Identification of transcription factor related to toxicity of dioxin

2378-四氯二苯并-对-二恶英（dioxin）及其相关化合物具有致畸、促肿瘤等多种毒性，但其毒性作用的基因表达机制尚不清楚。为了了解谷胱甘肽S-转移酶1（GSTP 1）基因表达的调控机制，我们利用CAT技术鉴定了GSTP 1基因5'侧翼序列上的一个调控元件。在原代培养的肝实质细胞中，GPEI元件被证明是PenCB刺激GSTP 1基因表达所必需的。已知GPEI含有以回文方式定向的佛波酯响应性元件样元件的二联体。提示PenCB激活的一条新的信号转导通路参与了GSTP 1基因的表达，不仅PenCB，表皮生长因子（EGF）也能诱导GSTP 1基因的表达。GPEI元件也是EGF和TGFα表达该基因所必需的。提示EGF和TGFα通过与PenCB相同的信号转导途径诱导GST 1。我们现在打算使用组成型表达GST 1的永生化大鼠肝实质细胞的核提取物作为起始材料来鉴定GPEI结合蛋白。用32 P标记的GPEI寡聚体与细胞核中的GPEI结合蛋白进行紫外线交联，并通过二维凝胶电泳进行分离。放射自显影检测后，分离与GPEI寡聚体结合的蛋白。通过MALDI-TOF/MS测定分离蛋白质的三肽段分子量，并结合蛋白质序列数据库对GPEI结合蛋白进行鉴定。

2378-tetrachlorodibenzo-p-dioxin (dioxin) and related compounds exert various toxicities such as teratogenicity and tumor promoting activity, but the mechanisms of gene expression accompanied with emerge of their toxic effect remain to be unclear. Previously, we showed that 3453'4'-pentachlorobiphenyl (PenC) a dioxin related compound, induces the expression of pi class glutathione S-transferase (GSTP1) gene in primary cultured rat liver parenchymal cells.In order to understand the regulation of GSTP1 gene expression, a regulatory element on 5' flanking sequence of this gene was identified by CAT assay. The GPEI element was shown to be required for stimulation of GSTP1 gene expression by PenCB in primary cultured liver parenchymal cells. GPEI is already known to contain a dyad of phorbol ester responsive element-like elements oriented palindromically. It is suggested that a novel signal transduction pathway activated by PenCB contributes to stimulation of GSTP1 expression.Not only PenCB but epidermal growth factor (EGF) induces the expression of GSTP1 gene. GPEI element is also required for the expression of this gene by EGF and TGFα. It is suggested that EGF and TGFα induce GSTP1 by the same signal transduction pathway as PenCB.We intend now to identify the GPEI binding protein using a nuclear extract of immortalized rat liver parenchymal cells which constitutively express GSTP1 as a starting material. GPEI binding proteins in the nucleous were cross-linked with 32P labeled GPEI oligomer by UV irradiation and were separated by 2D gel electrophoresis. Proteins bound to GPEI oligomer were isolated after detecting by autoradiography. After determining the molecular weights of triptic digests of isolated proteins by MALDI-TOF/MS, we have identified the GPEI binding proteins referring with the data base of protein sequences.