Analysis of the mechanism of transactivation of gene expression of malignant hypercalcemia factor (PTHrP) by HTLV-1

在本项目开始时，我们已经证明ATL的高钙血症的病理生理学是恶性体液性高钙血症（HHM），ATL的HHM因子是由HTLV-L感染的人T细胞系MT-2产生的，并且MT-2表达与甲状旁腺相关蛋白（PTHrP）的mRNA同源的mRNA。然后，我们通过cDNA克隆和序列测定，证明MT-2中表达的mRNA是PTHRP的mRNA。13通过对20余例ATL新鲜肿瘤细胞的分析，证实PTHRP MANA的结构性过表达，为PTHRP在高钙血症发生中的主要作用提供了证据。我们还揭示了该基因在HTLV-L携带者的PBMC中的表达的诱导，这表明HTLV-L的感染，而不是感染细胞的恶性转化，导致PTHRP在体内的表达。然后，我们通过CAT实验证明了PTHRP启动子的转录激活作用， ...更多信息 HTLV-1的vator，p40^<tax>（T Watanabe et aJ. J Exp Med 172：759-765，1990）。FfTLV 1 pX mRNA的表达与PTHRP mRNA的表达存在相关性。在对PTHRP前马达区反式激活顺式元件的鉴定过程中，我们发现前马达区的600 bp片段可以介导p40的反式激活作用<tax>。然而，没有已知的p40 α反式激活的顺式元件<tax>，如环AMP反应元件。相反，该区域富含GC，并且出现了锌指蛋白结合共有序列CGCCCGC的重复基序。因此，我们使用CAT测定测试了该基序增强p40 α反式激活的可能性<tax>。插入该基序的三个串联重复序列显示增强对p40的应答<tax>约三倍（T Watanabe，J Jpn Soc RES 31 ; 33-39，1991）。现在我们正在研究与这个序列基序相互作用的因子。迄今为止，已经证明锌指立即早期基因的主要成员EGR-L和EGA-2的表达与PTHRP的表达无关（T Watanabe等，手稿准备中）。少

At the time when this project started, we had shown that the pathophysiology of hypercalcemia of ATL Is that of humoral hypercalcemia of malignancy(HHM), and the HHM factor of ATL is produced by HTLV-L infected human T cell line, MT-2, and also that MT-2 expresses MRNA homologous to that of parathyrold hormone-related protein(PTHrP). Then, we have shown that the MRNA expressed in MT-2 is that of PTHRP by CDNA cloning and sequence determination. 13y the analysis of fresh tumor cells from more than 20 ATL cases, we demonstrated consitutive overexpression of PTHRP MANA, providing the evidence for main causative role of PTHRP in the development of hypercalcemia. We also revealed the induction of the expression of this gene in PBMCs of HTLV-L carriers, suggesting that infection of HTLV-L, not the malignant transformation of infected cells, leads to the expression of PTHRP In vivo. Then we demonstrated, by the CAT assay, the trans-activation of PTHRP promotor by the transcriptional transacti … More vator of HTLV-1, p40^<tax>(T Watanabe et aJ. J Exp Med 172 : 759-765, 1990). It was also shown that the expression of FfTLVl pX MRNA is correlated with the expression of PTHRP MRNA in vivo. In the process of identifying the cis-element for transactivation in the premotor of PTHRP, we found that the 600bp of premotor region can mediate more than ten fold trans-activation by p40^<tax>, . However, there were no well-known cis-element of trans-activation by p40^<tax>, such as cyclic AMP responsive element. Instead, this region was very rich in GC, and there appeared repeated motifs of consensus sequence of binding for Zn finger proteins, CGCCCCCGC. Therefore, we tested the possibility that this motifs enhance the trans-activation by p40^<tax> with the use of CAT assay. Insertion of three tandem repeats of this motifs was shown to enhance the response to p40^<tax> about three fold(T Watanabe, J Jpn Soc RES 31 ; 33-39, 1991). Now we are studying the factor(s)that interacts this sequence motif. So far, it has been demonstrated that the expression of EGR-L and EGA-2, the main members of Zn finger immediate early genes, does not correlate the expression of PTHRP(T Watanabe et al. manuscript in preparation). Less