Epithelial cells have a built-in propensity to proliferate, which is essential for the normal turnover and renewal of tissues. To maintain a constant cell number, cell division and apoptosis must be tightly coupled. Disruption of this balance predisposes the epithleium to hyperproliferative disorders, including cancerous growth. A constitutive checkpoint of this balance is mediated by direct cell-cell interactions, which is the basis of contact-dependent inhibition of growth. However, despite intense investigations on this wellcharacterized phenomenon, little is known about the relative contribution of different cell-cell interactions to contact-regulated epithelial cell growth. The TIGHT JUNCTION is an intercellular interaction that is universally found in all epithelial cells, but whose function in cell proliferation is almost completely unknown. The current proposal is designed to address the fundamental question of whether the TIGHT JUNCTION is directly involved in active cell-cell signaling events that lead to downstream regulation of cell growth control.
The specific aims of the current proposal are:(1) to directly assess the roles of the TIGHT JUNCTION in epithelial cell growth by systematic investigation of cell-cycle checkpoints and apoptosis-related events in TIGHT JUNCTION disrupted cells.(2) to comprehensively identify the molecular players at the TIGHT JUNCTION.(3) to analyze molecular mechanisms of action that relay TIGHT JUNCTION information to downstream cell-cycle and apoptosis pathways. T84 and HT-29 cell lines will be used as in vitro model systems for this project because they retain key functional characteristics of the intestinal epithelium including formation of intercellular junctions, generation of a polarized phenotype, exhibition of contact-inhibition of growth, responsiveness to cytokines and infectious agent. The functional roles of TIGHT JUNCTION will be studied by disruption of the TIGHT JUNCTION using anti-sense technology. The molecular composition of TIGHT JUNCTION will be characterized via a proteomics approach. The functions of novel TIGHT JUNCTION proteins in cell growth control will be analyzed systematically in parental and tight junction disrupted cells.