Multihormonal control of cell proliferation: opposite effects of two stimulators (17 beta-estradiol and L-triiodothyronine) and one inhibitor (dexamethasone) on F4Z2 pituitary tumor cells.

From the MtTF4 tumor of rat pituitary origin we established the F4Z2 cell line whose growth is stimulated by 17 beta-estradiol (E2). Taking E2 actions as references we investigated actions of other effectors on the proliferation, protein, and insulin growth factor-I (IGF-I) secretions of F4F2 cells. Dexamethasone (Dex) and L-T3 were chosen because they have also intracellular receptors and they act in pituitary cells. Cells were cultured in 96-well plates in RPMI 1640 medium supplemented either with charcoal-treated fetal calf serum (CT-FCS) or with BSA and transferrin. Hormones were added at the time of seeding and cells were counted 2-10 days later without renewing the culture medium. The accumulation of immunoreactive IGF-I in conditioned medium was used as an index of IGF-I secretion. For studies on protein secretion, cells were incubated for 24 h with [35S]methionine and labeled proteins were separated by polyacrylamide gel electrophoresis. We found that: 1) L-T3, like E2, stimulated in a dose-dependent and specific manner the proliferation of F4Z2 cells cultured in the presence of 5% CT-FCS; EC50 was: 1 X 10(-11) M and 0.2 X 10(-11) M for L-T3 and E2, respectively. In contrast, L-T3 but not E2 remained active in serum-free medium; 2) Dex was a strong inhibitor of cell proliferation in serum-free medium and in medium supplemented with 5% CT-FCS (EC50: 5 X 10(-9) M). The antiglucocorticoid RU 38 486 prevented this inhibitory effect; 3) when a stimulator (E2 or L-T3) was simultaneously incubated with the inhibitor (Dex) the number of cells depended on the ratio of hormone concentrations. When there was no large excess of one effector this number was intermediary between those counted in the presence of each hormone separately and L-T3 was more potent than E2 in preventing Dex inhibition; 4) Dex, E2, and L-T3 modified the electrophoretic patterns of secreted proteins but there was no evidence for a correlation between these modifications and the inhibition or the stimulation of cell proliferation, and 5) the accumulation of immunoreactive IGF-I was insensitive to E2, increased by L-T3, and markedly decreased by Dex. L-T3 but not E2 prevented the effect of Dex.(ABSTRACT TRUNCATED AT 400 WORDS)

Estradiol stimulation and inhibition of cell growth in new estrogen-sensitive cell lines and tumors established from the MtTF4 tumor.

Cell lines were established from the MtTF4 tumor, growth of which is inhibited by estradiol, in order to determine whether the effect observed in vivo was due to a direct action on tumor cells. Two different cell lines were obtained according to the medium in which tumor cells were dispersed and cultured. The F4P cells were obtained when the culture medium contained charcoal-treated fetal calf serum. The growth rate of these cells was slowed down by 17 beta-estradiol in animals and also in culture during the early passages. Thereafter, they became insensitive to 17 beta-estradiol in culture but remained negatively controlled in vivo. These cells, whatever their sensitivity to 17 beta-estradiol, secrete prolactin and carry functional D2 dopamine binding sites. The F4Z cells were established in medium containing fetal calf serum not treated with charcoal. The growth rate of these cells was stimulated by 17 beta-estradiol in animals but was 17 beta-estradiol insensitive in culture up to subculture 26. At this time, the growth rate of the subline also became stimulated by 17 beta-estradiol in culture, and this phenotype was still found at passage 108 (50% effective dose, 5 to 10 pmol; maximum stimulation, 180 to 300% of control). These cells neither secrete measurable amounts of prolactin nor have dopamine binding sites. Thus, according to the medium in which cells were dispersed and cultured, two different cell strains were derived from a tumor in which growth is inhibited by 17 beta-estradiol. The point of interest is that the growth rate of one strain was inhibited by 17 beta-estradiol, while the other was stimulated. Convergent data suggest that MtTF4 tumor was heterogeneous and that selection had occurred during the dispersion or the culture of cells. Since the growth of one of these cell lines was slowed down transiently in culture we conclude that the inhibition of tumor growth could be due to a direct action of 17 beta-estradiol on tumor cells. However, the dissociation between the response to 17 beta-estradiol in culture and in the animal observed at some time of cell evolution suggests that environment affects the sensitivity of cells to 17 beta-estradiol.

Non-classical antiestrogenic actions of dexamethasone in variant MCF-7 human breast cancer cells in culture.

The aim of this work was to determine whether dexamethasone (Dex), a synthetic glucocorticoid, counteracts the stimulatory effects of estradiol (E2) on MCF-7 cells. We have shown that Dex inhibits in a dose-dependent fashion the estradiol-stimulated cell proliferation. This inhibition (ID50 congruent to 5-10 nM), which is complete at 100 nM Dex, is prevented by the antiglucocorticoid RU 486 and is clearly different from that found with trans-4-OH-tamoxifen because the inhibition due to a fixed concentration of Dex is not abolished by a high concentration of estradiol. This inhibitory effect displays some degree of specificity. Progesterone and the progestins R 5020 and ORG 2058 are without effect and Dex does not alter the triiodo-L-thyronine-stimulated cell growth. To characterize further the antiestrogenic action of Dex, the effects of this drug on specific responses to estradiol were studied. (1) Among the positive responses to estradiol two are prevented by Dex (the increase of concentration of progestin receptors and that of immunoreactive insulin-like growth factor I, IR-IGF-I, in conditioned medium) and two are insensitive to Dex (the enhancement of the secretion of 52,000 and 160,000 Mr proteins). (2) A negative response to estradiol (the down-regulation of estrogen receptor) is not prevented but rather accentuated by Dex. Thus, Dex counteracts the stimulatory effects of estradiol on the proliferation of MCF-7 cell variants characterized by progestin insensitivity. This non-classical antiestrogenic effect could be due in part to the attenuation of the E2-induced IR-IGF-I secretion and, less probably, to the accentuation of the down-regulation of E2 receptors. It could account for certain therapeutic and/or side effects of glucocorticoids on estrogen target cells.

Size heterogeneity of affinity labeled estrogen receptors in the MtTF4 tumor whose growth is inhibited by estradiol, in pituitary gland and uterus.

UNLABELLED: Estrogen receptors (ER) of the MtTF4 tumor whose growth is inhibited by estradiol (E2) were analyzed and compared to those of tissues whose growth is stimulated by E2 (uterus and pituitary gland). Cytosol prepared in buffer containing protease inhibitors was incubated with [3H]tamoxifen aziridine ([3H]TAZ) in the presence or absence of non-radioactive competitor. The labeled proteins were precipitated, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in denaturing conditions and detected by fluorography. Two classes of ER were identified. The first class is of high molecular weight (Mr = 65,000-64,000). In normal tissues, it is indeed frequently made up of two subtypes as revealed by the presence of a doublet on autoradiograms. In the MtTF4 tumor these subtypes were only rarely suspected and never they were as marked and distinct as in normal tissues. The second class, of low molecular weight (Mr ! 54,000-52,000), is also frequently made up of two subtypes in the uterus and the proportion of this class is higher in the uterus of mature than of immature rats. The MtTF4 tumor contains this class of ER but, due to the presence of non-specifically labeled proteins in this region, its relative amount cannot be estimated and the doublet was exceptionally revealed. In the pituitary gland, this small receptor has not been found. CONCLUSIONS: (i) On the basis of molecular weight analyses, estrogen receptors are heterogeneous, (ii) the ER pattern depends on the type of tissue and the sexual maturity of rats but all the tissues examined contained at least one type of the "classic" high molecular weight receptor, and (iii) no evident correlation was found between the ER pattern and the positive or negative response to estradiol.