PROTO-ONCOGENE AND CYTOKINE REGULATION OF THE HUMAN GASTRIN PROMOTER

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。胃泌素是胃窦部G细胞释放的一种有效的多肽促分裂原。整个动物中胃泌素基因调控的机制尚不清楚。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有位点，包括可以结合c-Myc原癌蛋白复合物的E-box。这项拟议的研究将评估白细胞介素1、2、4和6、TNF α和EGF是否能够通过c-Myc原癌蛋白依赖性信号传导机制激活胃泌素启动子，这些因子存在于与HP感染相关的炎症过程中。在实验室中创建的含有有义或反义方向的c-myc cDNA的表达载体将用于改变胃腺癌（AGS）细胞内的c-Myc原癌蛋白水平。通过电穿孔将胃泌素报告基因构建体和c-myc表达载体共引入AGS细胞中。胃泌素报告基因构建体（包含胃泌素启动子序列和连接在无启动子荧光素酶报告基因上游的第1个外显子）将用于通过荧光素酶测定和蛋白质印迹法评价在刺激的AGS细胞中胃泌素启动子活化和c-Myc水平是否呈正相关。胃泌素启动子缺失将用于鉴定赋予c-Myc对胃泌素基因的反应性的区域。具有在胃泌素启动子中发现的E-box序列的生物素3'标记的寡核苷酸探针将用于荧光电泳迁移率变动测定，以评价c-Myc复合物的DNA结合。这项研究很重要，因为它将阐明c-Myc原癌蛋白在HP相关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