Hormone Receptor Analysis Research Articles

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor kappaB ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers' expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth.

Histological features of breast cancer, highly sensitive to chemotherapy

To establish tailor-made therapy for breast cancer, we investigated the possibility of predicting chemotherapy sensitive cases based on pre-therapeutic histological features. A total of 87 breast cancer patients underwent neoadjuvant chemotherapy with a paclitaxel (80 mg/m(2)/q1w, 12 courses)or an epirubicin regimen (90 mg/m(2)/q3wks, 4 courses). We investigated the chemo-sensitivity of invasive ductal carcinoma, solid-tubular carcinoma consisting of highly malignant cancer cells with many mitoses. We refer to this type of carcinoma as " chemo-sensitive carcinoma " and compared the histological therapeutic effects of chemo-sensitive and chemo-insensitive carcinomas. 1) Out of 87 patients, 20 cases (23%) showed the histological features of chemo-sensitive carcinomas on pre-therapeutic needle biopsy specimens. The remaining 67 cases (77%) were classified as chemo-insensitive carcinoma. 2) Histologically marked or complete response were observed in 50% (10/20) of chemo-sensitive carcinomas and 10% (7/67) of chemo-insensitive carcinomas (chi(2)=15.33, p=0.0001). Multivariate analysis of chemo-sensitive carcinoma, including HER2, hormone receptor and p53 status, revealed that chemo-sensitive carcinoma had a significant correlation with the histological therapeutic effects (p=0.01119). 3) Pathological complete response (pCR) was achieved in 35% (7/20) of chemo-sensitive carcinomas and 1.5% (1/67)of chemo-insensitive carcinomas (chi(2)=20.71, p<0.0001). Multivariate analysis revealed that chemo-sensitive carcinoma had a significant correlation with pCR (p=0.0091). The histological features of chemo-sensitive carcinoma were significant predictive factors for chemotherapeutic efficacy.

CDNA cloning and expression analysis of thyroid hormone receptor in the coho salmon Oncorhynchus kisutch during smoltification

Two types of cDNA clones encoding thyroid hormone receptor (TR) genes, TRα and TRβ, were isolated from the coho salmon Oncorhynchus kisutch. The two genes each consists of multiple isoforms, derived from different loci or by an alternative splicing. RT-PCR revealed a broad localization of the TRα and TRβ transcripts that both expressed in brain, gill, liver, muscle, kidney, ovary, skin and eyeball. During the early stages of the smoltification period, the mRNA level of the TR transcripts showed a large individual variability at the maximum of 10-fold. In contrast, as the plasma thyroid hormone level elevated at the middle of the period, the variation in the expression level became much smaller at 2-fold. After the completion of the smoltification, the mRNA accumulation level of the TR genes rapidly decreased. These findings suggest that the dynamics of the receptor (TR) should be taken account in addition to the availability of the hormone ligands (T3 and T4) to understand the underlying mechanisms of the smoltification causing numerous physiological and morphological changes in salmonids.

Transcriptional analysis of steroid hormone receptors in smooth muscle uterine leiomyoma tumors of postmenopausal patients

Smooth muscle tumors are histologically separated into benign leiomyomas and malignant leiomyosarcomas. Uterine leiomyomas represent benign clonal tumors often arising within the smooth muscle tissue of the human uterus. Uterine leiomyomas develop after the start of the menstrual cycle, become symptomatic during middle age, and in most postmenopausal patients tumor regression occurs. Rarely, leiomyomas progress to leiomyosarcomas, where many sarcomas have markedly reduced or no steroid hormone receptors, thus, evolve to a hormone non-responsive state. Premenopausal leiomyomas are known to express higher levels of estrogen receptor-alpha (ERα), estrogen receptor-beta (ERβ) and progesterone receptor (PGR) than control myometrium, whereas postmenopausal leiomyomas have not been so well characterized molecularly. In this present investigation, ERβ, ERα and PGR gene expression were assessed in leiomyomas and in matched adjacent myometrium from a cohort of 14 postmenopausal patients using semi-quantitative Realtime PCR and RT-PCR. The mean average results showed that ERβ was 2.5-fold statistically significantly over expressed in postmenopausal leiomyomas compared to patient matched myometrium ( p = 0.038), whereas ERα and PGR were not significantly differently expressed. These results showed that up-regulation of ERβ occurred at the transcriptional level in postmenopausal leiomyomas. Quantitation of steroid hormone receptors from benign uterine tumors may be important for a more tailored therapy. In addition, a role for steroid hormones, specifically ERβ, is discussed in terms of benign tumor regression or tumor maintenance in postmenopausal leiomyomas.

Markers of Cell Proliferation, Apoptosis, and Angiogenesis in Thyroid Adenomas: A Comparative Immunohistochemical and Genetic Investigation of Functioning and Nonfunctioning Nodules

To perform (i) an immunohistochemical investigation of cell proliferation, apoptosis, angiogenesis, and malignancy markers in 15 functioning and 15 nonfunctioning thyroid adenomas, and in normal adjacent tissue, and (ii) a genetic analysis of thyroid-stimulating hormone receptor (TSH-r), Gsalpha, and RAS mutations in the same group of adenomas, in order to describe their expression within tissues and to correlate them with the hormonal functioning. Thirty patients who underwent surgery for a solitary thyroid nodule were included in the study. Adenomas and normal adjacent tissues were evaluated by immunohistochemistry using the following antibodies: MIB-1 for proliferative activity, bcl-2 and mutant p53 for apoptosis control, vascular endothelial growth factor-A (VEGF-A) for angiogenic activity, and galectin-3 as a marker for malignancy. To calculate microvascular density, "hot spots" were selected and defined by cells positive for CD34 staining. Genetic analysis for TSH-r, Gsalpha, and H-, K-, and N-RAS mutations was performed on adenoma specimens. Our results evidenced that a proportion of both functioning and nonfunctioning adenomas showed immunohistochemical phenotypes similar to normal adjacent tissue. No differences were found between functioning and nonfunctioning thyroid adenomas with regard to the expression of markers associated to angiogenesis (VEGF-A, microvascular density) and apoptosis control (mutant p53, bcl-2). All adenomas resulted negative for galectin-3 immunostaining. MIB-1 was the only marker showing a substantial difference of expression between the two groups of adenomas. TSH-r mutations were found in 12 out of 15 functioning adenomas, whereas the absence of Gsalpha and H-, K-, and N-RAS mutations was demonstrated in all adenomas. Our data suggest that the differences between functioning and nonfunctioning thyroid adenomas are restricted to the genetic mutations of the TSH-r, to the hormonal status of tumors, and to the proliferative activity, not involving markers of apoptosis control and angiogenesis.

Development of Luciferase Reporter-Based Cell Assays

Using luciferase reporter constructs driven by specific promoter response elements, we developed a series of stable reporter cell lines for monitoring the activity of specific transcription factors (TFs). These TFs, which play essential roles in regulating diverse biological functions, include nuclear factor kappaB (NFkappaB), cyclic AMP response element-binding protein, activator protein 1, signal transducer and activator of transcription 1 and 3, nuclear factor of activated T cells, serum response factor, and hypoxia-inducible factor. The response of the stable reporter cells was highly specific. For example, tumor necrosis factor-alpha (TNFalpha) strongly activated NFkappaB reporter cells, but not other cell lines. The NFkappaB reporter was active in multiple cell lines, including 293T, HeLa, A549, and NIH3T3 cells, in response to TNFalpha, indicating that this system is useful to monitor specific TFs in different model cell lines. To facilitate high throughput screening of these cell lines, they were adapted to a 96-well format. These stable reporter cells are also applicable for the analysis of steroid hormone receptors, which bind directly to the response element after ligand binding. With the HeLa/glucocorticoid response element-luciferase stable reporter cells, we were able to discriminate pharmacological activity of different compounds for the glucocorticoid receptor. Taken together, these results demonstrate that the stable reporter cells are useful tools for: (1) detection of signaling pathway-specific ligands; (2) identification of novel ligands for specific TFs, and (3) screening for agonists and antagonists of specific ligands/receptors.

Entwicklung der operativ-gynäkologischen Versorgungsqualität in Hessen unter externer Qualitätssicherung

BACKGROUND AND RESEARCH GOAL: All German hospitals have to participate by law in comparative performance assessment, so called external quality assurance (QA). However, there is only limited research showing a positive impact of such state- and nationwide QA programmes. This article analyses whether the quality of surgical gynaecological care in Hessian hospitals changed after receiving regular feedback on the quality of care between 1998 to 2002. Secondary data analysis of data from all hospitals with gynaecological services in Hesse (n = 84) was performed. The requirement of a constant caseload and continuous data supply reduced cases from 218,217 to 148,549. Considering twelve quality indicators we recalculated the performance of all Hessian hospitals for each quarter during 1998 and 2002. Changes over time were analysed by using rank correlation coefficients and considering multiple testing. Two indicators (antibiotic prophylaxis in breast surgeries and hysterectomies) showed a significant quality improvement over time. Six indicators (post-operative infections, inappropriate removals of cyst of ovaries, hormone receptor analysis and x-ray of tumour in breast cancer, ovary-preserving procedures for benign tumours and heparin prophylaxis) demonstrated a slight quality improvement and another four indicators (unplanned revision of operation, organ injuries, adjuvant therapy and breast-preserving surgery in breast cancer) tended towards reduced quality. During 1998-2002, we observed only marginal improvements in the quality of care. The expected significant improvement of all quality indicators, based on external QA and comparative performance reporting of all Hessian gynaecological hospitals did not eventuate.

Testicular germ cell tumors exhibit evidence of hormone dependence

The aim of this investigation was to test the hypothesis that testicular germ cell tumors (TGCTs) are hormone-dependent cancers. Human TGCT cells were implanted in the left testis of male severe combined immunodeficient mice receiving either no treatment or hormone manipulation treatment [blockade of gonadotropin-releasing hormone secretion and/or signaling using leuprolide or leuprolide plus exogenous testosterone]. Real-time RT-PCR analysis was used to determine the expression profiles of hormone pathway-associated genes. Tumor burden was significantly smaller in mice receiving both leuprolide and testosterone. Real-time RT-PCR analysis of follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor and P450 aromatase revealed changes in expression in normal testis tissue related to presence of xenograft tumors and manipulation of hormone levels but a complete absence of expression of these genes in tumor cells themselves. This was confirmed in human specimens of TGCT. Reduced TGCT growth in vivo was associated with significant downregulation of LH receptor and P450 aromatase expression in normal testes. In conclusion, manipulation of hormone levels influenced the growth of TGCT in vivo, while the presence of xenografted tumors influenced the expression of hormone-related genes in otherwise untreated animals. Human TGCTs, both in the animal model and in clinical specimens, appear not to express receptors for FSH or LH. Similarly, expression of the P450 aromatase gene is absent in TGCTs. Impaired estrogen synthesis and/or signaling may be at least partly responsible for inhibition of TGCT growth in the animal model.

Loss of Heterozygosity on Chromosome 1 and 9 and Hormone Receptor Analysis of Metastatic Malignant Melanoma Presenting in Breast

Malignant melanoma (MM), the most common metastatic solid tumor to involve the breast, may present as a diagnostic problem, frequently requiring the use of ancillary studies for accurate diagnosis. The implication of hormonal interplay is strong since metastatic MM to the breast is seen nearly always in women. However, the role of hormonal status as a predisposing factor in the development of this entity is largely unresolved. A number of chromosomal loci, including 1p36 and 9p21-22, appear to harbor critical genes important to melanoma tumorigenesis, and additionally chromosome 9q22.3-31. We wanted to know if metastatic MM in breast showed chromosome 1p and 9p genetic alterations (loss of heterozygosity) similar to those that occur in primary cutaneous MM, and whether additional 9q LOH changes are present. Hormonal receptor status of the metastatic MM was also determined. We identified 20 patients with known MM metastatic to the breast, which we analyzed with the following genetic markers: D9S12 (9q22.3), D9S171 (9p21), IFNA (9p22), and D1S450 (1p). Visually directed microdissection was performed on archival histologic slides containing both tumor and adjacent normal breast epithelium, followed by single-step DNA extraction and polymerase chain reaction (PCR) amplification for evaluation of loss of heterozygosity (LOH) for the above-listed markers. Immunohistochemical (IHC) stains for estrogen receptor (ER) and progesterone receptor (PR) was performed on 10 of the cases. Twelve of the 20 cases contained DNA suitable for PCR amplification following direct visualization microdissection. Four of 8 (50%) informative cases showed LOH at 9p21 with D9S171. Ten cases were heterozygous for IFNA, with 2 cases (20%) showing LOH at this locus. These particular cases also showed LOH at 9p21. One of 9 (11%) informative cases showed LOH for D1S450 (1p36). Five cases were heterozygous for D9S12, and 2 (40%) showed LOH in the tumor at 9q22.3. IHC stains for ER and PR were negative in the 10 tumors studied. Metastatic MM presenting as a breast mass is an interesting entity often requiring IHC studies for diagnosis, particularly when the histologic features simulate breast carcinoma or when no primary tumor is known. These tumors are ER and PR negative. Metastatic MM involving the breast shows similar genetic allelic losses on chromosome 9p21-22 (50%) and 1p36 (11%), as previously described in primary cutaneous MM. Additional LOH was observed at the 9q22.3-31 locus (40%). We suggest this locus to be investigated for harboring potential genes important in the tumorigenesis of cutaneous MM.

Crystallization and preliminary X-ray diffraction analysis of the unliganded human growth hormone receptor

The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992. However, no information exists for the unliganded form of the receptor. The human growth hormone receptor's extracellular ligand-binding domain, encompassing amino-acid residues 1-238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants. The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 A and diffract to 2.5 A resolution using synchrotron radiation. The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.

Molecular cloning, sequence analysis and expression of the snake follicle-stimulating hormone receptor

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) control gonadal function in mammalian and many non-mammalian vertebrates through the interaction with their receptors, FSHR and LHR. Although the same is true for some reptilian species, in Squamata (lizards and snakes) there is no definitive evidence for the presence of either two distinct gonadotropins or two distinct gonadotropin receptors. Our aim was to characterize the gonadotropin receptor(s) of the Bothrops jararaca snake. Using a cDNA library from snake testis and amplification of the 5 ′-cDNA ending, we cloned a cDNA related to FSHR. Attempts to clone a cDNA more closely related to LHR were unsuccessful. Expression of FSHR mRNA was restricted to gonadal tissues. The snake FSHR is a G protein-coupled receptor with 673 amino acids, and the aminoterminal domain with 346 amino acids consists of a nine leucine-rich repeat-containing subdomain (LRR) flanked by two cysteine-rich subdomains. The β-strands in the LRR are conserved with exception of the third, a region that may be important for FSH binding. In contrast with mammalian, avian and amphibian FSHRs, the snake FSHR presents amino acid deletions in the carboxyterminal region of the extracellular domain which are also seen in fish and lizard FSHRs. cAMP assays with the recombinant protein transiently expressed in HEK-293 cells showed that the snake FSHR is more sensitive to human FSH (hFSH) than to human chorionic gonadotropin. Phylogenetic analysis indicated that the squamate FSHRs group separately from mammalian FSHRs. Our data are consistent with the apparently unique gonadotropin-receptor system in Squamata reptilian subgroup. Knowledge about the snake FSHR structure may help identify structural determinants for receptor function.

Relation between the rate of tumour cell proliferation and latency time in radiation associated breast cancer.

BackgroundPatients with possible radiation induced cancer could be used to study if the rate of tumour cell proliferation is related to latency time. Such a finding could help researcher to find time periods when other initiating risk factors operate.MethodsSeventeen women with breast cancer, with a prior history of radiation treatment towards the parts or the whole breast, exclusive of the primary treatment of a breast cancer were identified. Most women had received treatment for benign disorders as hemangiomas, shoulder pain or skin infections. Three patients had been treated with mantle radiation for Hodgkin's disease prior to developing breast cancer. DNA analysis were performed, on remaining tumour tissue after hormone receptor analysis had been done, measuring the fraction of tumour cells in S-phase. Latency time (time between diagnosis and previous radiation treatment) was calculated and related to the S-phase fraction.ResultsA significant inverse relationship between latency time and S-phase was found (p < 0.0025), indicating that tumours with a high S-phase had a short latency time and vice versa. Among the possible radiation induced tumours, median S-phase was 14%, comparable with a median latency time of 22 years. Very high S-phase values were associated with short latency times (eg a S-phase of 35% would be compatible with a latency time of 7 years).ConclusionOur preliminary results indicate that S-phase is related to latency time and that the median latency time maybe as long as 22 years. Our data may also explain why breast cancer is rare before 30 years of age and if patients are diagnosed at early ages, tumours often show high S-phase values and bad prognostic signs. We postulate that these results from radiation induced breast cancer may be used to extrapolate possible latency times in patients with non radiation induced breast tumours in order to isolate possible time periods for research after other initiating events.

Analysis of the human growth hormone receptor and IGF-I coding sequences in children with growth disorders.

Analysis of GHR and IGF-I coding sequences in 47 children with normal serum levels of GH, low IGF-I and growth disorders generally did not show mutation in the genes studied. Only one boy had a mutation located in the fifth exon of the GHR gene (C-->T in codon 88). This suggests that the growth disorders in this group of children might be due to a defect in a DNA region regulating expression of the GHR and IGF1 genes or genes involved in their regulation.

Clinical phenotype and infertility treatment in a male with hypogonadotropic hypogonadism due to mutations Ala129Asp/Arg262Gln of the gonadotropin-releasing hormone receptor

Objective To characterize the genotype and phenotype of a man with idiopathic hypogonadism with infertility. Design Molecular analysis and clinical description. Setting Medical school laboratory and reproductive endocrine clinic. Patient(s) A 40-year-old male with idiopathic hypogonadotropic hypogonadism. Intervention(s) Denaturing gradient gel electrophoresis analysis and DNA sequencing of the gonadotropin-releasing hormone receptor (GNRHR) gene were performed. The patient was treated with hCG and FSH. Main outcome measure(s) GNRHR mutation detection, genotype/phenotype correlation, and testicular response to exogenous gonadotropin therapy. Result(s) The proband demonstrated compound heterozygosity for Ala129Asp/Arg262Gln GNRHR mutations. He had a complete form of idiopathic hypogonadotropic hypogonadism, with descended testes and severe oligospermia but little response to exogenous gonadotropins. Conclusion(s) The phenotype of this patient differs from the one other family described with the same mutations. Exogenous gonadotropin therapy may not be as beneficial for increasing sperm concentration in older men with idiopathic hypogonadotropic hypogonadism.

Expression of thyroid hormone receptor/erbA genes is altered in human breast cancer.

The relation between thyroid status and diseases and cancer is unclear. No detailed analysis of thyroid hormone receptor (TR) expression in human breast cancer has been reported. We have analysed the expression and mutational status of the TRalpha1, encoded by the c-erbA proto-oncogene, TRbeta1 and TRbeta2 isoforms in 70 sporadic breast cancers. Alterations in the RNA level of TRbeta1, TRalpha1, or both were found in a number of patients. No expression of TRbeta2 RNA was detected. Western blotting analysis confirmed the differences in expression at the protein level in those cases where sufficient tumor sample was available. Additionally, tumor-specific truncated TRbeta1 RNA was found in six patients. Strikingly, three transcripts shared the same breakpoint. Only one tumor carried the corresponding deletion at the genomic DNA level, suggesting that the remaining abnormal TRbeta1 transcripts are aberrant splicing products. Though no significant correlation was found between TRbeta1 alteration and any clinical parameter, it showed a tendency to associate with early age of onset (<50 years). Our results reveal specific alterations in the expression of TRbeta and TRalpha genes in a subset of breast cancer patients, suggesting that deregulation of thyroid hormone target genes may be involved in the generation of this neoplasia.

MALT Lymphoma Originating in Breast and Uvula

A case of marginal zone B cell lymphoma of MALT type arising in the uvula and breast is reported. The patient, a 30-year-old woman who delivered a child and lactated in 1997, was suffering from Sjögren syndrome (SS). She was diagnosed with MALT lymphoma after a biopsy of the right breast and uvula. To investigate the relationship of the delivery, lactation and MALT lymphoma, we examined the immunohistochemical analysis of hormone receptors. As a result, lymphoid cells of the breast were stained with anti-progesterone receptor antibodies in the cytoplasm. Consequently, the MALT lymphoma of the uvula appeared to be associated with SS. Moreover, hormones such as progesterone may have influenced the breast involvement of MALT lymphoma in our case.

Candidate gene analysis of thyroid hormone receptors in metamorphosing vs. nonmetamorphosing salamanders.

We used two different experimental approaches to test the hypothesis that thyroid hormone receptor (TR) variation is associated with alternate life cycles modes in ambystomatid salamanders. In the first experiment, the inheritance of TRalpha and TRbeta genotypes was determined for metamorphic and non metamorphic offspring from backcrosses between Ambystoma mexicanum (an obligate metamorphic-failure species) and metamorphic F1 hybrids (A. mexicanum x A. tigrinum tigrinum). The segregation of TR genotype was independent of the expression of life cycle mode phenotype, and neither TR locus was linked to DNA markers that flank a major-effect locus for life cycle mode. In the second experiment, a portion of the ligand-binding domain of TRalpha and TRbeta was cloned and sequenced for DNA samples from 14 different ambystomatid salamander populations, including obligate metamorphic, facultative metamorphic, and obligate metamorphic-failure taxa. Nucleotide sequence variation was found for both TRalpha and TRbeta, with several nonsynonomous substitutions that presumably code for nonconservative amino acid replacements. However, no general relationship was found between TR allelic variation and life cycle mode among populations or species. These data do not implicate TRs as candidate loci involved in the current maintenance or past evolution of alternate life cycle modes in members of the tiger salamander complex.

Assessment of Tissue Estrogen and Progesterone Receptor Levels: A Survey of Current Practice, Techniques, and Quantitation Methods.

The assessment of steroid hormone receptors in resected breast carcinoma tissue is currently the standard of practice. The traditional method for assessment of receptor status is the ligand binding assay. More recently, immunohistochemistry (IHC) has become a popular method for such testing. Despite the widespread use of IHC and the availability of many antibodies, standardization of quantitative IHC for assessment of estrogen and progesterone receptors has not been achieved. While the College of American Pathologists (CAP) offers a Quality Assurance (QA) program for IHC quantitation of estrogen receptor (ER) and progesterone receptor (PgR), no universal standard is currently recognized in assessment of ER and PgR by IHC. We surveyed 300 laboratories within the United States for their current practices regarding the assessment of ER and PgR status in breast cancer tissue specimens. Eighty usable responses were received. Forty-nine (61%) laboratories performed the assay in-house, while the remainder sent the material out for assay. All responding laboratories performing their steroid receptor analysis in-house used the IHC technique. Forty-three (80%) laboratories answering the question on material accepted for analysis performed the assay only on paraffin-embedded material, three (6%) used either paraffin block or frozen material, and two (4%) used only frozen material. Eighty-eight percent of laboratories performing steroid receptor analysis in-house used a manual quantitation technique. Four (8%) used computer-assisted image analysis, and a single laboratory used laser scanning cytometry. Eight different antibodies were used among the 44 laboratories documenting the antibody supplier, and for any given commercially prepared antibody a wide variety of dilutions were used, with the exception of the standard solution used with the Ventana antibody. Of the laboratories using manual estimation techniques, 61% simply estimated the percentage of positive cells, 29% evaluated both the intensity of staining and percentage of nuclei staining, 6% used formal H-score analysis, 2% evaluated only intensity of nuclear staining, and 2% mainly counted the percentage of nuclei staining for ER but used a formal H score in the assessment of PgR. Cutoff points for the separation of positive and negative results varied widely, with some laboratories assessing any demonstrable positivity as a positive result, while others required as many as 19% of the nuclei to stain before a specimen was declared positive. Standardization techniques differed considerably among laboratories. Eighty-six percent used the CAP program for QA. While all laboratories utilized some form of intralaboratory control for assessment of ER and PgR, the nature of that control varied from laboratory to laboratory. Our survey indicates that a majority of laboratories perform their steroid hormone receptor analysis in-house using IHC. There is considerable variability in the antibodies utilized, the dilutions applied, and the quantitation method and level of expression used to dichotomize specimens into positive and negative groups. Finally, no universal control for interlaboratory standardization appears to exist.

Identification of an Upstream Promoter in the Human Gonadotropin-Releasing Hormone Receptor Gene

Analysis of the human gonadotropin-releasing hormone receptor (hGnRHR) gene 5′ flanking region revealed the presence of multiple TATA, CCAAT, and transcription start sites. In addition, at least three different transcripts (5.0, 2.5, and 1.5 kb) were detected by Northern blot analysis. Taken together, these data indicated the existence of multiple promoter elements in the hGnRHR gene, and these promoters are responsible for the multiplicity of regulation of human reproductive functions. In this report, by progressive 5′ and 3′ deletion (−2197 to −1351, relative to the ATG) and NotI linker scanning mutagenesis coupled to transient transfection into the mouse gonadotrope-derived αT3-1 cell, a distal promoter element was identified at −1705/−1674. The promoter was located immediately 5′ to a previously identified CAP site at −1673 in human pituitary and it drove a 17.6- ± 1.0-fold increase in reporter gene activity. Within the promoter, a pyrimidine-rich initiator element (Inr) (−1682) and a CCAAT box (−1702) were found and mutation of these elements abrogated both protein bindings and promoter activities. By 1- and 2-D SouthWestern blot assays, multiple nuclear factors (40 to 54 kDa) were found to interact specifically with this promoter element. These nuclear factors were also present in other cells, including COS-7, JEG-3, and SKOV-3 cells, and these findings were consistent with functional studies which showed that the promoter is also active in these cells.

Immunohistochemical analysis of the orphan steroid hormone receptor Nur77/Nurr1/Nor1 subfamily in the mouse brain

Immunohistochemical analysis of the orphan steroid hormone receptor Nur77/Nurr1/Nor1 subfamily in the mouse brain