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

在本项目启动时，我们已经证明了ATL高钙血症的病理生理机制是恶性体液高钙血症，ATL的HHM因子是由HTLV-L感染的人T细胞系MT-2产生的，MT-2表达与甲状旁腺激素相关蛋白(PTHrP)同源的基因。通过CDNA克隆和序列测定，证明MT-2中表达的mRNA为PTHrP。13通过对20多例ATL新鲜肿瘤细胞的分析，我们发现PTHrP MANA有一定的高表达，为PTHrP在高钙血症发生发展中的主要作用提供了证据。我们还揭示了该基因在L携带者外周血单核细胞中的诱导表达，提示体内导致甲状旁腺素受体基因表达的不是感染细胞的恶性转化，而是L病毒的感染。然后，我们通过CAT实验证明了转录反式…对pthrp启动子的反式激活。HTLV-1的更多支持者，p40^&lt；Tax&gt；(T Watanabe et AJ。J Exp Med 172：759-765,1990)。体内FfTLVl Px基因的表达与PTHrP基因的表达呈正相关。在寻找PTHrP前运动区反式激活顺式元件的过程中，我们发现该600bp的前运动区可以介导p40；Tax&gt；，然而，目前还没有已知的p40；Tax&gt；反式激活顺式元件，如cAMP反应元件。相反，该区域富含GC，并出现了锌指蛋白CGCCCCCGC共同结合序列的重复基序。因此，我们利用CAT实验验证了该基序增强p40；Tax&gt；反式激活的可能性。插入该基序的三个串联重复序列可将对p40^&lt；Tax&gt；的反应提高约三倍(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素(S)。到目前为止，锌指即刻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等人)。正在准备中的手稿)。较少

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