Analysis on the mechanisms of growth, differentiation and malignant transformation <of megakaryocytic cells

在这个项目中，我们研究了巨核细胞的生长、分化和恶性转化的机制。在巨核形成的后期，巨核细胞经历以DNA复制为特征的多倍体化，而没有伴随的细胞分裂。我们初步研究了AIM-1的作用，它是Aurora/Ipl1丝氨酸苏氨酸激酶家族的成员，在巨核细胞多倍体化过程中对有丝分裂至关重要。在增殖性造血细胞中，AIM-1 mRNA的表达仅限于细胞周期的G_2/M期。相反，在正常巨核细胞和红细胞/巨核细胞系（F-36P、K562和CMK）的多倍体化过程中，其表达持续受到抑制。通过诱导表达野生型AIM-1来补充AIM-1活性，可以消除tpa诱导的K562多倍体化。此外，通过诱导表达显性阴性（DN） AIM-1抑制AIM-1活性可导致K562和CMK的多倍体化。这些结果提示AIM-1的下调可能参与巨核细胞多倍体的形成。接下来，我们研究了BCR/ abl阳性红细胞/磁核细胞系K562的生长和存活机制。我们在K562中单独或联合诱导表达DN Ras （N17）、磷脂酰肌醇3-激酶（PI3-K）（Δp85）和STAT5 (694F)，诱导表达的N17、694F和Δp85分别抑制了90%、55%和40%的生长。此外，N17诱导少量K562细胞凋亡，而694F和Δp85几乎不起作用。相反，两种DN突变体在任何组合下共表达都会诱导严重的细胞凋亡。此外，尽管K562对IFN-α-和地塞米松诱导的细胞凋亡具有耐药性，但N17 694F或Δp85破坏其中一条通路使K562对这些药物敏感。这些结果表明，这些分子之间的合作是BCR/ABL充分发挥白血病活性所必需的。

In this project, we examined the mechanisms of growth, differentiation, and malignant transformation of megakaryocytic cells.During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization characterized by DNA duplication without concomitant cell division. We initially examined the roles of AIM-1 which is a member of Aurora/Ipl1 serine threonine kinase family and essential for mitosis in megakaryocytic polpyploidization. In a proliferative hematopoietic cells, the expression of AIM-1 mRNA was restrictedly observed at G_2/M phase of cell cycle. In contras its expression was continuously repressed during polyploidization in normal megakaryocytes as in erythro/megakaryocytic cell lines (F-36P, K562, and CMK). Supplement of AIM-1 activities by the induced express on of wild-type AIM-1 canceled TPA-induced polyploidizaTion of K562.Moreover, suppression of AIM-1 activities by the induced expression dominant-negative(DN) AIM-1 led to polyploidization of K562 and CMK. These results suggested that down-regulation of AIM-1 may be involved in polyploid formation of megakaryocytes.Next, we examined the mechanism of the growth and survival of a BCR/ABL-positive erythroid/magakaryocytic cell line, K562. We inducibly expressed DN Ras (N17), phosphatidylinositol 3-kinase(PI3-K)(Δp85) and STAT5 (694F) alone or ill combination in K562 The inducibly expressed N17, 694F and Δp85 inhibited the growth by 90% 55% and 40%, respectively. In addition, N17 induced apoptosis in a small proportion of K562, whereas 694F and Δp85 were hardly effective In contrast, coexpression of two DN mutants in any combinations induced severe apoptosis. Furthermore, although K562 was resistant to IFN-α- and dexamethasone-induced apoptosis, disruption of one pathway by N17 694F or Δp85 sensitized K562 to these reagents. These results suggest that cooperation among these molecules is required for full leukemogenic activities of BCR/ABL.