Thrombopoietin supports the continuous growth of cytokine-dependent human leukemia cell lines
Thrombopoietin supports the continuous growth of cytokine-dependent human leukemia cell lines
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DOI：
10.1038/sj.leu.2400621
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发表时间：
1997-04-01
期刊：
影响因子：
11.4
通讯作者：
Quentmeier, H
中科院分区：
文献类型：
--
作者：
Drexler, HG;Zaborski, M;Quentmeier, H
造血是一个复杂的细胞增殖和分化的过程，从原始干细胞到最终完全分化的细胞。长期和广泛的搜索，特别是调节巨核细胞生成因子导致克隆的激素，这里称为血小板生成素（TPO），专门促进增殖和分化的巨核细胞谱系。重组TPO的可用性及其即将到来的临床应用使得更详细地了解其对造血细胞的影响变得更加迫切。正常的巨核细胞和血小板生成主要发生在骨髓中，骨髓是一种难以原位研究的器官，特别是在人类中，这是由于巨核细胞祖细胞数量少以及随后难以分离为纯群体。因此，我们开发了一种体外系统，这可能使我们能够解决有关TPO生物学的问题。急性髓性白血病（AML）来源的细胞系HU-3、M-07 e、M-MOK和TF-1对粒细胞-巨噬细胞集落刺激因子（GM CSF）具有绝对依赖性。我们在TPO（省略GM-CSF）的连续存在下长期（>6个月）培养这些细胞。TPO单独支持这些姐妹细胞系HU-3/TPO、M-07 e/TPO、M-MOK/TPO和TF-1/TPO的维持和扩增，与用GMCSF生长的相应反培养物相比，这些姐妹细胞系显示出稍长的倍增时间、较大的细胞大小、以及较高百分比的多核巨细胞和轻微粘附细胞。在不存在TPO的情况下，细胞在几天内迅速死亡;因此，TPO生长的细胞系绝对依赖于该因子，但可以全部切换回GM-CSF生长。与GM-CSF处理的细胞相比，TPO处理的细胞中GM-CSF和白细胞介素-3（IL-3）的受体下调，干细胞因子（SCF）和TPO的受体上调。短期增殖试验显示TPO细胞系对促红细胞生成素、GM-CSF、IL-3、PIXY-321、SCF和TPO的反应比GM-CSF细胞系更强。对GM-CSF和TPO培养细胞系的流式细胞术分析显示，巨核细胞表面标志物CD 41、CD 42和CD 61上调，而后者细胞系中红细胞标志物血型糖蛋白A下调，这表明巨核细胞谱系中存在一定的分化，沿着巨核细胞谱系。因此，在长期暴露中，TPO似乎对响应细胞具有增殖和分化作用。在无血清培养条件下，TPO充当TPO细胞系的存活因子。两者合计，这些研究结果表明，TPO依赖的细胞系代表重要的生物试剂，进一步表征TPO的生物学，也应该提供一个很大的帮助，为未来的体外实验，旨在阐明巨核细胞和血小板生成。
Hematopoiesis is a complex process of regulated cellular proliferation and differentiation from the primitive stem cells to the final fully differentiated cell. The long and extensive search for a factor specifically regulating megakaryocytopoiesis led to the cloning of a hormone, here called thrombopoietin (TPO), that specifically promotes proliferation and differentiation of the megakaryocytic lineage. The availability of recombinant TPO and its imminent clinical use has made a more detailed understanding of its effects on hematopoietic cells more urgent. Normal megakaryocyto- and thrombopoiesis occurs predominantly in the bone marrow, a difficult organ to study in situ, particularly in humans, due to the low numbers of megakaryocytic progenitors and the consequent difficult isolation as pure populations. Thus, we developed an in vitro system which may allow us to address questions regarding the biology of TPO. The acute myeloid leukemia (AML)-derived cell lines HU-3, M-07e, M-MOK and TF-1 have absolute dependence on granulocyte-macrophage colony-stimulating factor (GM CSF). We cultured these cells long term (>6 months) in the continuous presence of TPO (omitting GM-CSF). TPO alone supported the maintenance and expansion of these sister cell lines, HU-3/TPO, M-07e/TPO, M-MOK/TPO and TF-1/TPO, that displayed somewhat longer doubling times, a larger cell size, and a higher percentage of polynucleated giant cells and slightly adherent cells than the corresponding countercultures grown with GMCSF. In the absence of TPO the cells died quickly, within a few days; thus, the TPO-grown cell lines have an absolute dependence on this factor, but could all be switched back to growth with GM-CSF. In comparison with the GM-CSF-treated cells, the receptors for GM-CSF and interleukin-3 (IL-3) were down-regulated and the receptors for stem cell factor (SCF) and TPO were up-regulated in the TPO-exposed cells. A short-term proliferation assay showed a stronger response of the TPO-cell lines to erythropoietin, GM-CSF, IL-3, PIXY-321, SCF and TPO than the GM-CSF-cell lines. Flow cytometric analysis of the GM-CSF- and TPO-cultured lines displayed an up-regulation of the megakaryocytic surface markers CD41, CD42 and CD61, and a down-regulation of the erythroid marker glycophorin A in the latter cell lines, suggesting some differentiation along the megakaryocytic lineage. Thus, in long-term exposure, TPO appears to have both a proliferative and a differentiative effect on responsive cells. Under serum deprived culture conditions, TPO acted as a survival factor on the TPO-cell lines. Taken together, these findings indicate that the TPO-dependent cell lines represent important biological reagents for further characterization of the biology of TPO and should also provide a great aid for future in vitro experiments aimed at elucidating megakaryocyto- and thrombopoiesis.