Growth hormone regulates growth hormone receptor gene transcription in primary human thyroid cells

In this study the regulation of GH-receptor gene (GHR/GHBP) transcription by different concentrations of GH (0, 12.5, 25, 50, 150, 500 ng/ml) with and without variable TSH concentrations (0.5, 2, 20 mU/l) in primary human thyroid cells cultured in serum-free hormonally-defined medium was studied. The incubation time was 6 h and GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification at hourly intervals. Correlating with the GH-concentrations added a constant and significant increase of GHR/GHBP gene transcription was found. After the addition of 12.5 ng/ml GH, GHR/GHBP mRNA concentration remained constant over the incubation period of 6 h but in comparison with the experiments where no GH was added there was a significant change of GHR/GHBP mRNA expression. Following the addition of 25 ng/ml GH a slight but further increase of GHR/GHBP transcription products was seen which increased even more in the experiments where higher GH concentrations were used. These data focusing on GHR/GHBP gene transcription derived from cDNA synthesis and quantitative PCR amplification were confirmed by run-on experiments. Furthermore, cycloheximide did not affect these changes supporting the notion that GH stimulates GHR/GHBP gene transcription directly. In a second set of experiments, in combination with variable TSH levels, identical GH concentrations were used and no difference in either GHR/GHBP mRNA levels or in transcription rate (run-on experiments) could be found. In conclusion, we report data showing that primary thyroid cells express functional GH-receptors in which GH has a direct and dose dependent effect on the GHR/GHBP gene transcription. Furthermore, TSH does not a have a major impact on GHR/GHBP gene regulation.

Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: the BIG 1-98 randomised clinical trial at 8·1 years median follow-up

Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up.

Growth hormone receptor polymorphism and growth hormone therapy response in children: a bayesian meta-analysis

Recombinant human growth hormone (rhGH) therapy is used in the long-term treatment of children with growth disorders, but there is considerable treatment response variability. The exon 3-deleted growth hormone receptor polymorphism (GHR(d3)) may account for some of this variability. The authors performed a systematic review (to April 2011), including investigator-only data, to quantify the effects of the GHR(fl-d3) and GHR(d3-d3) genotypes on rhGH therapy response and used a recently established Bayesian inheritance model-free approach to meta-analyze the data. The primary outcome was the 1-year change-in-height standard-deviation score for the 2 genotypes. Eighteen data sets from 12 studies (1,527 children) were included. After several prior assumptions were tested, the most appropriate inheritance model was codominant (posterior probability = 0.93). Compared with noncarriers, carriers had median differences in 1-year change-in-height standard-deviation score of 0.09 (95% credible interval (CrI): 0.01, 0.17) for GHR(fl-d3) and of 0.14 (95% CrI: 0.02, 0.26) for GHR(d3-d3). However, the between-study standard deviation of 0.18 (95% CrI: 0.10, 0.33) was considerable. The authors tested by meta-regression for potential modifiers and found no substantial influence. They conclude that 1) the GHR(d3) polymorphism inheritance is codominant, contrasting with previous reports; 2) GHR(d3) genotypes account for modest increases in rhGH effects in children; and 3) considerable unexplained variability in responsiveness remains.

Interpreting Breast International Group (BIG) 1-98: a randomized, double-blind, phase III trial comparing letrozole and tamoxifen as adjuvant endocrine therapy for postmenopausal women with hormone receptor-positive, early breast cancer

The Breast International Group (BIG) 1-98 study is a four-arm trial comparing 5 years of monotherapy with tamoxifen or with letrozole or with sequences of 2 years of one followed by 3 years of the other for postmenopausal women with endocrine-responsive early invasive breast cancer. From 1998 to 2003, BIG -98 enrolled 8,010 women. The enhanced design f the trial enabled two complementary analyses of efficacy and safety. Collection of tumor specimens further enabled treatment comparisons based on tumor biology. Reports of BIG 1-98 should be interpreted in relation to each individual patient as she weighs the costs and benefits of available treatments.

Re-evaluating adjuvant breast cancer trials: assessing hormone receptor status by immunohistochemical versus extraction assays

BACKGROUND: Tumor levels of steroid hormone receptors, a factor used to select adjuvant treatment for early-stage breast cancer, are currently determined with immunohistochemical assays. These assays have a discordance of 10%-30% with previously used extraction assays. We assessed the concordance and predictive value of hormone receptor status as determined by immunohistochemical and extraction assays on specimens from International Breast Cancer Study Group Trials VIII and IX. These trials predominantly used extraction assays and compared adjuvant chemoendocrine therapy with endocrine therapy alone among pre- and postmenopausal patients with lymph node-negative breast cancer. Trial conclusions were that combination therapy provided a benefit to pre- and postmenopausal patients with estrogen receptor (ER)-negative tumors but not to ER-positive postmenopausal patients. ER-positive premenopausal patients required further study. METHODS: Tumor specimens from 571 premenopausal and 976 postmenopausal patients on which extraction assays had determined ER and progesterone receptor (PgR) levels before randomization from October 1, 1988, through October 1, 1999, were re-evaluated with an immunohistochemical assay in a central pathology laboratory. The endpoint was disease-free survival. Hazard ratios of recurrence or death for treatment comparisons were estimated with Cox proportional hazards regression models, and discriminatory ability was evaluated with the c index. All statistical tests were two-sided. RESULTS: Concordance of hormone receptor status determined by both assays ranged from 74% (kappa = 0.48) for PgR among postmenopausal patients to 88% (kappa = 0.66) for ER in postmenopausal patients. Hazard ratio estimates were similar for the association between disease-free survival and ER status (among all patients) or PgR status (among postmenopausal patients) as determined by the two methods. However, among premenopausal patients treated with endocrine therapy alone, the discriminatory ability of PgR status as determined by immunohistochemical assay was statistically significantly better (c index = 0.60 versus 0.51; P = .003) than that determined by extraction assay, and so immunohistochemically determined PgR status could predict disease-free survival. CONCLUSIONS: Trial conclusions in which ER status (for all patients) or PgR status (for postmenopausal patients) was determined by immunohistochemical assay supported those determined by extraction assays. However, among premenopausal patients, trial conclusions drawn from PgR status differed--immunohistochemically determined PgR status could predict response to endocrine therapy, unlike that determined by the extraction assay.

Predictors of early relapse in postmenopausal women with hormone receptor-positive breast cancer in the BIG 1-98 trial

BACKGROUND: Aromatase inhibitors are considered standard adjuvant endocrine treatment of postmenopausal women with hormone receptor-positive breast cancer, but it remains uncertain whether aromatase inhibitors should be given upfront or sequentially with tamoxifen. Awaiting results from ongoing randomized trials, we examined prognostic factors of an early relapse among patients in the BIG 1-98 trial to aid in treatment choices. PATIENTS AND METHODS: Analyses included all 7707 eligible patients treated on BIG 1-98. The median follow-up was 2 years, and the primary end point was breast cancer relapse. Cox proportional hazards regression was used to identify prognostic factors. RESULTS: Two hundred and eighty-five patients (3.7%) had an early relapse (3.1% on letrozole, 4.4% on tamoxifen). Predictive factors for early relapse were node positivity (P < 0.001), absence of both receptors being positive (P < 0.001), high tumor grade (P < 0.001), HER-2 overexpression/amplification (P < 0.001), large tumor size (P = 0.001), treatment with tamoxifen (P = 0.002), and vascular invasion (P = 0.02). There were no significant interactions between treatment and the covariates, though letrozole appeared to provide a greater than average reduction in the risk of early relapse in patients with many involved lymph nodes, large tumors, and vascular invasion present. CONCLUSION: Upfront letrozole resulted in significantly fewer early relapses than tamoxifen, even after adjusting for significant prognostic factors.

Regulation of human growth hormone receptor gene transcription by triiodothyronine (T3)

In this study the hypothesis that triiodothyronine (T3) and growth hormone (GH) may have some direct or indirect effect on the regulation of GH-receptor/GH-binding protein (GHR/GHBP) gene transcription was tested. Different concentrations of T3 (0, 0.5, 2, 10 nmol/l) and GH (0, 10, 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally-defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification. GH at a concentration of 10 ng/ml resulted in a significant increase of GHR/GHBP gene expression whereas a supraphysiological concentration of GH (150 ng/ml) caused a significant decrease of GHR/GHBP mRNA levels. The simultaneous addition of 0.5 nmol/l T3 to the variable concentrations of GH did not modify GHR/GHBP mRNA levels whereas the addition of 2 nmol/l up-regulated GHR/GHBP gene expression already after 1 h, an increase which was even more marked when 10 nmol/l of T3 was added. Interestingly, there was a positive correlation between the increase of GHR/GHBP mRNA levels and the T3 concentration used (r: 0.8). In addition, nuclear run-on experiments and GHBP determinations were performed which confirmed the changes in GHR/GHBP mRNA levels. Cycloheximide (10 microg/ml) did not alter transcription rate following GH addition but blocked GHR/GHBP gene transcription in T3 treated cells indicating that up-regulation of GHR/GHBP gene transcription caused by T3 requires new protein synthesis and is, therefore, dependent on indirect mechanisms. In conclusion, we present data showing that T3 on its own has a stimulatory effect on GHR/GHBP gene transcription which is indirect and additive to the GH-induced changes.

Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

Androgens are essential for sexual development and reproduction. However, androgen regulation in health and disease is poorly understood. We showed that human adrenocortical H295R cells grown under starvation conditions acquire a hyperandrogenic steroid profile with changes in steroid metabolizing enzymes HSD3B2 and CYP17A1 essential for androgen production. Here we studied the regulatory mechanisms underlying androgen production in starved H295R cells. Microarray expression profiling of normal versus starved H295R cells revealed fourteen differentially expressed genes; HSD3B2, HSD3B1, CYP21A2, RARB, ASS1, CFI, ASCL1 and ENC1 play a role in steroid and energy metabolism and ANGPTL1, PLK2, DUSP6, DUSP10 and FREM2 are involved in signal transduction. We discovered two new gene networks around RARB and ANGPTL1, and show how they regulate androgen biosynthesis. Transcription factor RARB stimulated the promoters of genes involved in androgen production (StAR, CYP17A1 and HSD3B2) and enhanced androstenedione production. For HSD3B2 regulation RARB worked in cooperation with Nur77. Secretory protein ANGPTL1 modulated CYP17A1 and DUSP6 expression by inducing ERK1/2 phosphorylation. By contrast, our studies revealed no evidence for hormones or cell cycle involvement in regulating androgen biosynthesis. In summary, these studies establish a firm role for RARB and ANGPTL1 in the regulation of androgen production in H295R cells.

The cannabinoid CB2 receptor-selective phytocannabinoid beta-caryophyllene exerts analgesic effects in mouse models of inflammatory and neuropathic pain

The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB2). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB2 is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB2 receptor-dependent manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB2 agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions.

Three-dimensional consensus structure of sst2-selective somatostatin (SRIF) antagonists by NMR

The three-dimensional NMR structures of seven octapeptide analogs of somatostatin (SRIF), based on octreotide, with the basic sequence H-Cpa/Phe2-c[DCys3-Xxx7-DTrp/DAph(Cbm)8-Lys9-Thr10-Cys14]-Yyy-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Aph(Cbm)/Tyr/Agl(NMe,benzoyl) and Yyy being Nal/DTyr/Thr, are presented here. Most of these analogs exhibit potent and highly selective binding to sst2 receptors, and all of the analogs are antagonists inhibiting receptor signaling. Based on their consensus 3D structure, the pharmacophore of the sst2-selective antagonist has been defined. The pharmacophore involves the side chains of Cpa2, DTrp/DAph(Cbm)8, and Lys9, with the backbone for most of the sst2-selective antagonists comprised a Type-II' beta-turn. Hence, the sst2-selective antagonist pharmacophore is very similar to the sst2-selective agonist pharmacophore previously described.

A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits

We show that the five subunits of a gamma-aminobutyric acid type A receptor (GABA(A) receptor) can be concatenated to yield a functional receptor. This concatenated receptor alpha(1)-beta(2)-alpha(1)-gamma(2)-beta(2) has the advantage of a known subunit arrangement. Most of its functional properties are not significantly different from a receptor formed by individual subunits. Extent of expression amounted to about 40% of that of non-concatenated receptors in Xenopus oocytes, after injection of oocytes with comparable amounts of cRNA coding for concatenated and non-concatenated receptors. The ability to express receptors consisting of five subunits enables detailed studies of GABA(A) receptor subtype selective compounds.

Radiolabeled somatostatin receptor antagonists are preferable to agonists for in vivo peptide receptor targeting of tumors

Targeting neuroendocrine tumors expressing somatostatin receptor subtypes (sst) with radiolabeled somatostatin agonists is an established diagnostic and therapeutic approach in oncology. While agonists readily internalize into tumor cells, permitting accumulation of radioactivity, radiolabeled antagonists do not, and they have not been considered for tumor targeting. The macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to two potent somatostatin receptor-selective peptide antagonists [NH(2)-CO-c(DCys-Phe-Tyr-DAgl(8)(Me,2-naphthoyl)-Lys-Thr-Phe-Cys)-OH (sst(3)-ODN-8) and a sst(2)-selective antagonist (sst(2)-ANT)], for labeling with (111/nat)In. (111/nat)In-DOTA-sst(3)-ODN-8 and (111/nat)In-DOTA-[4-NO(2)-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH(2)] ((111/nat)In-DOTA-sst(2)-ANT) showed high sst(3)- and sst(2)-binding affinity, respectively. They did not trigger sst(3) or sst(2) internalization but prevented agonist-stimulated internalization. (111)In-DOTA-sst(3)-ODN-8 and (111)In-DOTA-sst(2)-ANT were injected intravenously into mice bearing sst(3)- and sst(2)-expressing tumors, and their biodistribution was monitored. In the sst(3)-expressing tumors, strong accumulation of (111)In-DOTA-sst(3)-ODN-8 was observed, peaking at 1 h with 60% injected radioactivity per gram of tissue and remaining at a high level for >72 h. Excess of sst(3)-ODN-8 blocked uptake. As a control, the potent agonist (111)In-DOTA-[1-Nal(3)]-octreotide, with strong sst(3)-binding and internalization properties showed a much lower and shorter-lasting uptake in sst(3)-expressing tumors. Similarly, (111)In-DOTA-sst(2)-ANT was injected into mice bearing sst(2)-expressing tumors. Tumor uptake was considerably higher than with the highly potent sst(2)-selective agonist (111)In-diethylenetriaminepentaacetic acid-[Tyr(3),Thr(8)]-octreotide ((111)In-DTPA-TATE). Scatchard plots showed that antagonists labeled many more sites than agonists. Somatostatin antagonist radiotracers therefore are preferable over agonists for the in vivo targeting of sst(3)- or sst(2)-expressing tumors. Antagonist radioligands for other peptide receptors need to be evaluated in nuclear oncology as a result of this paradigm shift.