Fåhraeus R, Palmqvist L, Nerdstedt A, Farzad S, Rymo L, Laín S
Department of Medical Biochemistry, Göteborg University, Gothenburg, Sweden.
EMBO J. 1994 Dec 15;13(24):6041-51. doi: 10.1002/j.1460-2075.1994.tb06950.x.
The expression of the Epstein-Barr virus LMP1 oncogene is regulated by viral and non-viral factors in a tissue dependent fashion. The virus encoded transcription factor EBNA2 induces its expression in human B-cells. However, this induction also requires the contribution of cellular and/or other viral factors. In nasopharyngeal carcinoma cells and in cells from Hodgkin's lymphoma, LMP1 gene transcription is independent of viral products. Here we show that the effect of a factor binding to a cAMP responsive-like element (CRE) in the LMP1 gene transcription regulatory sequence (LRS) is essential for efficient promoter activity in the DG75 B-cell line and that elevation of cAMP levels in the cells induces LRS-derived CAT activity in a CRE dependent fashion. Incubation of two EBV-immortalized B-cell lines expressing endogenous EBNA2A with 8-Br cAMP increased the levels of the latency associated 66 kDa LMP1 within 2 h. Interestingly, LMP1 expression in DG75 cells conferred resistance to the inhibitory effect of 8-Br cAMP on cell proliferation. The protein phosphatase 1 and 2A (PP1 and PP2A, respectively) inhibitor okadaic acid also stimulated LRS-CAT activity in DG75 cells. EBNA2A from an EBV-immortalized B-cell line co-immunopurified with a PP1-like protein. An EBNA2A fragment spanning residues 324-436 fused to the GST protein specifically rescued a PP1/PP2A-like component from DG75 cell extracts. This GST-EBNA2A fusion product inhibited a PP1-like activity in nuclear extracts from these cells.
爱泼斯坦-巴尔病毒LMP1癌基因的表达受病毒和非病毒因子以组织依赖性方式调控。病毒编码的转录因子EBNA2在人B细胞中诱导其表达。然而，这种诱导也需要细胞和/或其他病毒因子的参与。在鼻咽癌细胞和霍奇金淋巴瘤细胞中，LMP1基因转录独立于病毒产物。在此我们表明，一种因子与LMP1基因转录调控序列（LRS）中类似cAMP反应元件（CRE）结合的效应，对于DG75 B细胞系中有效的启动子活性至关重要，并且细胞内cAMP水平的升高以CRE依赖性方式诱导LRS衍生的CAT活性。用8-溴环磷腺苷（8-Br cAMP）孵育两个表达内源性EBNA2A的EBV永生化B细胞系，在2小时内增加了潜伏期相关的66 kDa LMP1的水平。有趣的是，DG75细胞中LMP1的表达赋予了对8-Br cAMP对细胞增殖抑制作用的抗性。蛋白磷酸酶1和2A（分别为PP1和PP2A）抑制剂冈田酸也刺激了DG75细胞中的LRS-CAT活性。来自EBV永生化B细胞系的EBNA2A与一种类似PP1的蛋白共免疫纯化。一个跨越324 - 436位残基的EBNA2A片段与GST蛋白融合，特异性地从DG75细胞提取物中拯救出一种类似PP1/PP2A的成分。这种GST-EBNA2A融合产物抑制了这些细胞的核提取物中的一种类似PP1的活性。
