Steroid regulation of transfected genes in mouse mammary tumour cells

The regulation of mouse mammary tumour virus (MMTV) RNA by glucocorticoid hormones is well-established and has provided much information on how steroid hormones work. However, we have shown that androgens can also control MMTV RNA accumulation in S115 mouse mammary tumour cells. This novel androgen action could be explained on the basis that the MMTV long terminal repeat (LTR) can respond to several classes of steroid if appropriate receptors are present in the cells. We have used transfection experiments to demonstrate that androgens can act directly on the LTR in S115 cells. Hormonal regulation of transfected chimaeric genes into these cells was effected by androgen and glucocorticoid but not by oestrogen or progesterone, corresponding to the receptor status of the cells. Furthermore, hormonal control was also conferred by the LTR on expression of an independent cotransfected adjacent gene under its own separate promoter, suggesting that effects of an LTR can stretch to neighbouring genes in a type of hormone-enhancer insertion mechanism.     

Glucocorticoid hormone interactions with cloned proviral DNA of mouse mammary tumor virus

The molecular details of glucocorticoid hormone regulation of expression of the mouse mammary tumor virus (MMTV) proviral gene have been investigated. Cloned proviral DNA was introduced into cultured cells by a gene transfer procedure. DNA acquired by transfection was shown to be expressed in a hormone regulated fashion. The proviral DNA was fragmented and recombined in vitro with an indicator gene to delimit the hormone response sequence. Inducibility of the indicator gene (thymidine kinase gene from Herpes Simplex Virus, tk) was observed upon recombination with the long terminal repeat (LTR) sequence of MMTV. Further delimitation of the LTR DNA demonstrated that 202 nucleotides located 5' of the RNA initiation site are sufficient to confer glucocorticoid regulation. In vitro interaction of LTR DNA with glucocorticoid hormone receptor complex, showed a preferential affinity to the same sequence which mediated hormonal regulation in transfected cells. Evidence for a direct receptor gene interaction in the process of gene induction was gained by the measurement of the kinetics of induction and the use of a glucocorticoid antagonist (RU 486). The induction of the transfected gene is very rapid, independent of simultaneous protein synthesis and requires a functional glucocorticoid receptor hormone complex.     

Differential In Vivo Regulation of Steroid Hormone Receptor Activation by Cdc37p

The CDC37 gene is essential for the activity of p60^v-src when expressed in yeast cells. Since the activation pathway for p60^v-src and steroid hormone receptors is similar, the present study analyzed the hormone-dependent transactivation by androgen receptors and glucocorticoid receptors in yeast cells expressing a mutant version of the CDC37 gene. In this mutant, hormone-dependent transactivation by androgen receptors was defective at both permissive and restrictive temperatures, although transactivation by glucocorticoid receptors was mildly defective only at the restrictive temperature. Cdc37p appears to function via the androgen receptor ligand-binding domain, although it does not influence receptor hormone-binding affinity. Models for Cdc37p regulation of steroid hormone receptors are discussed.     

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.     

Medroxyprogesterone acetate: receptor binding and correlated effects on steroidogenesis in rat granulosa cells

Medroxyprogesterone acetate (MPA), a widely used synthetic steroid, was studied to determine both its effects on steroid receptors and steroidogenesis in the well-characterized rat ovarian granulosa cell model. Initial receptor binding studies showed MPA was as potent as progesterone and 10-fold less potent than R-5020 (an active synthetic progestin) in binding to progesterone cytosolic receptors in rat ovarian granulosa cells. MPA was 20-fold less potent than testosterone, and 10-fold less potent than dexamethasone in binding to the androgen and glucocorticoid cytosolic receptors, respectively. The binding of MPA to progestrone, androgen and glucocorticoid receptors predicted direct effects of MPA on FSH-stimulated estrogen (E), progesterone (P), and 20 alpha-dihydroprogesterone (DHP) production by cultured rat ovarian granulosa cells. MPA at 10(-7) to 10(-6) M significantly augmented FSH-stimulated P and DHP production (a previously documented progestin, androgen and glucocorticoid effect). This augmentation was blocked by the concurrent addition to cell culture of 10-fold excess RU-486 (a potent anti-progestin and anti-glucocorticoid). At concentrations greater than 10(-6) M, MPA inhibited the production of P and DHP (a progestin effect), and the production of E (a progestin and glucocorticoid effect). MPA, structurally a progestin, has complex steroid hormone effects predicted by its interaction with progesterone, androgen and glucocorticoid receptors.     

The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction.

Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk-promoter or the MMTV-promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone-responsive element in mammary cells.     

Arachidonic acid as a possible modulator of estrogen, progestin, androgen, and glucocorticoid receptors in the central and peripheral tissues

In an attempt to learn whether modulation of steroid hormone receptor by arachidonate is generalized or not, the arachidonate effect was examined in cytosol estrogen (ER), progestin (PR), androgen (AR) and glucocorticoid receptors (GCR) from the central and peripheral tissues of rats by sucrose density gradient centrifugation, and gel filtration on LH20 columns or dextran-coated charcoal absorption. Arachidonate and other long-chain fatty acids appear to inhibit the specific binding of estrogen ([3H]R2858), progestin ([3H]R5020), androgen ([3H]R1881) and glucocorticoid ([3H]dexamethasone) to the respective receptors in brain (neonatal cerebral cortex and hypothalamus-preoptic area, HPOA), uterus and prostate, with the exception of the potentiating effect on the brain estrogen receptors. The potency of the unsaturated fatty acids paralleled to some degree the number of cis double bonds and carbon, in that oleate (C18:1) arachidonate (C20:4) docosahexaenoate (C22:6). The arachidonate inhibition was dose-dependent in the tissue steroid hormone receptors, except for dose-dependent potentiation of the brain cortical estrogen receptors. Inhibitory potency as expressed by the concentration for 50% maximum inhibition (Ki) was in the range of 11-18 microM for the receptors other than the uterine estrogen receptors with the value of 44 microM, suggesting lower sensitivity for the estrogen receptor to the arachidonate effect in the uterus. Analysis on kinetics and Scatchard plot revealed the non-competitive type of the inhibition. In addition, arachidonate lowered dose-dependently the peak of labelled progestin or estrogen binding to the 8S receptor proteins, which were collected from fractions in the 8S region of the cytosols from intact or diethylstibestrol-primed rat uteri. These results suggest the generalized modulatory effect of arachidonate on the steroid hormone receptors in the central and peripheral tissues. Arachidonate could affect, negatively or positively, the estrogen receptors, and negatively the progestin, androgen and glucocorticoid receptors, through a possibly direct but weak binding at sites different from steroid binding sites on the receptor molecules. A potential messenger role of arachidonate itself has been implicated in the regulation or modulation of the steroid hormone receptors.     

Functional abnormality of glucocorticoid receptor in Shionogi carcinoma 115 cells as evidenced by gene transfer experiments.

The assay systems for steroid receptor functions in steroid-sensitive cells (SC-3 cells) were developed in which hormone-responsive element linked to a reporter gene [chloramphenicol acetyl transferase (CAT) gene] was transfected by the electroporation technique. Stimulation with androgen of SC-3 cells transfected with mouse mammary tumor virus promoter-CAT gene (MMTV-CAT) resulted in clear enhancement of CAT activity, whereas glucocorticoid required abnormally high concentrations to obtain significant stimulation. The simultaneous addition of glucocorticoid surprisingly inhibited androgen-induced CAT activity in SC-3 cells, whereas glucocorticoid and androgen acted together synergistically to activate CAT activity in T 47D cells. When SC-3 cells were cotransfected with the expression vector of human glucocorticoid receptor (GR) gene, inhibition with glucocorticoid of androgen-enhanced CAT activity was abolished. These results would suggest that SC-3 cells contain functionally abnormal GR.     

The Mitochondrion as a Primary Site of Action of Glucocorticoids: Mitochondrial Nucleotide Sequences,Showing Similarity to Hormone Response Elements,Confer Dexamethasone Inducibility to Chimaeric Genes Transfected in LATK−Cells

The hypothesis of a primary action of steroid hormones on mitochondrial gene expression has been supported by the detection of the glucocorticoid receptor in liver mitochondria and the demonstration of the interaction of the receptor with putative mitochondrial HREs. We now show that two putative mitochondrial glucocorticoid response elements present within the cytochrome oxidase subunit I gene (GREI and GREII), linked to a thymidine kinase promoter and to the CAT gene, transfected to LATK⁻cells, confer dexamethasone inducibility to the CAT gene. As the plasmids were stably transfected, hormone induction was analysed in the nuclear background. This effect is dose dependent and is abolished by the glucocorticoid antagonist RU38486.