) Colorectal cancer (CRC) is the second most common fatal malignancy in the Western world. While significant advances have been made in understanding genetic alterations associated with this malignancy, biochemical pathways involved in the development of CRC are less well understood. Recent evidence suggests, however, that the inducible cyclooxygenase (COX), COX-2, and its products, prostaglandins (PGs), may play a key role in the pathogenesis of CRC. We have reported that production of COX-2 and PGs is regulated by the epidermal growth factor receptor in polarized HCA-7 CRC cells (PNAS 94:657, 1997). We now have found that lipid-extracted conditioned medium from parental HCA-7 cells stimulates proliferation of HCA-7 cells engineered to express an inducible COX-2 antisense cDNA, as well as in the PG-deficient CRC cell line, HCT-15. Moreover, we have observed that cyclopentenone PGs, which are dehydration products of PGD2 and PGE2, induce proliferation at concentrations in the low nanomolar or subnanomolar range in these cell lines. Studies are now proposed to further explore the role of cyclopentenone PGs in colonic epithelial proliferation. We will examine the ability of various cyclopentenone PGs to induce proliferation in other colon cancer cell lines that do not make PGs, including ALA, FET, and HCT-15 cells, and also in other gastrointestinal epithelial cell lines. To complement these studies, we will overexpress COX in ALA, FET, and HCT-15 cells and determine whether they form cyclopentenone PGs and whether proliferation is altered. We have found that parent HCA-7 cells produce the cyclopentenone PG, PGJ2. We will utilize chromatographic and mass spectrometric methods to definitively identify other cyclopentenone PGs. We have obtained indirect evidence that cyclopentenone PGs induce proliferation by interaction with a putative receptor. To obtain direct support for this hypothesis, we have synthesized a radiolabeled cyclopentenone PG, [tritium-labeled]-15-deoxy-delta 12,14-prostaglandin J2, with a high specific activity and we will perform radioligand binding studies in sub-cellular extracts from HCA-7 cells. Finally, we will study the formation of cyclopentenone PGs in animal models of CRC and in human colon tumor samples by quantifying these compounds utilizing mass spectrometric assays that we will develop. These studies will provide insight into the role of cyclopentenone PGs in the development of CRC and further clarify the contributions of COX to the pathogenesis of CRC.
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