Transition of human breast cancer cells from an oestrogen responsive to unresponsive state

An in vitro model system is described for studying the problem of loss of steroid sensitivity in breast cancer cells. Growth of cloned oestrogen-sensitive human breast cancer cells in the long-term absence of steroid gives rise to a population of oestrogen-insensitive cells. In ZR-75-1 cells, the effect is clonal but occurs at high frequency suggesting a mechanism affecting a wide proportion of the cell population synchronously. This does not involve any reduction in oestrogen receptor number. Furthermore, there is no coordinated loss of oestrogen-sensitive molecular markers, showing that oestrogen receptors remain not only present but functional. These growth changes are not accompanied by any loss of growth inhibition by antioestrogen. Although steroid deprivation does not result in loss of oestrogen-sensitive markers, this does not hold true for other steroids. There was a reduction in progestin, androgen and glucocorticoid regulation on transfected LTRs. Loss of steroid-sensitive growth was accompanied by changes in response to exogenous growth factors and altered endogenous growth factor mRNA production. Steroid-deprived T-47-D cells acquire sensitivity to stimulation by TGFβ and have raised TGFβ₁ and TGFβ₂ mRNA levels. ZR-75-1 cells are growth inhibited by TGFβ and have reduced TGFβ₁mRNA levels. In MCF-7 cells, increased IGFII mRNA, following transfection, can result in an increased basal cell growth rate in the absence of steroid. These findings are discussed in relation to possible autocrine mechanisms in the loss of steroid sensitivity of breast cancer cells.     

Differential, multihormonal regulation of the mouse major urinary protein gene family in the liver.

The hormonal requirements for the regulation of the major urinary protein (MUP) mRNA levels in mouse liver have been examined. Previous experiments have shown that administration of testosterone to female or castrated male mice increases MUP mRNA levels approximately fivefold to normal male levels. We have found that thyroxine and the peptide hormone, growth hormone, each had a pronounced effect on MUP mRNA levels. MUP mRNA was reduced 150-fold in growth-hormone-deficient mutant mice (little). The administration of growth hormone and thyroxine induced MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. testosterone administration. When administered separately to these mice, growth hormone and thyroxine induced with MUP mRNA approximately 150-fold, and when administered together, they induced MUP mRNA approximately 1,000-fold. Testicular feminized mice, which lack a functional major testosterone receptor protein, can also be induced to male levels by treatment with both growth hormone and thyroxine. In addition, we present evidence which indicates that growth hormone, thyroxine, and testosterone differentially regulate the levels of distinct MUP mRNA species.     

Effects of growth and insulin treatment on the levels of insulin receptors and their mRNA in Hep G2 cells

We have studied the variations in the number of insulin receptor and insulin receptor mRNA levels in (Hep G2) cells in response to growth and insulin treatment. The levels of insulin receptors are relatively low in growing cells. After approximately 5 days in culture, if cells are not refed they cease to divide and the number of receptors/cell increases, reaching 4 times the initial values by the 9th day. Refeeding the cells completely prevented both growth arrest and the increase in insulin receptor number. Insulin added daily to cells at 0.33 microM caused receptor down-regulation but did not prevent a 3-fold increase in binding with growth arrest. Pulse-chase studies of metabolically labeled ([35S]methionine) cells showed that the receptor degradation rate (apparent t 1/2, 18-20 h) was comparable in rapidly growing versus growth-arrested cells. The increased receptor level in non-refed cells is not due to generation of a soluble factor by confluent cells, nor is it caused by depletion of insulin, glucose, or insulin-like growth factor I from the culture medium. The levels of insulin receptor mRNA measured on Northern blots increased in growth-arrested cells in parallel to the increase in receptor number. The mRNA value begins to increase from the 3rd day in culture and by the 9th day reaches a level 6.0 times that on the 3rd day. Chronic insulin-induced receptor down-regulation did not alter insulin receptor mRNA levels at any time point studied. These data demonstrate that the increase in insulin receptor number/cell in growth-arrested cells is paralleled by an increase in insulin receptor mRNA content with no change in the receptor degradation rates. This suggests that the increase in the number of insulin receptors is due to enhanced receptor synthesis due to increased receptor mRNA content. Conversely, down-regulation of the insulin receptor does not affect the level of insulin receptor mRNA and thus must be due to increased receptor degradation.     

Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.     

Stimulatory role of dopamine on fibroblast growth factor-2 expression in rat striatum

We have previously shown that systemic injection of (-)nicotine produces a selective up-regulation of fibroblast growth factor (FGF)-2 mRNA levels in rat striatum. Because (-)nicotine can increase striatal release of dopamine and glutamate, in the present study we have investigated the contribution of these neurotransmitters in the modulation of FGF-2 expression. We found that coinjection of dopaminergic D1 (SCH23390) or D2 (haloperidol) receptor antagonists prevents nicotine-induced elevation of FGF-2 expression. However, injection of the NMDA receptor antagonist MK-801 produced a significant increment of FGF-2 mRNA and protein levels in rat striatum similar to the effect produced by (-)nicotine alone. Interestingly this effect of MK-801 could also be prevented by D1 or D2 receptor antagonists, suggesting that an elevation of dopamine levels may be required for the regulation of the trophic molecule. Accordingly we found that the non-selective dopaminergic agonist apomorphine can similarly increase striatal FGF-2 mRNA levels. Despite the observation that both D1 and D2 receptors appear to contribute to the modulation of FGF-2 expression, only a direct activation of D2 receptors, through quinpirole administration, was able to mimic the effect of apomorphine. On the basis of FGF-2 neurotrophic activity, these results suggest that direct or indirect activation of dopaminergic system can be neuroprotective and might reduce cell vulnerability in degenerative disorders.     

Estrogen regulation of epidermal growth factor receptor messenger ribonucleic acid

Previous studies have demonstrated that 17 beta-estradiol (E2) causes a 3-fold increase in epidermal growth factor (EGF) receptors in uterine membranes. We now report that the increase in uterine EGF receptor levels is due to an increase in the steady-state levels of EGF receptor mRNA. After a single E2 injection, EGF receptor mRNA levels, as determined by RNA blots, increase 3- to 4-fold between 1 and 3 h, remain elevated at 6 h, and decline between 12 and 18 h. The effect is specific for E2 since the nonestrogenic hormones progesterone, dexamethasone, 5 alpha-dihydrotestosterone, and the inactive stereoisomer of E2, 17 alpha-estradiol, are without effect. E2-Mediated increases in EGF receptor mRNA levels are blocked by actinomycin D but not by puromycin. Taken together, these results indicate that E2 regulates the level of EGF receptor by increasing the steady-state concentration of EGF receptor mRNA in vivo.     

Increases in cytosolic Ca++ down regulate thyrotropin receptor gene expression by a mechanism different from the cAMP signal.

Thyrotropin (TSH) receptor mRNA levels in rat FRTL-5 thyroid cells are decreased by treatment with the calcium ionophores, A23187 or ionomycin, as well as with TSH, cholera toxin, forskolin, and 8-bromo-cAMP. Down regulation is, in each case, associated with a decrease in [125I]TSH binding and a decreased ability of TSH to increase cAMP levels. The ionophore does not alter cAMP levels and ethylene glycol-bis-(beta-aminoethyl ether) N, N'-tetraacetic acid (EGTA) in the medium prevents down regulation of TSH receptor mRNA levels by the ionophore, but not by TSH; the EGTA action is reversed by the simultaneous addition of Ca++. Whereas down regulation by TSH and its cAMP signal requires the presence of insulin and/or serum in the medium; down regulation by a calcium ionophore is still evident in their absence. Down regulation of TSH receptor mRNA levels and receptor desensitization by TSH/cAMP or an ionophore is lost in cells transfected with a full length TSH receptor cDNA devoid of regulatory elements, but able to reconstitute TSH receptor signal generation.