Rearranged NFKB2 gene in the HUT78 T-lymphoma cell line codes for a constitutively nuclear factor lacking transcriptional repressor functions.
Rearranged NFKB2 gene in the HUT78 T-lymphoma cell line codes for a constitutively nuclear factor lacking transcriptional repressor functions.
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发表时间：
1994-07
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影响因子：
8
通讯作者：
Jiandong Zhang;C. Chang;Luigia Lombardi;Riccardo Dalla-Favera
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作者：
Jiandong Zhang;C. Chang;Luigia Lombardi;Riccardo Dalla-Favera
NFKB 2基因的重排与淋巴恶性肿瘤有关，但这些改变的功能意义尚不清楚。在这里，我们从结构和功能上描述了T细胞淋巴瘤系HUT 78中鉴定的重排NFKB 2基因。重排在3'锚蛋白结构域内具有截短的NFKB 2序列，导致分别通过北方印迹和免疫沉淀分析检测到的截短的mRNA种类和蛋白质的产生。相应cDNA的克隆和测序表明，通过选择性剪接，重排的基因编码两个蛋白质的84和85 kD（p84/85），保留了DNA结合的rel结构域和前五个锚蛋白重复，但失去了其羧基端，包括第七个锚蛋白重复。HUT 78细胞的免疫荧光和免疫沉淀分析表明，p84/85以未加工的形式异常地位于细胞核中，这表明这些蛋白质在被加工成p52之前可以逃脱正常NFKB 2 p100蛋白质典型的细胞质滞留。对HUT 78细胞核提取物进行的电泳迁移率变化分析表明，异常NF κ B 2蛋白特异性结合κ B位点，并改变HUT 78细胞中NF-κ B复合物的组成。涉及NFKB 2表达载体和κ B驱动的报告质粒的瞬时共转染测定表明NFKB 2 p85已经失去了正常NFKB 2 p52典型的转录抑制子功能。这些数据表明，在HUT 78细胞中检测到的NF κ B 2基因重排导致能够改变NF-κ B转录系统功能的异常NF κ B 2蛋白的产生。由于在淋巴恶性肿瘤中发现了类似的重排，这些发现进一步支持了NFKB 2改变在肿瘤发生中的作用。
Rearrangements of the NFKB2 gene are associated with lymphoid malignancies, but the functional significance of these alterations is not known. Here we characterize structurally and functionally a rearranged NFKB2 gene identified at the T cell lymphoma line, HUT78. The rearrangement has truncated NFKB2 sequences within the 3' ankyrin domain, leading to the production of truncated mRNA species and proteins as detected by Northern blot and immunoprecipitation analysis, respectively. Cloning and sequencing of the corresponding cDNAs indicates that, via alternative splicing, the rearranged gene codes for two proteins of 84 and 85 kD (p84/85) which retain the DNA-binding rel domain and the first five ankyrin repeats, but have lost their carboxy-terminus including the seventh ankyrin repeat. Immunofluorescence and immunoprecipitation analysis of HUT78 cells indicate that p84/85 are abnormally located in the nucleus in an unprocessed form, suggesting that these proteins can escape the cytoplasmic retention typical of the normal NFKB2 p100 protein before it is processed into p52. Electrophoretic mobility shift assays performed on HUT78 nuclear extracts indicate that the abnormal NFKB2 proteins bind kappa B sites specifically and alter the composition of NF-kappa B complexes in HUT78 cells. Transient co-transfection assays involving NFKB2 expression vectors and kappa B-driven reporter plasmids indicate that NFKB2 p85 has lost the transcriptional repressor functions typical of normal NFKB2 p52. These data indicate that the NFKB2 gene rearrangement detected in HUT78 cells leads to the production of abnormal NFKB2 proteins capable of altering the function of the NF-kappa B transcription system. Since analogous rearrangements are found in lymphoid malignancies, these findings further support a role of NFKB2 alterations in tumorigenesis.