Yamaguchi Hiroki, Hanawa Hideki, Uchida Naoya, Inamai Mitsuharu, Sawaguchi Kazuhiro, Mitamura Yoshio, Shimada Takashi, Dan Kazuo, Inokuchi Koiti
Division of Hematology, Department of Internal Medicine, Nippon Medical School, Bunkyo-Ku, Tokyo, Japan.
Exp Hematol. 2009 Jun;37(6):701-14. doi: 10.1016/j.exphem.2009.02.007.
Concerning MLL-AF4 leukemogenesis, previous mouse models suggest that the tumorigenesis capacity of MLL-AF4 alone is insufficient for causing leukemia. Based on the finding that an Fms-like tyrosine kinase 3 (Flt3) gene mutation in the tyrosine kinase domain (TKD) was observed in approximately 15% of mixed lineage leukemia (MLL), we investigated synergistic leukemogenesis effects of the two genes in vitro.
In a mouse interleukin-3 (IL-3)-dependent cell line, 32Dc, expression of MLL-AF4 and mutant Flt3 was induced using a lentiviral vector. We analyzed apoptosis induction in the absence of IL-3 and the granulocyte colony-stimulating factor-related induction of differentiation, gene expression profiling, and the mechanism involved in the synergistic effects of MLL-AF4 and Flt3-TKD.
Neither Flt3-expressing 32Dc (32Dc(Flt3-TKD)) nor MLL-AF4-expressing 32Dc (32Dc(MLL-AF4)) acquired IL-3-independent proliferative capacity in semisolid/liquid media. However, Flt3-TKD+MLL-AF4-expressing 32Dc (32Dc(Flt3-TKD+MLL-AF4)) acquired a non-IL-3-dependent proliferative capacity by inhibiting apoptosis in the two media. The 32Dc(Flt3-TKD) and 32Dc(MLL-AF4) cells differentiated into granulocytes in the presence of granulocyte colony-stimulating factor. However, in the 32Dc(Flt3-TKD+MLL-AF4) cells, there was no differentiation. Subsequently, we performed gene expression profiling. The enhancement of Hox genes expression was not identified. However, expression of S100A6 was synergistically enhanced in the presence of both MLL-AF4 and Flt3-TKD genes. Moreover, anti-S100A6 small interfering RNA downregulated leukemic proliferation.
We conclude that their synergistic enhancement of S100A6 expression plays an important role in MLL-AF4-associated leukemogenesis.
关于MLL-AF4白血病发生，先前的小鼠模型表明，单独的MLL-AF4致瘤能力不足以引发白血病。基于在大约15%的混合谱系白血病（MLL）中观察到酪氨酸激酶结构域（TKD）中的Fms样酪氨酸激酶3（Flt3）基因突变这一发现，我们在体外研究了这两个基因的协同白血病发生效应。
在小鼠白细胞介素-3（IL-3）依赖细胞系32Dc中，使用慢病毒载体诱导MLL-AF4和突变型Flt3的表达。我们分析了在无IL-3情况下的凋亡诱导以及粒细胞集落刺激因子相关的分化诱导、基因表达谱分析，以及MLL-AF4和Flt3-TKD协同效应所涉及的机制。
表达Flt3的32Dc（32Dc(Flt3-TKD)）和表达MLL-AF4的32Dc（32Dc(MLL-AF4)）在半固体/液体培养基中均未获得不依赖IL-3的增殖能力。然而，表达Flt3-TKD+MLL-AF4的32Dc（32Dc(Flt3-TKD+MLL-AF4)）通过在两种培养基中抑制凋亡而获得了不依赖IL-3的增殖能力。32Dc(Flt3-TKD)和32Dc(MLL-AF4)细胞在粒细胞集落刺激因子存在的情况下分化为粒细胞。然而，在32Dc(Flt3-TKD+MLL-AF4)细胞中，没有分化。随后，我们进行了基因表达谱分析。未发现Hox基因表达增强。然而，在同时存在MLL-AF,4和Flt3-TKD基因的情况下，S100A6的表达协同增强。此外，抗S100A6小干扰RNA下调白血病增殖。
我们得出结论，它们对S100A6表达的协同增强在MLL-AF4相关白血病发生中起重要作用。
