We have previously shown that estrogen-dependent growth enhancement of murine transformed Leydig cells (B-1 F) is mediated through inhibition of arachidonic acid metabolite formation. In the present study, the growth-inhibitory ability of leukotrienes (LTs) on B-1 F cells in serum-free culture was directly addressed. All peptidyl LTs (LTC4, LTD4, and LTE4) inhibited B-1 F cell growth in a dose-dependent manner and exhibited maximum inhibition of DNA synthesis (60–80%) compared with that of untreated cells in a range of 10−9 to 10−8 m. To examine the mechanism of this LT-dependent inhibition, binding studies of LTD4 toward plasma membrane were conducted. Specific binding sites for LTD4 were identified. Scatchard analyses indicated the presence of a single class of high-affinity sites (K4 = 0.9 ± 0.2 nm; maximum binding sites, 61 ± 18 fmol/mg protein). This binding of LTD4 to the high-affinity site was markedly inhibited by ICI 198615, a specific inhibitor for LTD4. These results would suggest that inhibitory effects of LTs, at least LTD4, are elicited as a receptor-mediated event. In addition, this LT-dependent growth inhibition could not be blocked by simultaneous exposure of cells to estrogen, whereas estrogen partially protected arachidonic acid-dependent growth inhibition. Furthermore, treatment of cells with estrogen resulted in marked suppression of 5-lipoxygenase activity. Collectively, the present data clearly show that LTs play an important role as intermediates in an autocrine loop for B-1 F cells to exhibit estrogen-dependent growth.
This investigation was supported by Grants-in-Aid from the Ministry of Education, Tokyo, Japan, by the Cancer Research Promotion Fund, Tokyo, Japan, by the Foundation of the Growth Science, Osaka Cancer Research Fund, and by the Hirai Cancer Research Fund.