The effects of estrogen on prolactin gene methylation in normal and neoplastic rat pituitary tissues.

The effects of estrogen treatment on rat prolactin (PRL) gene methylation were analyzed in normal pituitaries and in three transplantable rat pituitary tumors. Northern analysis showed increased PRL mRNA expression in estrogen-treated pituitary and in MtT/F4 and MtT/F-DMBA tumors. Prolactin mRNA amounts in MtT/W15 tumor were decreased by estrogen treatment. There was an inverse relationship between changes in PRL mRNA expression and changes in gene methylation in the coding regions of the PRL gene after estrogen treatment. The amounts of the 4.6-Kb and 1.8-Kb restriction fragments generated by HpaII digestion in pituitary tissues were influenced by estrogen, with an increase in these fragments in normal pituitary, MtT/F4, and MtT/F-DMBA tumors after estrogen treatment. In contrast, the amounts of the 4.6-Kb and 1.8-Kb fragments were decreased in MtT/W15 tumors after estrogen treatment. Most of the internal -CCGG- sites in the PRL gene were methylated in the liver, and the PRL gene was not expressed in the liver. These data suggest that there is a tissue-specific pattern of DNA methylation of the PRL gene and that PRL gene methylation is influenced by estrogen in vivo in normal and tumorous pituitary tissues. These results also suggest that estrogen may influence PRL expression by multiple mechanisms, including changes in the level of DNA methylation.     

Detection of prolactin messenger RNA in rat anterior pituitary by in situ hybridization.

The effects of chronic diethylstilbestrol treatment on rat prolactin mRNA was analyzed by in situ hybridization histochemistry. Forty-day-old female rats were treated with 10 mg diethylstilbestrol in Silastic tubes for 3, 6, and 9 weeks. Estrogen treatment for 9 weeks increased pituitary wet weight (51.6 +/- 2.4 versus 7.9 +/- 0.31 mg for controls), serum prolactin (4155 +/- 571 versus 47.1 +/- 8.9 ng/ml for controls), and the percentage of immunoreactive prolactin cells (69% +/- 3% versus 34% +/- 2% for controls). In situ hybridization studies showed an increase in rat prolactin mRNA with increasing duration of estrogen treatment. After 9 weeks of estrogen treatment, there was a 2.3-fold increase in rat prolactin mRNA. 3H-cDNA was distributed diffusely throughout the anterior pituitary in both normal and hyperplastic pituitaries. There were no separate foci of adenomatous pituitary with increased labeling or with increased immunoreactive PRL cells. Although transplantable pituitary MtT/W15 tumors secreted very large amounts of PRL, compared with pituitaries from DES-treated rats, rat prolactin mRNA as evaluated by mean grain counts was considerably less in the MtT/W15 tumor than in DES-treated pituitary cells. These results show that in situ hybridization histochemistry can be used to detect changes in rat prolactin mRNA in tissue sections from the anterior pituitary with chronic estrogen treatment and that these pituitaries show a diffuse increase in immunoreactive prolactin cells and cellular prolactin mRNA, rather than distinct adenomatous areas within the glands.     

TGF-B and Estrogen Regulate P27(Kip1) and Cyclin D(1) in Normal and Neoplastic Rat Pituitary Cells

Pituitary hyperplasia and tumor growth are regulated by various hormones and growth factors. Estrogen (E₂) stimulates pituitary cell proliferation and prolactin (PRL) production. Estrogen also regulates transforming growth factor-β (TGF-β) effects in the pituitary. TGF-β in turn regulates various cell cycle proteins including p15 and p27^Kip1 (p27). To better understand the regulatory role of growth factors and hormones in the cell cycle we analyzed cyclin D₁, cyclin E, and p27 expression in normal and neoplastic rat pituitary cells. An in vitro analysis using cultured normal pituitary cells and GH₃ tumor cells and an in vivo analysis of estrogen-treated normal pituitary and implanted GH₃ cells were performed. Semiquantitative RT-PCR was used to analyze mRNA expression for cyclin D₁, cyclin E, and p27 in cultured pituitary cells and E₂-treated pituitaries in vivo. Cyclin D₁ and p27 were localized in the nuclei of normal pituitary cells by immunocytochemistry (ICC). Very weak or absent immunostaining for cyclin D₁ and p27 was present in GH₃ cells. Both normal pituitary and GH₃ cells had strong nuclear staining for cyclin E. Normal pituitary had a 20-fold greater amount of cyclin D₁ mRNA and a 3-fold greater amount of p27 mRNA compared to GH₃ cells, whereas GH₃ cells had slightly (1.5-fold) more cyclin E than normal pituitary cells. E₂ treatment in vivo stimulated cell proliferation and decreased cyclin D₁ mRNA levels in normal pituitary. GH₃ tumor cells, implanted subcutaneously in the same animal, showed increased proliferation after E₂ treatment, but there was no change in cyclin D₁ mRNA in GH₃ cells. Cyclin E and p27 mRNA levels did not change significantly in normal pituitary or in GH₃ cells after E₂ treatment in vivo. Treatment of normal pituitary cells with 10⁻⁹ M TGF-β1 for 3 d in vitro led to significant decreases in cyclin D₁ and p27 mRNAs (p < 0.05), whereas cyclin E levels were unchanged. These results indicate that cyclin D₁ and p27 mRNAs are present at significantly higher levels in normal pituitary compared to GH₃ cells, and that both E₂ and TGF-β1 can downregulate cyclin D₁ mRNA levels in normal pituitary cells, suggesting that these factors regulate G1 to S phase transition in pituitary cells. The lower levels of specific cell cycle regulators in GH₃ cells may explain the decreased regulatory control by E₂ in GH₃ tumor cells.     

Analysis of S-100 protein positive folliculo-stellate cells in rat pituitary tissues.

The effects of diethylstilbestrol (DES) treatment and withdrawal on the folliculo-stellate (FS) cells in hyperplastic pituitaries were examined in Fischer 344 (F344) and Wistar Furth (W/F) rats by immunochemistry and electron microscopy. The presence of S-100 protein positive cells was examined by immunostaining in spontaneous and in transplantable rat pituitary tumors. The pituitaries of F344 rats showed a fivefold greater increase in weight in response to 5 weeks of DES treatment compared to pituitaries from W/F rats. S-100 protein (S-100) positive cells were significantly increased in both strains with hyperplastic pituitaries (P less than 0.05) 2 days after DES withdrawal. Ultrastructural studies revealed increased phagocytic activity in FS cells. S-100 positive cells were absent in both MtT/W15 and MtT/F4 transplantable tumors even after treatment with DES. Some spontaneous pituitary tumors in aged female rats had S-100 positive cells within the tumors at the periphery of the tumor nodules (3 of 12 cases), while most of these neoplasms did not contain S-100 positive cells. Incubation of normal dissociated pituitary cells with S-100 protein increased PRL secretion in vitro. The effectiveness of S-100 protein in increasing PRL secretion in vitro was less than thyrotropin releasing hormone (TRH). These results indicate that S-100 positive cells are present in normal and hyperplastic pituitaries, but not in spontaneous or in transplantable rat pituitary tumors and also suggest that the FS cells may exert a paracrine type regulation on PRL secretion.     

The effects of estrogens on tumor growth and on prolactin and growth hormone mRNA expression in rat pituitary tissues.

Estrogens inhibit tumor growth and modify PRL and GH expression in the MtT/W15 transplantable rat pituitary tumor. The effects of estradiol (E2) and diethylstilbestrol (DES) on PRL and GH mRNA levels were investigated. Estrogens increased GH mRNA levels and decreased PRL mRNA levels as detected by in situ hybridization and Northern blot hybridization with oligonucleotide probes, while inhibiting tumor growth. Similar changes in immunoreactive GH and PRL were seen in the tumor cells. The pituitary glands of tumor-bearing rats treated with estrogen for 3 weeks were increased in weight with a concurrent increase in pituitary PRL mRNA when analyzed by dot blot hybridization. These results indicate that estrogens have an inhibitory effect on the growth of the MtT/W15 tumor and increase GH protein and mRNA levels, while causing PRL protein and mRNA levels to decrease. The pituitaries of tumor-bearing rats concomitantly undergo PRL cell hyperplasia with an increase in PRL mRNA. These results also demonstrate a paradoxical effect of estrogens on different pituitary tissues.     

Regulation of prolactin gene expression in a DMBA-estrogen-induced transplantable rat pituitary tumor.

A new transplantable rat pituitary tumor was induced in F344 female rats with dimethylbenz(a)anthracene and estrogen (MtT/F-DMBA) and studied for 20 serial transplant generations. The tumor grew without estrogen supplements in female rats by the second transplant generation. Sensitivity to estrogens, as indicated by a prolonged latency period for tumor development, was seen at the 20th, but not the 5th transplant generation. MtT/F-DMBA tumors expressed prolactin (PRL), growth hormone (GH), and adrenocorticotropin (ACTH) mRNAs. A decrease in the percentage of cells expressing PRL mRNA, PRL protein, and in the number of secretory granules per cell occurred with serial transplantation. S-100 protein-positive folliculostellate cells were present in the hyperplastic pituitary but not in the transplantable tumors. Estrogen treatment at the 20th transplant generation prolonged the tumor latency period, increased the number of cells expressing PRL mRNA greater than 5-fold by in situ hybridization analysis (14 +/- 2% versus 77 +/- 5%), increased PRL secretion (132 +/- 40 ng/ml versus 3762 +/- 890 ng/ml), and increased the number of cytoplasmic secretory granules per cell. These results indicate that hyperplastic pituitary and true pituitary neoplasms differ in their ability to grow readily after transplantation. The presence of S-100 protein-positive folliculostellate cells, which are present in hyperplastic but not in neoplastic pituitary tissues, may serve as a morphologic marker to separate hyperplastic and neoplastic rat pituitary tissues. Transplantable tumors remained responsive to estrogen with expression of a more differentiated phenotype, including an increased number of cells expressing PRL mRNA and increased numbers of PRL secretory granules.     

Analysis of prolactin and growth hormone production in the MtT/F4 transplantable pituitary tumor by the reverse hemolytic plaque assay.

The reverse hemolytic plaque assay (RHPA) was used to detect hormone secretion from normal pituitary cells and from the transplantable MtT/F4 pituitary tumor cells. Aliquots of the same cell suspensions were analyzed by immunocytochemistry (ICC). Normal pituitaries had more growth hormone (GH)-producing cells than tumors when analyzed by both the RHPA and ICC. However, the MtT/F4 tumor had significantly more prolactin (PRL)-secreting cells. Mammosomatotropic (MS) cells, which produced both PRL and GH, were identified in both normal and tumorous pituitaries with the RHPA and ICC. A combined procedure of RHPA followed by ICC staining on the same slide also revealed MS cells in both normal and tumorous pituitary cells, although the percentage of MS with this technique was less than with the other two methods. These results show that MS cells from a significant population of cells in the MtT/F4 tumor and that the RHPA and ICC can be used to study the regulation of this cell type.     

DNA Methylation Regulates p27Kip1 Expression in Rodent Pituitary Cell Lines

We previously reported loss of expression of p27^Kip1 (p27) protein in rat GH₃ and mouse GHRH-CL1 pituitary tumor cells compared with normal pituitary (NP). The molecular basis for the loss of expression of p27 protein in GH₃ and GHRH-CL1 cells is unknown. To determine the role of p27 gene methylation in the regulation of the expression of this cell cycle protein, the methylation patterns of p27 in normal and neoplastic pituitary cells was analyzed. Inhibition of DNA methyltransferase (DNA-MTase) with 5-aza-2′-deoxycytidine (AZAdC) induced expression of both p27 protein and mRNA when GH₃ and GHRH-CL1 cells were treated for 7 days in vitro. DNA methylation correlated inversely with the expression of p27 gene products in NP and pituitary tumor cell lines. Bisulfite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH₃ and GHRH-CL1 cells. After treatment of GH₃ and GHRH-CL1 cells with 10 μmol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%) with variable methylation patterns observed by bisulfite genomic sequencing. Analysis of genomic DNA with methylation-sensitive enzymes showed that all SmaI, HhaI, and AvaI enzyme sites of the p27 gene in exon 1 were methylated in GH₃ cells but not in NP, confirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed abundant p27, had a methylation pattern similar to the NP. DNA-MTase activity was elevated fourfold in GH₃ cells and twofold in GHRH-CL1 cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silencing of the p27 gene in some pituitary tumors and possibly in other types of neoplasms.     

Distribution of estrogen receptor β mRNA-containing cells in ovariectomized and estrogen-treated female rat brain

Estrogen receptor (ER)-β is a member of the nuclear receptor superfamily and mediates various estrogenic actions. Changes in ER-α mRNA expression induced by estrogen have been well documented, whereas those with regard to ER-β have only been reported for a part of the hypothalamus. In the present study, we examined the effect of estrogen on ER-β mRNA expression in the female rat brain. Detection of ER-β mRNA using the in situ hybridization method with a digoxigenin-labeled RNA probe was performed in two groups of female rats: ovariectomized (OVX) and estrogen (E₂)-treated. A wide distribution of ER-β mRNA-containing cells was demonstrated in both groups. In the E₂-treated group compared with the OVX group, the number of ER-β mRNA-containing cells was significantly reduced in the external plexiform layer of the olfactory bulb, entorhinal cortex, intermediate part of the lateral septal nucleus, nucleus of the horizontal limb of the diagonal band, amygdala (lateral, medial and basolateral part), thalamus (anteroventral, laterodorsal and lateral posterior part), medial geniculate nucleus, suprachiasmatic nucleus and Purkinje cells in the cerebellum. These results reveal that ER-β mRNA-containing cells were decreased by estrogen in several brain regions in the female rat brain, suggesting that ER-β mRNA is downregulated by the physiological level of estrogen in a region-specific manner.     

Estrogen modulates central and peripheral responses to cold in female rats

The aim of this study was to determine whether estrogen modulates central and peripheral responses to cold in female rats. In ovariectomized female rats with and without administered estrogen [E₂ (+) and E₂ (−), respectively], the counts of cFos-immunoreactive cells in the medial preoptic nucleus (MPO) and dorsomedial hypothalamic nucleus (DMH) in the hypothalamus were greater in the E₂ (+) rats than in the E₂ (−) rats at 5°C. Examination of the response of normal female rats to exposure to 5°C at different phases of the estrus cycle revealed that counts of cFos-immunoreactive cells in the MPO, DMH, and posterior hypothalamus and the level of uncoupling protein 1 mRNA in the brown adipose tissues were greater in the proestrus phase than on day 1 of the diestrus phase. This result was linked to the level of plasma estrogen. The body temperature during cold exposure was higher in the E₂ (+) rats than in the E₂ (−) rats and was also higher in the proestrus phase than on day 1 of the diestrus phase. We conclude that estrogen may affect central and peripheral responses involved in thermoregulation in the cold.     

Effects of estrogens on pituitary cell and pituitary tumor growth

Estrogens have been known to induce PRL cell hyperplasia in the anterior pituitary of some species for many decades. Recent studies have shown variable susceptibility to estrogen-induced hyperplasia in different strains of rats. The distinction between hyperplastic pituitaries and adenomas is usually not made by most investigators in this field, although true neoplasms can usually be propagated by serial transplantation. The growth of transplantable tumors is usually inhibited by estrogen in vivo. Estrogens have a biphasic effect on pituitary cell proliferation in vitro with higher concentrations of estradiol inhibit cell growth, and lower concentrations stimulating PRL secretion. Estrogens can regulate PRL gene methylation in vivo thus affecting PRL mRNA expression. Recent studies have suggested that estrogen regulates signal transduction by stimulating protein kinase C. Estrogens also regulate specific proto-oncogenes such as c-myc and c-fos. These observations may help to explain some of the regulatory effects of estrogens on cell proliferation and tumor development.     

雌激素及其受体对低氧性肺血管重建的作用

 目的：探讨雌激素及其受体对低氧性肺血管重建的作用。 方法:采用间断常压低氧法建立大鼠低氧性肺动脉高压模型。将30只雄性SD大鼠随机分成：对照组、雌激素预防组、低氧组、雌激素治疗组、治疗对照组。以右心导管法测定平均肺动脉压(mPAP)；测定右心室肥厚指数［RV/(LV+S)］；图像分析技术测定肺小血管管壁厚度；用放射免疫法测定血清17-β雌二醇浓度。用RT-PCR方法检测肺组织中ERβ mRNA的表达，用免疫组化染色法检测肺小血管壁的ERβ蛋白表达。 结果:①低氧组、治疗对照组大鼠mPAP、RV/(LV+S)、WT%和WA%均高于对照组；雌激素预防组、雌激素治疗组大鼠mPAP、RV/(LV+S)、WT%和WA%均低于低氧组、治疗对照组。②低氧组、治疗对照组大鼠ERβ mRNA表达量和ERβ蛋白表达与正常对照组无差异。经雌激素干预后，雌激素预防组、雌激素治疗组的ERβ mRNA表达和ERβ蛋白表达显著高于低氧组、治疗对照组。 结论:17-β雌二醇能有效降低低氧性肺动脉高压大鼠的肺动脉压力、阻抑右心室肥厚,对低氧性肺动脉高压血管重建具有一定的预防和治疗作用，其作用可能是通过雌激素受体β实现的。     

Estrogen action: A historic perspective on the implications of considering alternative approaches

In the 50 years since the initial reports of a cognate estrogen receptor (ER), much has been learned about the diverse effects and mechanisms of estrogens, such as 17β-estradiol (E₂). This expert narrative review briefly summarizes perspectives and/or recent work of the authors, who have been addressing different aspects of estrogen action, but take a common approach of using alternative considerations to gain insight into mechanisms with clinical relevance, and inform future studies, regarding estrogen action. Their “Top Ten” favorite alternatives that are discussed herein are as follows. 1 — E₂ has actions by binding to a receptor that do not require its enzymatic conversion. 2 — Using a different strategy for antibody binding could make the estrogen receptor (ER) more discernible. 3 — Blocking ERs, rather than E₂ production, may be a useful strategy for breast cancer therapy. 4 — Secretion of α-fetoprotein (AFP), rather than only levels of E₂ and/or progesterone, may influence breast cancer risk. 5 — A peptide derived from the active site of AFP can produce the same benefits of the entire endogenous protein in endocrine cancers. 6 — Differential distribution of ER subtypes in the body and brain may underlie specific effects of estrogens. 7 — ERβ may be sufficient for the trophic effects of estrogen in the brain, and ERα may be the primary target of trophic effects in the body. 8 — ERβ may play a role in the trophic effects of androgens, and may also be relevant in the periphery. 9 — Downstream of E₂'s effects at ERβ, there may be consequences for biosynthesis of progestogens and/or androgens. 10 — Changes in histones and/or other factors, which may be downstream of ERβ, potentially underlie the divergent effects of E₂ in the brain and peripheral tissues.     

Evidence That Folliculo-Stellate Cells Mediate the Inhibitory Effect of Japanese Kampo Medicine,Unkei-To,on Growth Hormone Secretion in Rat Anterior Pituitary Cell Cultures

PROBLEM: Cytokine-induced neutrophil chemoattractant (CINC) was reported to influence anterior pituitary hormone release. We recently found that Unkei-to, one of the Japanese Kampo medicines, stimulated CINC secretion by rat anterior pituitary cells and the pituitary folliculo-stellate (FS)-like cell line (TtT/GF). Therefore, the effect of Unkei-to on growth hormone (GH) secretion by rat anterior pituitary cells was investigated. METHOD OF STUDY: Dispersed normal anterior pituitary cells, the folliculo-stellate-like cell line TtT/GF, and the GH₃ cell were used to test the effect of Unkei-to on GH secretion. In reconstitutive coculture experiments, TtT/GF cells were mixed with GH₃ cells at a ratio (TtT/GF cells: GH₃ cells) of 1:99. From this mixture, cells were seeded onto plates at a density of 10⁴ cells/well and were cultured for 5 days. The cells were then used in the experiments. RESULTS: Unkei-to at 20 μg/ml significantly inhibited GH secretion by normal anterior ptuitary cells within 12 hr of incubation. In contrast Unkei-to stimulated GH secretion by GH₃ cells in a time- and dose-dependent manner, suggesting that an accessory cell type was involved. To assess the contribution of CINC as a paracrine factor, an experiment using a reconstitutive coculture system was performed, and Unkei-to was found to inhibit GH secretion when GH₃ cells were cocultured with TtT/GF cells. The addition of anti-CINC antibody to the reconstitutive coculture system antagonized Unkei-to-inhibited GH secretion. CONCLUSION: CINC, which was secreted from FS cells by Unkei-to, may be responsible for mediating the inhibitory effect of Unkei-to on GH secretion by rat anterior pituitary cells.     

The Expression of Wnt4 Is Regulated by Estrogen via an Estrogen Receptor Alpha-dependent Pathway in Rat Pituitary Growth Hormone-producing Cells

Wnt signaling is important in many aspects of cell biology and development. In the mouse female reproductive tract, Wnt4, Wnt5a, and Wnt7a show differential expression during the estrus cycle, suggesting that they participate in female reproductive physiology. Although the pituitary is a major gland regulating reproduction, the molecular mechanism of Wnt signaling here is unclear. We elucidated the subcellular distribution of Wnt4 in the pituitary of estrogen-treated ovariectomized female rats. Expression of Wnt4 mRNA increased dramatically, particularly in proestrus compared with estrus and metestrus. Wnt4 protein was observed in the cytoplasm of almost all growth hormone (GH)-producing cells and in only a few thyroid-stimulating hormone β (TSHβ)-producing cells. In rat GH-producing pituitary tumor (MtT/S) cells, estrogen-induced expression of Wnt4 mRNA was completely inhibited by estrogen receptor antagonist ICI 182,780 in vitro. Thus, rat pituitary GH cells synthesize Wnt4 and this is induced by estrogen mediated via an estrogen receptor alpha-dependent pathway.     

Aberrant DNA methylation of cyclin D2 and p27 genes in rodent pituitary tumor cell lines correlates with specific gene expression

We previously reported that increased DNA methylation was an important mechanism of silencing the p27 gene in some pituitary tumor cell lines [1]. DNA methylation correlated inversely with p27 gene expression. The p27 and cyclin D2 genes are located in the same region of mouse chromosome 6, rat chromosome 4, and human chromosome 12p13. Because both genes are located in the same gene cluster, we investigated whether methylation was a principal mechanism regulating cyclin D2 as well as p27 expression in rodent pituitary cell lines. Bisulfite genomic sequencing showed that the normally unmethylated cytosines of the p27 gene in normal pituitary (NP) were extensively methylated in GH3 and GHRH-CL1 cells, but not in AtT 20, αT₃-1 and LβT₂ cells; but cyclin D2 was extensively inactivated in various pituitary tumor cell lines by increased DNA methylation. These abnormalities of methylation in p27 and cyclin D2 genes occurred with different frequencies in five pituitary tumor cell lines with 100% (5/5) methylation of the cyclin D2 gene and 40% (2/5) methylation of the p27 gene. Treatment with the methyl transferase inhibitor 5′-aza-2′-deoxycytidine (AZAdC) increased expression of cyclin D2 and p27 in GH3 and GHRH-CL1 pituitary tumor cells. There was a correlation between hypermethylation and gene expression. GH₃ tumors implanted into Wistar-Furth rats in vivo did not change the methylation status of the p27 and cyclin D2 genes. These data indicate a coordinately reduced expression of these two linked genes in most rodent pituitary tumor cell lines and suggest that methylation of cyclin D2 and p27 might occur in a “hot spot” in this gene-rich cluster. Supported in part by NIH CA 37231 and CA 42951     

A study of the relationship of metabolic MR parameters to estrogen dependence in breast cancer xenografts

¹H MRS, ³¹P MRS and diffusion‐weighted MRI (DW‐MRI) were applied to study the metabolic changes associated with estrogen dependence in estrogen receptor (ER)‐positive BT‐474 and triple‐negative HCC1806 breast cancer xenografts supplemented with or without 17β‐estradiol (E₂) at a dose of 0.18 or 0.72 mg/pellet. Furthermore, the effect of estrogen withdrawal on the metabolism of BT‐474 and HCC1806 breast cancer xenografts was studied on day 0, day 2 and day 10. Increasing the dose of E₂ resulted in a rapid growth and increases in the lactate level and phosphomonoester/β‐nucleoside triphosphate (PME/βNTP), phosphocreatine/inorganic phosphate (PCr/Pi) and βNTP/Pi ratios in BT‐474 breast cancer xenografts; however, no significant changes were found in HCC1806 breast cancer xenografts. Estrogen withdrawal resulted in a marked decrease in lactate level and PME/βNTP ratio and an observed increase in βNTP/Pi, PCr/Pi and apparent diffusion coefficient (ADC) values of BT‐474 breast cancer xenografts on day 10. These data suggest that the lactate level and PME/βNTP, PCr/Pi and βNTP/Pi ratios of ER‐positive tumors are closely related to ER dependence. Copyright © 2015 John Wiley & Sons, Ltd.     

Cell type influences the molecular mechanisms involved in hormonal regulation of ERG K<Superscript>+</Superscript> channels

While the thyrotropin-releasing hormone (TRH) effect of raising intracellular Ca²⁺ levels has been shown to rely on G_q/11 and PLC activation, the molecular mechanisms involved in the regulation of ERG K⁺ channels by TRH are still partially unknown. We have analysed the effects of βγ scavengers, Akt/PKB inactivation, and TRH receptor (TRH-R) overexpression on such regulation in native and heterologous expression cell systems. In native rat pituitary GH₃ cells β-ARK/CT, Gα_t, and phosducin significantly reduced TRH inhibition of rERG currents, whereas in HEK-H36/T1 cells permanently expressing TRH-R and hERG, neither of the βγ scavengers affected the TRH-induced shift in V _1/2. Use of specific siRNAs to knock Akt/PKB expression down abolished the TRH effect on HEK-H36/T1 cell hERG, but not on rERG from GH₃ cells. Indeed, wortmannin or long insulin pretreatment also blocked TRH regulation of ERG currents in HEK-H36/T1 but not in GH₃ cells. To determine whether these differences could be related to the amount of TRH-Rs in the cell, we studied the TRH concentration dependence of the Ca²⁺ and ERG responses in GH₃ cells overexpressing the receptors. The data indicated that independent of the receptor number additional cellular factor(s) contribute differently to couple the TRH-R to hERG channel modulation in HEK-H36/T1 cells. We conclude that regulation of ERG currents by TRH and its receptor is transduced in GH₃ and HEK-H36/T1 cell systems through common and different elements, and hence that the cell type influences the signalling pathways involved in the TRH-evoked responses.