afimoxifene and Pituitary-Neoplasms

This paper studies the modulatory effects of two antiestrogens, the steroid ICI 164 384 and the nonsteroidal compound 4-hydroxytamoxifen (4-OH-tam), on the proliferation of L-T3-stimulated cell lines. Three cell lines known to be stimulated by thyroid hormones and to contain estrogen receptors but to have a different estradiol sensitivity to estradiol were used; F4Z2 and MCF-7 cells were stimulated, whereas GH3 cells were insensitive. Cells were counted 6-7 days after hormones and antihormones were added to the culture medium, separately or in association (a fixed hormone concentration and increasing antihormone concentrations and vice versa). In F4Z2 and MCF-7 cells, antiestrogens prevented noncompetitively the stimulatory effect of L-T3 and, as expected, competitively the stimulatory effect of estradiol. In GH3 cells, antiestrogens had proper inhibitory effects, but they did not prevent significantly the proliferative effect of L-T3. To investigate the mechanisms of the modulatory effects in F4Z2 cells we examined the consequences of antiestrogens on thyroid hormone receptors (nuclear binding of L-T3 and mRNAs of thyroid hormone receptors, i.e. c-erbA alpha and -beta) and insulin-like growth factor-I (IGF-I; secretion and mRNAs). Antiestrogens neither competed with L-[125I]T3 nor reproducibly decreased the number and affinity of thyroid hormone-binding sites. While 4-OH-tam frequently decreased the amount of c-erbA beta transcripts, ICI 164 384 did not alter the distribution of alpha and beta cDNA transcripts. Further, neither antiestrogen prevented the increases in IGF-I accumulation in conditioned medium and IGF-I mRNA concentrations induced by L-T3 (0.1 nM). In conclusion, 1) antiestrogens are potent noncompetitive inhibitors of the action of L-T3 on the proliferation of cells whose growth is responsive to estradiol (F4Z2 and MCF-7), but not of the action on a cell line whose growth is insensitive to estradiol (GH3). 2) The loss of L-T3 sensitivity is not due to a loss of thyroid receptors or a decrease in IGF-I production. 3) In addition to estrogen receptors, factors involved in the estradiol control of cell proliferation appear to be required for the antiestrogen inhibition of L-T3 action.

Topics: Breast Neoplasms; Cell Division; DNA, Neoplasm; Estradiol; Estrogen Antagonists; Female; Humans; Insulin-Like Growth Factor I; Pituitary Neoplasms; Polyunsaturated Alkamides; Receptors, Estrogen; Receptors, Thyroid Hormone; RNA, Messenger; Tamoxifen; Transcription, Genetic; Triiodothyronine; Tumor Cells, Cultured

Chronic administration of the catecholestrogens 2-OH-estrone (2-OH1) and 2-OH-estradiol (2-OHE2), of tamoxifen and its metabolites and of high concentrations of estradiol have been previously shown to inhibit the growth of the estrogen/progesterone receptor-positive transplantable prolactin (PRL)-secreting rat pituitary tumor 7315a. The mechanism of action of these inhibitory effects on tumor growth is unknown. In the present study we investigated the direct effects of these compounds on PRL release by a tumor cell clone derived from the 7315a tumor. E2 stimulated PRL release in FCSABS (10% estrogen-stripped fetal calf serum)-cultured tumor cells in a biphasic manner: at low concentrations (0.1-100 nM) there was a dose-dependent stimulation of PRL release, which decreased in response to 1 microM E2 and which was greatly inhibited by 10 microM E2. Both 2-OHE2 (100 nM and 1 microM) and 2-OHE2 (1 microM) inhibited PRL release by FCS-cultured tumor cells. In FCSABS-cultured tumor cells, 0.1-10 nM 2-OHE1 and 1 microM 2-OHE2 inhibited PRL release, but 1-100 nM 2-OHE2 stimulated PRL release. Tamoxifen (TMX) and its metabolites dihydroxy (di-OH-TMX) and 4-hydroxytamoxifen (4-OH-TMX) inhibited PRL in a dose-dependent manner. The PRL release inhibiting effect of 4-OH-TMX was 100 times more potent that those of TMX and di-OH-TMX, which were similar in their effect. The inhibitory effects of micromolar concentrations of the catecholestrogens on PRL release could be overcome by estradiol, while the inhibitory effects of high concentrations of tamoxifen were not prevented by estradiol. Both "endogenous" (catecholestrogens) and "exogenous" (tamoxifen and its metabolites) antiestrogens and very high concentrations of estradiol directly inhibit PRL secretion by cultured pituitary tumor cells. The mechanism of their anti-tumor effects, however, seems to differ. The catecholestrogens have direct anti-estrogenic effects on cultured tumor cells, which can be antagonized by estradiol. The final effect of their mixed antagonistic/agonistic action depends on the presence or absence of estrogens in the culture medium. Tamoxifen also affects tumor growth probably mainly via a direct effect, partly involving anti-estrogenic and partly direct toxic effects.

Topics: Animals; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Estrogens, Catechol; Female; Hydroxyestrones; Pituitary Neoplasms; Prolactin; Rats; Rats, Inbred BUF; Secretory Rate; Tamoxifen; Tumor Cells, Cultured

Nuclear [3H]4-OHTAM-ER complexes extracted by 0.6 M KCl from the MCF-7 human breast cancer and the GH3 rat pituitary tumor cell lines, sedimented as a 5S form on sucrose gradients after 1 to 24 h exposure to ligand (20 nM). The nuclear [3H]4-OHTAM-ER from MCF-7 cells increased from 2 +/- 0.2 pmoles/mg DNA at 1 h to approximately 4 +/- 0.1 pmoles/mg DNA at 6 and 24 h. In the GH3 cells the nuclear binding of [3H]4-OHTAM increased rapidly, and there was no significant difference in the receptor levels at 1 and 6 h (10 +/- 1.3 and 8.9 +/- 1.1 pmoles/mg DNA, respectively). At 24 h there was a decrease in [3H]4-OHTAM-ER levels (7.0 +/- 0.2 pmoles/mg DNA); however, this was due primarily to an increase in DNA synthesis, which occurred in these cells by 24 h. It appears that in both cell lines there is no processing of nuclear [3H]4-OHTAM-ER, and it is not associated with changes in sedimentation coefficient of the complex. When both cell lines were incubated with [3H]E2 (20 nM), processing of the nuclear [3H]E2-ER occurred over 6 h. In the MCF-7 cells there was a decrease in receptor content within 6 h from 3.2 +/- 0.2 to 0.9 +/- 0.2 pmoles/mg DNA. The nuclear [3H]E2-ER in the GH3 cells decreased from 7.3 +/- 0.5 to 2.8 +/- 0.3 pmoles/mg DNA. Although these cell lines appeared to process the [3H]E2-ER complex, the nuclear forms of [3H]E2-ER were different.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Breast Neoplasms; Cell Line; Cell Nucleus; Centrifugation, Density Gradient; Estradiol; Female; Humans; Pituitary Neoplasms; Rats; Receptors, Estrogen; Tamoxifen; Time Factors

E2 (1 nM) stimulated the synthesis of PRL in GH3 cells. OH TAM (100 nM) did not affect basal PRL synthesis, but completely inhibited the increase produced by 1 nM E2. [3H]E2 and [3H]OH TAM both bound to the cytosolic 8S ER and these were split into 4S subunits on sucrose gradients containing 0.4 M KCl. By comparison, ER complexes extracted from nuclei of GH3 cells cultured in media containing [3H]E2 or [3H]OH TAM both sedimented at 5S on sucrose gradients containing 0.4 M KCl. Both 4S and 5S ER complexes were recognized by the monoclonal antibody D547 which increased their sedimentation coefficients to 8-9S. In contrast, a polyclonal antibody raised to calf uterine ER in the goat, interacted with the cytosolic ER so that the binding of [3H]E2 was inhibited but the binding of [3]OH TAM was only slightly reduced. A molecular model is proposed to describe the binding of E2 and OH TAM to the ER that might contribute to an understanding of estrogen and antiestrogen action.

Topics: Animals; Cell Line; Centrifugation, Density Gradient; Pituitary Neoplasms; Prolactin; Rats; Receptors, Estrogen; Tamoxifen