COLLAGEN TYPE I/SPHINGOLIPID PATHWAYS IN REGULATION OF LEIOMYOMA GROWTH

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Uterine leiomyomas, also known as fibroid tumors, are the most common tumors of the uterus, occurring with a frequency of 20-25% in women of reproductive age. Their occurrence has been linked to diverse symptoms including excessive uterine bleeding, pelvic pressure and pain, premature births, and infertility. The increased incidence of leiomyomas after the onset of menarche, the growth of tumors during pregnancy and after administration of steroids as well as the regression of tumors after menopause gives clinical evidence to support the role of ovarian steroids in the growth of uterine fibroids. These tumors also exhibit growth- factor dependent growth potential. Three human uterine cell lines designated ST1 MT1 and HT1 have been established in our laboratory. The long-term objectives of this research are to identify the factors involved in the regulation of the proliferation of uterine leiomyomas utilizing these cell lines and to determine the mechanism of action of these factors. The specific aims of the proposed research are to: I. Determine if the presence of collagen type I in the extracellular matrix of leiomyomas has an effect on the response of these tumors to known mitogens such as estradiol, EGF and mitogenic factors from conditioned medium from ST1 cells. In these studies ST1 and HT1 cells will be grown on defined matrices. The level of collagen type I secretion by ST1 and HT1 cells grown on these matrices will be determined by ELISA. The effect of estradiol, EGF and the mitogenic fraction of conditioned medium on the growth kinetics of these cell lines will also be assessed, when cells are grown on defined matrices. II. Purify the low molecular weight mitogenic factor from conditioned medium of ST1 cell lines and determine if the factor is a member of the sphingolipid class of growth factors. The content of sphingosine 1- phosphate in the mitogenic fraction of conditioned medium will be determined by HPLC to determine any correlation between this activity and sphingosine 1-phosphate will also be assessed. III. Determine if growth factors for uterine leiomyomas act via a sphingolipid signaling pathways in the regulation of leiomyoma growth. In the proposed studies, ST1 and HT1 cell lines will be exposed to mitogens. Levels of all intermediates in the putative signal transduction pathway (ceramide, sphingosine and sphingosine 1-phosphate) will be monitored by thin-layer chromatography and/or HPLC.