Li Q, Frestedt J L, Kersey J H
University of Minnesota Cancer Center and Departments of Pediatrics and Laboratory Medicine/Pathology, University of Minnesota, Minneapolis, MN, USA.
Blood. 1998 Nov 15;92(10):3841-7.
Acute leukemia with t(4;11)(q21,q23) translocation results from the in-frame fusion of the MLL to the AF4/FEL gene. In previous studies, we and others demonstrated that AF4 transcripts are present in a variety of hematopoietic and nonhematopoietic human cells. To further study the wild-type and leukemia fusion AF4, we used glutathione S-transferase (GST)-fusion proteins as immunogens to produce rabbit polyclonal antibodies that were specific for normal and chimeric AF4 proteins. Using Western blotting analysis, we demonstrated that the AF4 gene encodes proteins with apparent molecular weight of 125 and 145 kD. A 45-kD protein coprecipitated with AF4 protein in immunoprecipitation. Also, the anticipated MLL-AF4-encoded 240-kD protein was detected in all cell lines with t(4;11) translocations; fusion proteins were present in lesser quantity than the wild-type AF4. The proteins recognized by the antibodies are of the predicted sizes of the AF4 and MLL-AF4-encoded proteins based on previous DNA sequencing analysis. The MLL-AF4 fusion protein had a similar subcellular distribution as AF4. Both t(4;11) and non-t(4;11) leukemic cells showed a similar pattern of punctate nuclear staining in all cell lines tested using confocal immunofluorescence microscopy. AF4 antibodies should be useful for further elucidation of the function of AF4 in normal cellular physiology, as well as the function of MLL-AF4 in leukemogenesis. The antibodies should also be helpful for the diagnosis of the MLL-AF4 fusion proteins in t(4;11) leukemias.
伴有t(4;11)(q21,q23)易位的急性白血病是由MLL与AF4/FEL基因的读码框内融合所致。在先前的研究中，我们和其他人证明AF4转录本存在于多种造血和非造血人类细胞中。为了进一步研究野生型和白血病融合型AF4，我们使用谷胱甘肽S-转移酶（GST）融合蛋白作为免疫原，制备了对正常和嵌合AF4蛋白具有特异性的兔多克隆抗体。通过蛋白质印迹分析，我们证明AF4基因编码表观分子量为125和145 kD的蛋白质。在免疫沉淀中，一种45-kD的蛋白质与AF4蛋白共沉淀。此外，在所有伴有t(4;11)易位的细胞系中均检测到预期的MLL-AF4编码的240-kD蛋白；融合蛋白的含量低于野生型AF4。基于先前的DNA测序分析，抗体识别的蛋白质大小与AF4和MLL-AF4编码的蛋白质预测大小相符。MLL-AF4融合蛋白的亚细胞分布与AF4相似。使用共聚焦免疫荧光显微镜在所有测试的细胞系中，伴有t(4;11)和不伴有t(4;11)的白血病细胞均显示出类似的点状核染色模式。AF4抗体对于进一步阐明AF4在正常细胞生理学中的功能以及MLL-AF4在白血病发生中的功能应是有用的。这些抗体对于诊断t(4;11)白血病中的MLL-AF4融合蛋白也应是有帮助的。
