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

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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