PROTO-ONCOGENE AND CYTOKINE REGULATION OF THE HUMAN GASTRIN PROMOTER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 胃泌素是一种从胃窦区的G细胞释放出来的强有力的多肽有丝分裂原。胃泌素基因在整个动物体内的调控机制尚不清楚。在幽门螺杆菌(HP)感染期间观察到的持续和夸大的血浆胃泌素水平增加了患胃癌和结直肠癌的风险，这可能是由于胃泌素基因表达的变化，而不是G细胞数量的变化。胃泌素启动子包含多种转录因子的共同位点，其中包括能与c-Myc原癌蛋白复合体结合的E-box。这项拟议的研究将评估幽门螺杆菌感染相关炎症过程中存在的白细胞介素1、2、4和6、肿瘤坏死因子α和表皮生长因子是否能够通过c-Myc原癌蛋白依赖的信号机制激活胃泌素启动子。在实验室建立的含有c-myc正义或反义基因的表达载体将用于改变胃腺癌(AGS)细胞中c-Myc原癌蛋白的水平。胃泌素报告基因的构建和c-myc表达载体将通过电穿孔的方式共同导入AGS细胞。胃泌素报告构建体包含胃泌素启动子序列和无启动子荧光素酶报告基因上游连接的第一个外显子，将通过荧光素酶分析和Western blotting来评估刺激的AGS细胞中胃泌素启动子激活和c-Myc水平是否正相关。胃泌素启动子的缺失将被用来识别赋予胃泌素基因c-Myc反应性的区域。在胃泌素启动子中发现E-box序列的生物素3‘标记寡核苷酸探针将用于化学发光电泳迁移率改变分析，以评估c-Myc复合体的DNA结合。这项研究很重要，因为它将阐明c-Myc原癌蛋白在幽门螺杆菌相关性高胃泌素血症期间招募的信号机制中的功能作用，并参与胃泌素基因的上调。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Gastrin is a potent polypeptide mitogen released from the G-cells in the antral region of the stomach. Mechanisms for gastrin gene regulation in the whole animal are not clear. The sustained and exaggerated plasma gastrin level observed during Helicobacter pylori (HP) infection that increases the risk for gastric and colorectal cancers is likely due to changes in gastrin gene expression, rather than a change in G-cell number. The gastrin promoter contains the consensus sites for a variety of transcription factors, which include the E-box that can bind the c-Myc proto-oncoprotein complex. This proposed study will evaluate whether interleukins 1, 2, 4, and 6, TNF alpha, and EGF, which are present during the inflammation associated with HP infection, are capable of activating the gastrin promoter via c-Myc proto-oncoprotein dependent signaling mechanisms. Expression vectors created in the laboratory that contain the c-myc cDNA in the sense or antisense orientation will be used to vary the c-Myc proto-oncoprotein level within gastric adenocarcinoma (AGS) cells. Gastrin reporter constructs and c-myc expression vectors will be co-introduced into the AGS cells by electroporation. The gastrin reporter constructs, which contain gastrin promoter sequences and the 1st exon linked upstream of a promoterless luciferase reporter gene, will be used to evaluate whether gastrin promoter activation and c-Myc levels are positively correlated in stimulated AGS cells via luciferase assays and Western blotting. Gastrin promoter deletions will be used to identify the regions that confer c-Myc responsiveness to the gastrin gene. Biotin 3' labeled oligo probes with the E-box sequences found in the gastrin promoter will be used in chemiluminescent electrophoretic mobility shift assays to evaluate DNA binding of c-Myc complexes. This study is important since it will elucidate the functional role of the c-Myc proto-oncoprotein in signaling mechanisms that are recruited during HP associated hypergastrinemia and involved in upregulating the gastrin gene