Eicosatraenoic acids (mostly arachidonic acid, 20:4,omega-6) are metabolized by many cells to a variety of signalling compounds (eicosanoids) that act locally. This metabolic event occurs in response to a variety of stimuli including hormones and environmental insults, and arachidonate can be channelled through several enzymatic pathways, including prostaglandin endoperoxide synthases (cyclo-oxygenases) to yield ultimately the prostanoids. Evidence suggesting the importance of prostanoids in the cinogenesis of human colonic epithelium include: a) their increased concentration in tumor tissue; b) their ability to decrease tumor proliferation rate in vitro; and c) clinical trials suggesting that drugs with a primary effect of inhibiting cyclo- oxygenases (and therefore prostanoid synthesis) are associated with regression of polyps in pre-malignant conditions as well as in the general population without known predisposing factors. However, little is known of the mechanisms by which these effects occur, nor the ability of normal or transformed epithelial cells to use a particular signalling pathway after binding a prostanoid via a specific receptor. Several recent advances now allow these issues to be addressed: 1) Procurement of sequence information/cloning of receptors for prostanoids EP2, EP3, EP4, IP, FP and TP; 2) A cDNA clone for a second (inducible) cyclo- oxygenase with potential importance in the intestinal mucosa, in addition to cyclo-oxygenase 1 already available; 3) Sequence information on a prostaglandin transporter for the intestinal epithelial cell, thought to be important in terminating cellular effects of prostanoids. All probes available can be used with quantitative techniques (nuclease protection assays) as well as for methodology localizing macromolecules in complex tissues (in situ hybridization).
The aim of this proposal, then, is to determine mechanisms whereby responses to prostanoids by epithelial cells of the intestinal mucosa are regulated, as influenced by the array of receptors for prostanoids which epithelial cells express inherently or the level of prostanoid-generated cyclo-oxygenase enzyme, and how this contributes to or inhibits growth of intestinal colonic epithelial cell tumors. Specifically, we propose to test the hypotheses that: (1) Colonic epithelial cells """"""""in transition"""""""" (to adenocarcinoma) respond to prostanoids in a unique way, compared with normal colonic epithelium, due to their expression of a novel set of prostanoid receptors/signalling proteins: (2) Continuing signals exist for up-regulation of cyclo-oxygenase-2 (COX- 2) in colonic mucosal epithelium and in selected cells of the lamina propria; that COX-2 in mucosa is inhibited by certain (of seven) NSAIDS proposed for study; and that the cell distribution/regulation of COX will differ considerably between subjects with epithelium in transition to adenocarcinoma and those with normal epithelium (no current or past polyps). Epithelial cells """"""""in transition"""""""" will be from the colon of both an animal model (azoxymethane-treated rats) as well as humans with known tumor predisposition (familial adenomatous polyposis, history of multiple recurrent polyps), and our analyses will use molecular probes developed and present in our laboratory (DNA's coding for known members of the prostanoid receptor family as well as for COX-1 and 2); individual prostanoid receptor-specific immunoglobulin, and in situ techniques (for hybridization and immunocytochemistry).
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