Highly potent LH-releasing hormone (LHRH) agonists have been recently introduced in therapy for the treatment of the carcinoma of the prostate, an androgen-dependent pathology. These peptides are believed to act mainly by inhibiting the pituitary-testicular axis and, consequently, by reducing testosterone levels. The recent observation that binding sites for LHRH analogs are present on prostatic tumor tissue suggests that these drugs could also act directly on the tumor. To verify this hypothesis, the effects of two potent LHRH agonists [Zoladex (Z) and Buserelin (B)] have been studied on the proliferation of the human prostatic cancer cell line LNCaP (lymph node carcinoma of the prostate). LNCaP cells were treated for 9 days with different doses of either Z or B (concentrations from 10(-12)-10(-6) M). Both analogs significantly inhibited cell proliferation at doses between 10(-9)-10(-6) M. The antiproliferative action of the two LHRH agonists was shown to be dose dependent, with IC50 values of 0.82 and 1.79 nM for Z and B, respectively. A similar treatment with B was without any significant effect on the proliferation of a mouse embryo fibroblast cell line (Swiss 3T3), which was used as a nontumoral control. The inhibitory action of both LHRH agonists (10(-8) M) on LNCaP cell proliferation was completely antagonized by the simultaneous treatment of the cells with a potent LHRH antagonist (Nal-Arg-LHRH; 10(-8) M); when given alone at the dose selected, the antagonist did not affect cell growth. These results clearly suggest that the antiproliferative effect of LHRH agonists on LNCaP cells may be mediated by specific receptors. The presence of binding sites for [125I]B was consequently demonstrated on the membranes of LNCaP cells cultured in a medium containing charcoal-stripped fetal calf serum, i.e. in the absence of steroids. The affinity of these binding sites for the ligand was lower than that observed for the same receptors on rat pituitary membranes. To clarify the mechanism of the antiproliferative action of the LHRH agonists, the effects of both Z and B on the incorporation of [3H]thymidine and [14C]methionine into LNCaP cells were investigated. During a short incubation period (3 h), the two LHRH agonists rapidly inhibited [3H]thymidine incorporation into the cells. The same treatment did not affect the incorporation of [14C]methionine into proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
The development of two cell lines (GT1 and GN) of immortalized LHRH neurons has allowed an accurate study of the mechanisms controlling the synthesis and the secretion of LHRH. These cell lines, obtained in mice by genetic targeted tumorigenesis, retain many of the phenotypic characteristics of LHRH neurons. Of interest, GT1 cells derive from an hypothalamic tumor, whereas GN cells were obtained from a tumor localized in the olfactory bulb. The different origin of these cell lines lead to hypothesize that they might represent hypothalamic postmigratory neurons (GT1 cells), or LHRH neurons blocked at an early stage of their migration (GN cells). Using different experimental procedures, we found that the two cell subclones GT1-7 and GN11 express a different morphology and migratory behavior in vitro. In particular, we found that GN11 cells, but not GT1-7 cells, show the morphological shape of migrating neurons. When analyzing the spontaneous motility we found that only GN11 cells express a high capacity of migrating in a matrix of collagen gel. Moreover, in a chemomigratory assay GN11 cells did show a significant response to the chemotactic stimulus represented by the FBS. On the contrary, GT1-7 cells show very low spontaneous motility and appear insensitive to the FBS stimulus. These results suggest that the simultaneous use of the GT1-7/GN11 cells may represent an experimental tool for screening the factors possibly involved in the control of the migratory processes of LHRH neurons in normal and in pathological conditions, such as those due to their impaired migration, like it happens in Kallmann's syndrome.
These experiments were performed to analyze whether treatments of ovariectomized female rats with ovarian steroid regimens able to induce either an increase (positive feedback effect) or a decrease (negative feedback effect) of serum levels of luteinizing hormone (LH) have some impact on the characteristics of mu-opioid binding sites in circumscribed areas of the brain. The increase of serum levels of LH elicited by a treatment with estradiol benzoate (EB) plus progesterone (P; positive feedback effect) was accompanied by a significant decrease in the number of mu-binding sites in the hypothalamus and in the corpus striatum. The decrease in serum levels of LH induced by a treatment with EB alone (negative feedback effect) brought about a significant increase of the number of mu-binding sites in the thalamus and in the hippocampus. These results seem to suggest that the release of LH induced by EB plus P may involve a decrease of hypothalamic mu-binding sites. Apparently, the inhibitory effect on LH release exerted by EB alone does not involve any change of the density of these binding sites in the hypothalamus.
LHRH synthesis and release are modulated in vivo by gonadal steroids. Although immunocytochemical and autoradiographic studies failed to detect appreciable amounts of estrogen or androgen receptor in LHRH-producing neurons, the recent finding that the promoter region of the LHRH gene contains several steroid hormone-responsive elements indicates a possible direct effect of sex steroids on these specialized neurons. The immortalized LHRH-producing neuronal cell line, GT1, which became recently available, may allow the study of LHRH dynamics. The presence of specific binding sites for estrogen and androgens as well as the presence of the two major enzymatic pathways involved in modulation of androgen action (the 5 alpha-reductase/3 alpha-hydroxysteroid dehydrogenase and the aromatase) have been studied in the GT1-1 clone. High affinity, low capacity binding sites for [3H]estradiol (Kd, 0.11 nM; binding capacity, 6.2 fmol/mg protein) and for a ligand of the androgen receptor, [3H]R1881 (Kd, 0.054 nM; binding capacity, 9.58 fmol/mg protein), have been identified in this cell line. A 2-fold induction of androgen-binding sites has been observed after 3 days of treatment of GT1-1 cells with estradiol (1 microM), indicating that the estradiol binding is probably linked to a functional estrogen receptor. Aromatase and 5 alpha-reductase/3 alpha-hydroxysteroid dehydrogenase activities have been also tested in GT1-1 cells. Under the culture conditions adopted, no detectable aromatization of [1 beta 3H]delta 4-androstenedione to estrone was observed using the tritiated water method. On the other hand, GT1-1 cells efficiently converted testosterone into dihydrotestosterone and subsequently into 5 alpha-androstan-3 alpha,17 beta-diol. In conclusion, GT1-1 cells possess several elements of the machinery through which sex steroids may influence LHRH dynamics.
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