The long-term goals of this proposal are to identify and characterize novel paradigms of gastrointestinal transformation. Our initial objectives are to study the process of cancer development in the stomach where predisposing factors include the absence of gastric acid secretion (achlorhydria) and longstanding Helicobacter pylori infection. The current paradigms describing transformation in the stomach have not adequately described in molecular terms how these predisposing factors cause disease. Rather current understanding primarily reflects direct application of what is known regarding transformation in the colon. We have taken the approach that the stomach expresses several known growth factors, including EGF receptor ligands that increase with gastric transformation. The zinc finger transcription factor called ZBP-89 is a potent repressor of EGF receptor activation and inhibits transcription of the ornithine decarboxylase promoter and the stomach specific gene gastrin through specific CACCC/GTGGG elements. Recently, we have found that ZBP-89 is a transcriptional activator of the cell cycle inhibitor p21 in the presence of sodium butyrate. ZBP-89 resides on chromosome 3q21 which is a site of reciprocal translocations in both leukemia and gastric cancer. ZBP-89 is differentially expressed in some gastric cancers and somatic mutations of its C- terminal domain have been identified in gastrointestinal cancers. Therefore the specific aims of this application are to examine the role of ZBP-89 in cell cycle progression and proliferation; to characterize related ZBP-89 molecules and to examine its relevance in vivo by targeted gene disruption. Recombinant ZBP- 89 and various mutations of this protein will be used to examine how this transcription factor regulates cellular proliferation in transfected cells. Mutations of ZBP-89 that have lost their ability to regulate cell growth will be created to determine whether this protein interacts with known cell cycle regulators. Inactive mutants that bind DNA will be subcloned into an inducible promoter and used to disrupt ZBP-89 function in cell lines in a dominant negative manner. The domain mediating cell cycle regulation will be used to isolate and clone interacting molecules. Related ZBP-89 molecules will be identified by low stringency screening and characterized in terms of their ability to stimulate or inhibit cell growth. To study the role of ZBP-89 in vivo, the mouse ZBP-89 gene will be targeted for disruption by inserting the bacterial beta-galactosidase gene into the mouse locus. Insertion of the beta-galactosidase gene will permit the study of ZBP-89 expression during mouse development and to assess in which tissues ZBP-89 is critical for function.
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