Development and clinical application of molecular diagnosis in leukemias

T(3；21)(q26；q22)易位是慢性粒细胞白血病(CML)急变期中发现的一种持续的染色体异常，被认为在CML向急变期发展的过程中起着重要作用。位于急性髓细胞白血病t(8；21)(q22；q22)易位断裂点的AML1基因也被t(3；21)(q26；q22)易位重排。对携带t(3；21)的白血病细胞系细胞(SKH1)的cDNA文库进行筛选，分离到AML1/EVI-1嵌合cDNAs。SKH1细胞表达了180kD的AML1/EVI-1融合蛋白，该融合蛋白含有AML1的氨基末端半部分，包括一个矮小的同源结构域，与整个锌指EVI-1蛋白融合在一起。这些发现有力地表明，t(3；21)易位导致了一种新的嵌合转录因子的形成，这种嵌合转录因子可能通过…促进慢性粒细胞白血病的进展。对细胞的生长和分化有更多的干扰。生化分析表明，AML1/EVI-1本身不改变通过PEBP2位点的反式激活水平，但主要抑制完整的AML1的反式激活，AML1被认为是髓系细胞分化的刺激因子。AML1/EVI-1与PEBP2结合的亲和力高于AML1，DNA结合竞争是AML1/EVI-1这种显性负效应的一种可能机制。此外，AML1/EVI-1刺激c-fos启动子反式激活，提高AP-1活性。利用AML1/EVI-1的缺失突变体进行的实验表明，这两个功能是相互独立的，因为对完整AML1活性的主要负面影响和对AP-1活性的刺激分别依赖于C末端附近的矮小结构域和锌指结构域。此外，我们还发现AML1/EVI-1阻断了G-CSF诱导的32Dc13髓系细胞的粒细胞分化。也有人认为，融合蛋白的AML1和EVI-1来源的部分在这种分化阻断中发挥了关键作用。结论：t(3；21)白血病细胞转化可能是由于AML1/EVI-1嵌合蛋白的双重功能所致。较少

The t (3 ; 21) (q26 ; q22)translocation, which is one of consistent chromosomal abnormalities found in blastic crisis of chronic myelocytic leukemia(CML) , is thought to play an important role in leukemic progression of CML to an acute blastic crisis phase. The AML1 gene, which is located at the translocation breakpoint of the t (8 ; 21) (q22 ; q22) translocation found in acute myelocytic leukemia, was also rearranged by the t (3 ; 21) (q26 ; q22) translocation. Screening of a cDNA library of the t (3 ; 21) -carrying leukemic cell line cells (SKH1) resulted in the isolation of AML1/EVI-1 chimeric cDNAs. SKH1 cells expressed the 180 kD AML1/EVI-1 fusion protein containing an amino-terminal half of AML1 including a runt homology domain which is fused to the entire of zinc finger EVI-1 protein. These findings strogly suggest that the t (3 ; 21) translocation results in the fromation of a new class of a chimeric transcription factor which could contribute to leukemic progression of CML thr … More ough interference with cell growth and differentiation. Biochemical analyzes revealed that AML1/EVI-1 itself does not alter the transactivation level through PEBP2 sites but dominantly suppresses the transactivation by intact AML1, which is assumed to be a stimulator of myeloid cell differentiation. The DNA-binding competition is a putative mechanism of such dominant negative effects of AML1/EVI-1 because it binds to PEBP2 sites in the higher affinity than AML1.Furthermore, AML1/EVI-1 stimulated the c-fos promoter transactivation and raised the AP-1 activity. Experiments, using deletion mutants of AML1/EVI-1, showed that these two functions are mutually independent because the dominant negative effects upon intact AML1 and the stimulation of AP-1 activity are dependent on the runt domain and the zinc finger domain near the C-terminus, respectively. Furthermore we showed that AML1/EVI-1 blocks granulocytic differentiation, otherwise induced by G-CSF,of 32Dc13 myeloid cells. It was also suggested that both AML1-derived and EVI-1-derived portions of the fusion protein play crucial roles for such differentiation block. We conclude that the leukemic cell transformation in t (3 ; 21) leukemias is probably caused by those dual functions of AML1/EVI-1 chimeric protein. Less