004; 83: 137–152.
Sakashita K, Koike K, Kinoshita T, Shiohara M, Kamijo T, Taniguchi S et al. Dynamic DNA methylation change in the CpG island region of p15 during human myeloid development. J Clin Invest 2001; 108: 1195–1204.
Taylor SM, Jones PA . Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell 1979; 17: 771–779.
Creusot F, Acs G, Christman JK . Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-2′-deoxycytidine. J Biol Chem 1982; 257: 2041–2048.
Christman JK, Mendelsohn N, Herzog D, Schneiderman N . Effect of 5-azacytidine on differentiation and DNA methylation in human promyelocytic leukemia cells (HL-60). Cancer Res 1983; 43: 763–769.
Pinto A, Attadia V, Fusco A, Ferrara F, Spada OA, Di Fiore PP . 5-Aza-2′-deoxycytidine induces terminal differentiation of leukemic blasts from patients with acute myeloid leukemias. Blood 1984; 64: 922–929.
Momparler RL, Bouchard J, Samson J . Induction of differentiation and inhibition of DNA methylation in HL-60 myeloid leukemic cells by 5-aza-2′-deoxycytidine. Leuk Res 1985; 9: 1361–1366.
Momparler RL, Dore BT, Momparler LF . Effect of 5-aza-2′-deoxycytidine and retinoic acid on differentiation and c-myc expression in HL-60 myeloid leukemic cells. Cancer Lett 1990; 54: 21–28.
Attadia V . Effects of 5-aza-2′-deoxycytidine on differentiation and oncogene expression in the human monoblastic leukemia cell line U-937. Leukemia 1993; 7 (S1): 9–16.
Dore BT, Chomienne C, Momparler RL . Effect of 5-aza-2′-deoxycytidine and vitamin D3 analogs on growth and differentiation of human myeloid leukemic cells. Cancer Chemother Pharmacol 1998; 41: 275–280.
Niitsu N, Hayashi Y, Sugita K, Honma Y . Sensitization by 5-aza-2′-deoxycytidine of leukaemia cells with MLL abnormalities to induction of differentiation by all-trans retinoic acid and 1alpha,25-dihydroxyvitamin D3. Br J Haematol 2001; 112: 315–326.
Milhem M, Mahmud N, Lavelle D, Araki H, DeSimone J, Saunthararajah Y et al. Modification of hematopoietic stem cell fate by 5aza 2′deoxycytidine and trichostatin A. Blood 2004; 103: 4102–4110.
Engelhardt M, Winkler J, Waller C, Lange W, Mertelsmann R, Henschler R . Blood progenitor cell (BPC) mobilization studied in multiple myeloma, solid tumor and non-Hodgkin's lymphoma patients after combination chemotherapy and G-CSF. Bone Marrow Transplant 1997; 19: 529–537.
Engelhardt M, Bertz H, Afting M, Waller CF, Finke J . High-versus standard-dose filgrastim (rhG-CSF) for mobilization of peripheral-blood progenitor cells from allogeneic donors and CD34(+) immunoselection. J Clin Oncol 1999; 17: 2160–2172.
Bosse R, Kulmburg P, von Kalle C, Engelhardt M, Dwenger A, Rosenthal F et al. Production of stem-cell transplants according to good manufacturing practice. Ann Hematol 2000; 79: 469–476.
Gaipa G, Dassi M, Perseghin P, Venturi N, Corti P, Bonanomi S et al. Allogeneic bone marrow stem cell transplantation following CD34+ immunomagnetic enrichment in patients with inherited metabolic storage diseases. Bone Marrow Transplant 2003; 31: 857–860.
Guo Y, Follo M, Geiger K, Lübbert M, Engelhardt M . Side-population cells from different precursor compartments. J Hematother Stem Cell Res 2003; 12: 71–82.
Engelhardt M, Kumar R, Albanell J, Pettengell R, Han W, Moore MA . Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood 1997; 90: 182–193.
Daskalakis M, Nguyen TT, Nguyen C, Guldberg P, Köhler G, Wijermans P et al. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-aza-2′-deoxycytidine (decitabine) treatment. Blood 2002; 100: 2957–2964.
Gothot A, van der Loo JC, Clapp DW, Srour EF . Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34(+) cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998; 92: 2641–2649.
Teofili L, Rutella S, Chiusolo P, La Barbera EO, Rumi C, Ranelletti FO et al. Expression of p15INK4B in normal hematopoiesis. Exp Hematol 1998; 26: 1133–1139.
Wijermans P, Lübbert M, Verhoef G, Bosly A, Ravoet C, Andre M et al. Low-dose 5-aza-2′-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndrome: a multicenter phase II study in elderly patients. J Clin Oncol 2000; 18: 956–962.
Furukawa Y, Kikuchi J, Nakamura M, Iwase S, Yamada H, Matsuda M . Lineage-specific regulation of cell cycle control gene expression during haematopoietic cell differentiation. Br J Haematol 2000; 110: 663–673.
Teofili L, Morosetti R, Martini M, Urbano R, Putzulu R, Rutella S et al. Expression of cyclin-dependent kinase inhibitor p15(INK4B) during normal and leukemic myeloid differentiation. Exp Hematol 2000; 28: 519–526.
Teofili L, Martini M, Di Mario A, Rutella S, Urbano R, Luongo M et al. Expression of p15(ink4b) gene during megakaryocytic differentiation of normal and myelodysplastic hematopoietic progenitors. Blood 2001; 98: 495–497.
Liang G, Gonzales FA, Jones PA, Orntoft TF, Thykjaer T . Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2′-deoxycytidine. Cancer Res 2002; 62: 961–966.
Lübbert M, Wijermans P, Kunzmann R, Verhoef G, Bosly A, Ravoet C et al. Cytogenetic responses in high-risk myelodysplastic syndrome following low-dose treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine. Br J Haematol 2001; 114: 349–357.
Dodge JE, Munson C, List AF . KG-1 and KG-1a model the p15 CpG island methylation observed in acute myeloid leukemia patients. Leuk Res 2001; 25: 917–925.
Schmidt M, Bies J, Tamura T, Ozato K, Wolff L . The interferon regulatory factor ICSBP/IRF-8 in combination with PU.1 up-regulates expression of tumor suppressor p15Ink4b in murine myeloid cells. Blood 2004; 103: 4142–4149.
Lübbert M, Daskalakis M, Kunzmann R, Engelhardt M, Guo Y, Wijermans P . Nonclonal neutrophil responses after successful treatment of myelodysplasia with low-dose 5-aza-2′-deoxycytidine (decitabine). Leuk Res 2004; 28: 1267–1271.
Acknowledgements
We thank Eva Samek for excellent technical support and Dr Jonathan Dodge, Boston, for valuable suggestions during the performance of experiment. We are grateful to Professor Dr Fusenig (German Cancer Center) for providing the HaCat cell line. We greatly acknowledge Drs Florian Otto, Ralph Wäsch, Tobias Berg and Michael Stock for valuable suggestions and critical reading of the manuscript, appreciate Professor Dr Roland Mertelsmann's continuous support, and Professor Dr Thomas Licht's helpful discussion. We specially thank Dr Diane Church (Dartmouth Medical School) for expert assistance. This work was supported by a grant from Deutsche Forschungsgemeinschaft (Lu 429/5-1).
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Guo, Y., Engelhardt, M., Wider, D. et al. Effects of 5-aza-2′-deoxycytidine on proliferation, differentiation and p15/INK4b regulation of human hematopoietic progenitor cells. Leukemia 20, 115–121 (2006). https://doi.org/10.1038/sj.leu.2404019
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DOI: https://doi.org/10.1038/sj.leu.2404019
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