Hematopoietic Cell Maturation Is Regulated by Maintaining a Balance against Cytokine Induced-Cell Proliferation - Clinical Application for Differentiation-inducing Therapy of Leukemia-

利用人巨核细胞系MO7E中稳定转导的人促红细胞生成素(EPO)受体基因的因子依赖性细胞株MO7ER，研究了这些细胞因子对细胞生长和分化的生物学效应。促血小板生成素(TPO)、促红细胞生成素(EPO)和钢铁因子(SLF)均以剂量依赖方式刺激MO7ER细胞增殖。SLF+TPO或EPO单独作用对MO7ER细胞生长有显著的协同促进作用，而TPO+EPO单独作用仅表现为相加作用。在细胞分化方面，TPO或EPO均可诱导血小板膜糖蛋白(GP)IIb/IIIa和GPIb表达增强。SLF可诱导GPIIb/IIIa和GPIb的表达，但作用弱于EPO或TPO。然而，将SLF添加到tpo-o…中更多的含有rEPO的培养物(在MO7ER细胞中诱导强大的有丝分裂)导致这些巨核细胞特异性抗原的抑制。加入低剂量的阿糖胞苷(Ara-C)(1~10 ng/ml)可促进TPO或EPO诱导的MO7ER细胞的巨核细胞分化，同时轻度抑制细胞生长。小剂量Ara-C+TPO+SLF可抑制SLF的促增殖作用，并诱导GPIIb/IIIa和GPIB的表达，与单独使用TPO的效果相当。用乙酰胆碱酯酶染色阳性细胞数和巨核细胞核多倍体来评价TPO和SLF联合刺激对正常小鼠骨髓细胞TPO诱导的巨核细胞成熟的抑制作用。此外，与单独应用G-CSF相比，小剂量Ara-C与G-CSF联合应用可促进WEHI-3B细胞向粒细胞分化。从不同类型的急性髓系白血病患者分离的原代培养的白血病细胞也复制了这一现象，这些结果表明，造血细胞的成熟至少部分是通过对抗细胞因子诱导的细胞增殖来调节的。较少

Using a factor-dependent cell line MO7ER, which contains a stably transduced human erythropoietin (EPO) receptor gene in human megakaryoblastic cell line MO7e and which resulted in concomitant expression of EPO receptor, c-Mpl and c-Kit, we investigated the biological effects of these cytokines in terms of cell growth and differentiation. Thrombopoietin (TPO), EPO and Steel factor (SLF) all stimulated MO7ER cell proliferation in a dose-dependent manner. Combined stimulation of cells with SLF plus either TPO or EPO resulted in striking synergistic enhancement of MO7ER cell growth as compared with each cytokine alone, whereas combination of TPO plus EPO showed only an additive effect on cell proliferation. With regards * cell differentiation, either TPO or EPO treatment induced enhancement of platelet glycoprotein (GP) IIb/IIIa and GPIb expression. SLF induced GPIIb/IIIa and GPIb expression, but the effect was much weaker than that of EPO or TPO. However, addition of SLF to either TPO- o … More r EPO-containing cultures (which induced potent mitogenesis in MO7ER cells) resulted in suppression of these megakaryocyte specific antigens. Addition of low-dose cytosine arabinoside (Ara-C)(1 to 10 ng/ml) enhanced TPO- or EPO- induced megakaryocytic differentiation in MO7ER cells while mildly suppressing cell growth. Treatment the cells with low-dose Ara-C plus TPO plus SLF overrode the proliferative enhancing effects of SLF and induced GPIIb/IIIa and GPIb expression as efficient as TPO alone. Retardation of TPO-induced megakaryocytic maturation was also observed in normal murine bone marrow cells by combined stimulation with TPO and SLF as assessed by the numbers of acetylcholinesterase staining-positive cells and megakaryocyte nuclear polyploidy. In addition, combination of low-dose Ara-C plus G-CSF enhanced granulocytic differentiation in WEHI-3B cells, as compared with the cells treated with G-CSF alone. This phenomenon was also reproduced using the primary cultured leukemia cells which were isolated from the patients with various types of acute myelogenous leukemias,These results suggest that hematopoietic cell maturation is, at least in part, regulated by countering cytokine-induced cell proliferation. Less