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Y. Amir-Zaltsman, Y. Ausher, B. Gayer, S. Lichter, F. Serour, S. Birkenfeld, and F. Kohen


The age-related changes in tissue response to chronic treatment for 1 month with a potent LHRH agonist were investigated in the ageing male rat, and the observed pharmacological effects were compared with orchidectomy. In both young (4 months) and old (22 months) rats, treatment resulted in a significant decrease in the weights of prostates and testes, a decrease in plasma LH and testosterone levels, a loss of LH receptors in the testes and in a complete depletion of prostatic nuclear androgen receptors, reaching levels observed after castration. In young rats, treatment with an LHRH agonist or orchidectomy induced a three- or sixfold increase in prostatic creatine kinase (CK) activity which may have been induced by the local stimulatory effect of oestradiol arising from the conversion of precursor steroids secreted by the adrenal. On the other hand, in old rats, 7 days after orchidectomy or after treatment with an LHRH agonist a twofold increase or no change was induced in prostatic CK activity respectively. SDS gel electrophoresis patterns of cytosolic prostatic proteins of young rats treated with an LHRH agonist or young rats orchidectomized 7 days previously revealed the presence of several intensified proteins, two of them having apparent molecular weight of 67 kDa and 43 kDa, whereas in the old rats treated with LHRH agonist or old rats castrated 7 days previously, these two proteins were not intensified. The results of this study confirmed that continuous treatment with an LHRH agonist to young and old rats induces medical castration since the pharmacological effects observed were the same as those induced with surgical castration. However, in the old rats, the lack of an increase in prostatic CK activity upon treatment with LHRH agonist, and the moderate increase in CK activity upon orchidectomy, suggest that prostatic cells in older rats have decreased sensitivity to hormonal manipulation.

Journal of Endocrinology (1990) 124, 261–268

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D Sömjen, Y Amir-Zaltsman, G Mor, B Gayer, S Lichter, G Barnard, and F Kohen


Previous studies indicated that the anti-idiotypic antibody (clone 1D5) significantly increased the specific activity of creatine kinase (CK) activity in the rat uterus, and in vitro in skeletal cells capable of responding to oestradiol (E2), suggesting that the antibody has oestrogenic-like activity. Moreover, the F(ab′)2 dimer of clone 1D5 acted like an antagonist and completely inhibited the increase in CK specific activity by either E2 or clone 1D5 in these skeletal cells. In the present study, we examined the in vivo effects of clone 1D5 and its proteolytic fragment, the F(ab′)2 dimer, E2 and dihydrotestosterone (DHT) on CK specific activity in the epiphyseal cartilage, diaphyseal bone, uterus, prostate, thymus and pituitary of immature or gonadectomized female and male rat animal models. In the intact immature animals, clone 1D5 caused an increase in CK in all organs of the female except in the pituitary. In the diaphyseal bone and prostate of male rats there was no stimulation by 1D5. The CK response in the uterus, epiphysis, and diaphysis of immature female rats was dose-dependent and was blocked by either the anti-oestrogen tamoxifen or the F(ab′)2 dimer of clone 1D5. E2, DHT, as well as clone 1D5, stimulated CK specific activity in both the diaphysis and epiphysis of ovariectomized female and castrated male rats, whereas sex specificity in the CK response was observed also in the uterus and the prostate of gonadectomized animals. Collectively, these results suggest that, as in cell culture, an intact antibody is necessary for the observed stimulation of CK specific activity and the F(ab′)2 dimer can act as an antagonist. Furthermore, the observed biological effects of clone 1D5 which are absolutely parallel to E2, imply that the anti-idiotypic antibody is able to penetrate the cell and reach the nuclear oestrogen receptor and transduces a signal to the nucleus, by as yet uncharacterized mechanisms.

Journal of Endocrinology (1996) 149, 305–312

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Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel

(Received 12 August 1975)

A specific radioimmunoassay for oestradiol was used to examine at which stage of postnatal development the ovary is capable of secreting oestrogen in response to stimulation by gonadotrophins, prostaglandin E2(PGE2), or the 8-bromo-derivative of cyclic AMP (8-Br-cAMP) in vitro.

Ovaries from Wistar-derived rats (6–9 days of age) were placed in incubation flasks (two ovaries per flask, at least six flasks for each experimental group) and incubated for 30 min at 37°C in 1 ml Krebs–Ringer bicarbonate buffer (KRB), pH 7·4, containing glucose (1 mg/ml) under an atmosphere of 95% O2 : 5% CO2. The medium was replaced by 1 ml fresh KRB, containing albumin (100 μg/ml), hormones, PGE2 or 8-Br-cAMP as indicated. The incubation was continued for another 4 h. The medium was extracted with 5 vol. of ether, and the ether extract was analysed for

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D Somjen, N Stern, E Knoll, O Sharon, B Gayer, T Kulik, and F Kohen

Carboxy derivatives of isoflavones that exhibit oestrogenic/anti-oestrogenic properties were used as carriers for affinity drug targeting to H295R adrenocortical carcinoma cells that express transcripts of oestrogen receptor (ER) alpha and beta. These derivatives were prepared by introducing a carboxymethyl group at the 6-position of genistein and of biochanin A, yielding 6CG and 6CB respectively. In transactivation assays, 6CG displayed mixed agonist/antagonist activity for ERalpha, whereas 6CB displayed only weak antagonist activity. Low concentrations of oestrogen, 6CG and 6CB were capable of inducing proliferation in H295R cells and of stimulating creatine kinase (CK) specific activity, suggesting that these cells were sensitive to oestrogenic compounds. In in vivo experiments, both 6CG and 6CB were capable of inhibiting oestrogen-induced CK specific activity in rat tIssues. For affinity drug targeting, the cytotoxic drug daunomycin was coupled to 6CB and 6CG, yielding 6CB-Dau and 6CG-Dau respectively. These conjugates were tested for their antiproliferative ability to inhibit DNA synthesis as assessed by incorporation of [(3)H]thymidine in H295R cells. A dose-dependent cytoxicity was observed with both conjugates. At 0.3-3 nM, both conjugates were 10 to 30 times more potent than daunomycin. At 30 nM these conjugates were two to three times more potent than daunomycin. At concentrations ranging between 300 and 3000 nM, no difference in cytotoxicity was observed between the conjugates and daunomycin. When the cells were treated over a wide range of concentrations with a combination of 6CG plus daunomycin, the observed cytotoxicity was less than with daunomycin alone. When non-transformed rat enterocytes, which do not express ER, were treated with 6CG-Dau or daunomycin, the antiproliferative effect of 6CG-Dau was the same as that of daunomycin over the concentration range tested. These pilot studies suggest that the ready availability of oestrogenic binding sites in H295R cells can be exploited for site-directed chemotherapy.

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D Somjen, F Kohen, B Gayer, T Kulik, E Knoll, and N Stern

We have reported previously that dihydrotestosterone (DHT) induces a biphasic effect on DNA synthesis in human vascular smooth muscle cells (VSMC), i.e. stimulation at low concentrations and inhibition at high concentrations. In contrast, DHT dose-dependently stimulated [(3)H]thymidine incorporation in a human endothelial cell line (ECV304). Additionally, DHT increased the specific activity of creatine kinase (CK) in both vascular cell types. In the present study, we have determined whether some of these effects are exerted via membrane-binding sites. We measured changes in DNA synthesis and CK after treatment with DHT and the membrane-impermeant testosterone-3-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (T-BSA). High concentrations of either DHT or T-BSA inhibited VSMC proliferation (by 52+22% and 51+25% respectively). DHT as well as T-BSA increased DNA synthesis in ECV304 cells dose-dependently. In contrast, T-BSA did not affect CK in either cell type. In both cell types, DHT as well as T-BSA increased mitogen-activated protein kinase (MAPK) kinase activity as measured by total phosphorylated MAPK. Further, the inhibitory effect of either the free or protein-bound androgens on DNA synthesis was blocked by UO126, an inhibitor of MAPK kinase activity. T-BSA conjugate labeled with Europium showed binding to whole VSMC, which could be displaced by excess T-BSA, but not by estradiol-BSA or the free hormones. Finally, using T-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for androgen in VSMC. Hence, the inhibitory effects of testosterone on DNA synthesis in VSMC are apparently exerted by membrane-binding sites for androgen, do not require intracellular entry of the hormone and its binding to the classical nuclear receptors and are linked to MAPK activation.

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D Sömjen, Y Amir-Zaltsman, B Gayer, G Mor, N Jaccard, Y Weisman, G Barnard, and F Kohen


Previous studies indicated that the anti-idiotypic antibody (clone 1D5) caused an increase in uterine creatine kinase (CK) activity when administered in vivo to immature female rats, indicating that the antibody has oestrogenic-like activity. It was, therefore, of interest to investigate the structural requirements of clone 1D5 to act as an oestrogen mimetic in an in vitro model system. In the present study, the effect of clone 1D5 and its proteolytic fragments, F(ab′)2, Fab′ and Fc on CK activity was examined in cultured skeletal cells having functional oestrogen receptor (ER). Incubation of female-derived calvaria cells or epiphyseal cartilage cells with clone 1D5 (8·33 nm) or oestradiol (E2) (30 nm) for 24 h caused a significant increase in CK activity, indicating that clone 1D5 acted as an agonist. On the other hand, incubation of male-derived calvaria cells devoid of a functional ER with clone 1D5 or E2 did not have any effect on CK activity. Incubation of female-derived calvaria cells with clone 1D5 and E2 did not result in any further increase in CK activity, whereas dihydrotestosterone (DHT) did not alter the response to clone 1D5. The CK response to clone 1D5, in female-derived calvaria cells was time- and dose-dependent and could be inhibited in a dose-dependent manner by the oestrogen antagonist tamoxifen. In contrast, the proteolytic fragments of clone 1D5, the F(ab′)2 dimer (12 nm) and the Fab′ monomer (24 nm), and the Fc fragment (28 nm) did not have E2-like activity in these cells. However, while the Fab′ monomer or the Fc fragment, as well as clone 1D5, did not affect the response of the female-derived calvaria cells to E2, the F(ab′)2 dimer acted like an antagonist and completely inhibited the stimulatory effect of E2 or 1D5, but was unable to block the stimulatory effect of DHT on CK in male-derived calvaria cells. Collectively, these results imply that a bivalent antibody is necessary for the observed physiological responses, and that the anti-idiotypic antibody can be converted from an agonist to an antagonist by removal of the Fc portion of the antibody molecule. Furthermore, the anti-idiotypic antibody has an oestrogenic-like effect inhibited by tamoxifen only in skeletal cells capable of responding to E2.

Journal of Endocrinology (1995) 145, 409–416

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D Somjen, Y Amir-Zaltsman, B Gayer, T Kulik, E Knoll, N Stern, LJ Lu, L Toldo, and F Kohen

The novel genistein (G) derivative, 6-carboxymethyl genistein (CG) was evaluated for its biological properties in comparison with G. Both compounds showed oestrogenic activity in vitro and in vivo. On the other hand G and CG differed in the following parameters: (i) only CG displayed mixed agonist-antagonist activity for oestrogen receptor (ER) alpha in transactivation assays and (ii) only CG was capable of attenuating oestrogen (E(2))-induced proliferation in vascular smooth muscle cells and of inhibiting oestrogen-induced creatine kinase (CK) specific activity in rat tissues. On the other hand only G enhanced the stimulatory effect on CK specific activity in the uterus. In comparison to the selective oestrogen receptor modulator (SERM) raloxifene (RAL), CG showed the same selectivity profile as RAL in blocking the CK response to E(2) in tissues derived from both immature and ovariectomized female rats. Molecular modelling of CG bound to the ligand binding domain (LBD) of ERbeta predicts that the 6-carboxymethyl group of CG almost fits the binding cavity. On the other hand, molecular modelling of CG bound to the LBD of ERalpha suggests that the carboxyl group of CG may perturb the end of Helix 11, eliciting a severe backbone change for Leu 525, and consequently induces a conformational change which could position Helix 12 in an antagonist conformation. This model supports the experimental findings that CG can act as a mixed agonist-antagonist when E(2) is bound to its receptors. Collectively, our findings suggest that CG can be considered a novel SERM with unique effects on the vasculature, bone and uterus.