PROTO-ONCOGENE AND CYTOKINE REGULATION OF THE HUMAN GASTRIN PROMOTER

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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