Cytosolic Estrogen Receptor Research Articles

Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane receptor.

Impaired insulin secretion is a hallmark in both type I and type II diabetic individuals. Whereas type I (insulin-dependent diabetes mellitus) implies ss-cell destruction, type II (non-insulin dependent diabetes mellitus), responsible for 75% of diabetic syndromes, involves diminished glucose-dependent secretion of insulin from pancreatic beta-cells. Although a clear demonstration of a direct effect of 17beta-estradiol on the pancreatic ss-cell is lacking, an in vivo insulinotropic effect has been suggested. In this report we describe the effects of 17beta-estradiol in mouse pancreatic ss-cells. 17beta-Estradiol, at physiological concentrations, closes K(ATP) channels, which are also targets for antidiabetic sulfonylureas, in a rapid and reversible manner. Furthermore, in synergy with glucose, 17beta-estradiol depolarizes the plasma membrane, eliciting electrical activity and intracellular calcium signals, which in turn enhance insulin secretion. These effects occur through a receptor located at the plasma membrane, distinct from the classic cytosolic estrogen receptor. Specific competitive binding and localization of 17beta-estradiol receptors at the plasma membrane was demonstrated using confocal reflective microscopy and immunocytochemistry. Gaining deeper knowledge of the effect induced by 17beta-estradiol may be important in order to better understand the hormonal regulation of insulin secretion and for the treatment of NIDDM. receptor.

Time-dependent relationship between the estrogen receptors and the matrix metalloproteinases following deciduoma induction in rats

The purpose of this study was to investigate time-related interactions between the estrogen receptors, mediators of steroidal regulation of uterine growth, and an extracellular regulatory enzyme, the matrix metalloproteinases (MMPs) engaged in connective tissue degradation and remodeling that are fundamental to implantation and placentation. Pseudopregnant rats, in which the decidual response, the basis for decidualization, was surgically induced on day 4 of pseudopregnancy (PG), were sacrificed on PG days 3, 6, 9, and 15 for retrieval of uterine tissues for assays: the radioligand binding assay for the estrogen receptors and substrate zymography for the MMPs. Following increases on PG day 3, there were time-dependent decreases in the cytosolic low and high capacity estrogen receptors during deciduoma development (PG days 6–9) and regression (PG day 15) in both the endometrium and myometrium. Moreover, whereas the low capacity estrogen receptor levels were only slightly decreased (PG days 6–15), the high capacity receptors were reduced on day 6 ( P<0.001) and were completely diminished during PG days 9 and 15. In contrast, the MMPs (92 and 72 kDa) activities were increased from PG days 6–15 ( P<0.05) over the pre-decidual induction values on PG day 3 in both uterine compartments. The results suggest that deciduoma induction can modulate the concentration of cytosolic estrogen receptor subtypes and MMP activities in rats. The inverse time-dependent interrelationship between these cellular and extracellular components during deciduoma development and regression imply that the remodeling role of the MMPs may be enhanced by the reduced cytosolic estrogen receptor/estrogen action.

Development of oestrogen receptor expression and hormone response in the uterus of the bovine fetus.

The objective of the present study was to determine the temporal expression of oestrogen receptor alpha in the uterus of the developing bovine fetus. Bovine fetuses were collected and approximate gestational age was determined by crown-rump measurement. Fetal uteri were either snap frozen in dry ice-ethanol, or placed in sterile Dulbecco's modified Eagle's medium. Fetal uteri (n = 20) were homogenized and cytosolic oestrogen receptor measured by [3H]ligand binding assay. Total RNA was extracted from fetal uteri (n = 53) and amplified by reverse transcription-polymerase chain reaction using primers specific for the oestrogen receptor, progesterone receptor, interleukin 1 alpha, interleukin 6, transforming growth factor beta, prolactin receptor, epidermal growth factor receptor, retinoic acid receptor isoforms alpha, beta, and gamma, or glyceraldehyde-3-phosphate dehydrogenase (loading control). Expressed as a ratio with glyceraldehyde-3-phosphate dehydrogenase, mRNA encoding oestrogen receptor was identified in fetal uteri throughout the period from day 65 to day 200, and was increased from day 100 to day 185 (P < 0.003); uterine samples from day 100 to day 200 expressed interleukin 1 alpha, interleukin 6, transforming growth factor beta, prolactin receptor, epidermal growth factor receptor and retinoic acid receptor isoforms alpha, beta, and gamma, but did not express detectable mRNA encoding progesterone receptor. Despite the presence of mRNA encoding oestrogen receptor, [3H]oestradiol binding was not detected until after day 155. Fetal uterine explants collected at days 100-110 (n = 3) or at days 185-200 (n = 3) were cultured in the presence of oestradiol (1.0 nmol l-1, or vehicle); there was a significant effect of oestradiol treatment on specific mRNA expression at days 185-200 (P < 0.014), but not at days 100-110 (P = 0.71). It is concluded that mRNA encoding oestrogen receptor is constitutively expressed at least from day 65 in the uterus of the bovine fetus, but that oestrogen receptor alpha protein and a functional oestrogen response are not present until late in the second third of pregnancy.

Effect of cdri-85/287 on uterine estradiol and progesterone receptor levels/ morphometric measurements during preimplantation period in rat

Studies with 85/287, a potent nonsteroidal antiestrogen /antiimplantation agent were taken up. In this paper we report alterations in uterine morphometric measurements and estrogen/progesterone receptor levels under the influence of this compound. Results showed 32% decline in stromal absolute volume density on day 5 post-coitum(p.c.) only, whereas eosinophilic leucocyte number decreased both on days 3 and 5 p.c. (41%) in treated rat uterus. Epithelial mitotic activity showed complete cessation both in control and treated rats on day 5 p.c. Under the influence of the compound both cytosolic and nuclear estrogen receptor (ERc and ERn) levels decreased on day 3 p.c., but on day 5 p.c. ERn increased significantly. A significant increase was however noticed in progesterone nuclear receptors (PRn) on day 5 p.c On the whole our studies showed overall significant changes in uterine morphometric measurement/estrogen and progesterone receptor levels during the preimplantation period in rat under the influence of compound 85/287, causing asynchrony of events and thus failure of implantation.

The effects of delayed hormone replacement therapy on estrogen receptors of the cynomolgus monkey bladder and vagina.

The aim of this study was to determine cytosolic estrogen receptor content of the cynomolgus monkey bladder and vagina after hormone replacement therapy. Animals maintained without hormone therapy for 2 years after surgical menopause were randomized to receive either no hormones (OVX), conjugated equine estrogens (CEE), or estrogen/medroxyprogesterone acetate (CEE + MPA) treatment for 30 months. Estrogen receptor content of bladder and vagina cytosolic extracts was determined using a commercial enzyme-linked immunosorbent assay. Estrogen receptors were uniformly present, although the vaginal concentration was 100-fold greater than in the bladder. Estrogen and combination therapy significantly decreased cytosolic receptor content in both sites compared with controls. The cynomolgus urogenital tract remains estrogen sensitive 2 years after surgical menopause. Prolonged exposure to estrogens decreases cytosolic estrogen receptor content in a manner similar to that described for short-term estrogen therapy. These results suggest that the effects of hormone replacement on the urinary tract can be identified even if initiated years after menopause.

The effects of delayed hormone replacement therapy on estrogen receptors of the cynomolgus monkey bladder and vagina

The aim of this study was to determine cytosolic estrogen receptor content of the cynomolgus monkey bladder and vagina after hormone replacement therapy. Animals maintained without hormone therapy for 2 years after surgical menopause were randomized to receive either no hormones (OVX), conjugated equine estrogens (CEE), or estrogen/medroxyprogesterone acetate (CEE + MPA) treatment for 30 months. Estrogen receptor content of bladder and vagina cytosolic extracts was determined using a commercial enzyme-linked immunosorbent assay. Estrogen receptors were uniformly present, although the vaginal concentration was 100-fold greater than in the bladder. Estrogen and combination therapy significantly decreased cytosolic receptor content in both sites compared with controls. The cynomolgus urogenital tract remains estrogen sensitive 2 years after surgical menopause. Prolonged exposure to estrogens decreases cytosolic estrogen receptor content in a manner similar to that described for short-term estrogen therapy. These results suggest that the effects of hormone replacement on the urinary tract can be identified even if initiated years after menopause. Neurourol. Urodynam. 17:241–247, 1998. © 1998 Wiley-Liss, Inc.

CYTOSOLIC ESTROGEN RECEPTOR CONCENTRATIONS IN THE LUMBOSACRAL SPINAL CORD FLUCTUATE DURING THE ESTROUS CYCLE

Estrogen responsive neurons have been anatomically identified with autoradiographic and immunohistochemical techniques and their distribution mapped in the lumbosacral spinal cord of female rats. Such neurons contain estrogen receptors (ERs). The present study was undertaken to: 1) quantify cytosolic estrogen receptor (ER) concentrations in the lumbosacral spinal cord and 2) determine if there is a relationship between cytosolic ER concentrations and fluctuations in serum estradiol (SE2) levels during the estrous cycle. Lumbosacral spinal segments were removed from intact cycling rats during the morning of proestrus, the afternoon of proestrus, and the morning of estrus, metestrus and diestrus. Trunk blood was collected at euthanasia and SE2 levels were determined using radioimmunoassay. Cytosolic ER concentrations were measured using a dextran-charcoal coated tube method. Concentrations of cytosolic ERs were low during estrus and metestrus, increased during diestrus with maximum concentrations during the afternoon of proestrus. These changes in ER concentrations paralleled SE2 levels measured in intact cycling animals; i.e., during estrus SE2 levels were low, but began to rise during metestrus, diestrus, and during the morning of proestrus with a maximum peak increase during the afternoon of proestrus. These data indicate there are fluctuations of cytosolic ER concentrations during the estrous cycle and that these changes coincide with changing SE2 concentrations suggesting that ER content is influenced by SE2.

Establishment and characterization of a highly tumorigenic human diploid endometrial cancer cell line

A new cell line designated CUME-1 has been established from a poorly differentiated endometrial adenocarcinoma of the uterus. This cell line grew well without interruption for more than 88 months and 110 serial passages were successively carried out. The cells were highly tumorigenic in nude mice (85%). Repeated karyotype analyses from early (4th) to late (55th) passages of this cell line revealed a diploid stable clone in each passages without any noticeable structural or numerical aberrations. But from the 80th passage, a subpopulation with reciprocal translocation between chromosomes 1q and 9q consistently appeared and was observed in about 30% of the cells. This cell line is one of the rare examples of experimentally proved tumorigenic cells of human solid tumor origin that retains the diploid karyotype in vitro. HLA typing indicated the presence of DR4, DR13, DQ3, and DQ6. Cytosol estrogen and progesterone receptors were found both in fresh primary tumor and in this cell line. Gonadotropin-releasing hormone (Gn-RH) receptor mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in cultured cells. Using the single-strand conformation polymorphism (SSCP) technique, we have screened CUME-1 cells for p53 mutation in exons 4 to 9. No mobility shift was observed. This cell line may be useful in studying the in vitro chromosomal evolution of the cell line and the in vivo properties of human endometrial adenocarcinoma.

Hydroxylated Polychlorinated Biphenyls (PCBs) as Estrogens and Antiestrogens: Structure–Activity Relationships

The effects of structure on the estrogenicity and antiestrogenicity of hydroxylated polychlorinated biphenyls were investigated using the following estrogen-sensitive assays: competitive binding to the rat and mouse cytosolic estrogen receptor (ER); immature rat and mouse uterine wet weight, peroxidase and progesterone receptor (PR) levels; induction of luciferase activity in HeLa cells stably transfected with a Gal4:human ER chimera and a 17mer-regulated luciferase reporter gene; proliferation of MCF-7 human breast cancer cells; induction of chloramphenicol acetyl transferase (CAT) activity in MCF-7 cells transiently transfected with a full-length human ER expression plasmid and a plasmid containing an estrogen-responsive vitellogenin A2 promoter linked to a CAT reporter gene. The chemicals synthesized for this study contained a 4-hydroxy group in one ring, a 2- or 3-chloro substituentmetaororthoto the hydroxyl group, and variable substitution (2′,3′,4′,5′-, 2′,3′,4′,6′-, 2′,3′,5′,6′-tetrachloro and 2′,4′,6′-trichloro) in the chlorophenyl ring. The compounds included: 2,2′,3′,4′,5′- (A), 2,2′,3′,4′,6′- (B), and 2,2′,3′,5′,6′-pentachloro- (C); 2,2′,4′,6′-tetrachloro-4-biphenylol (D); 2′,3,3′,4′,5′- (E), 2′,3,3′,4′,6′- (F), and 2′,3,3′,5′,6′-pentachloro (G); and 2′,3,4′,6′-tetrachloro-4-biphenylol (H). With the exception of 2′,3,4′,6′-tetrachloro-4-biphenylol (H), all of the compounds competitively bound to the mouse and rat ER with relative binding affinities [compared to 17β-estradiol (E2)] varying from 1.4 × 10−3to 5.3 × 10−5. The structure–ER binding relationships for the hydroxy-PCB congeners were different in the rat and mouse, and no dose-dependent estrogenic activities were observed in the mouse or rat uterus. Several hydroxy-PCB congeners exhibited antiestrogenic activity (primarily in the mouse uterus) and two compounds, 2,2′,3′,5′,6- and 2,2′,3′,4′,6′-pentachloro-4-biphenylol, inhibited E2-induced uterine wet weight, PR binding, and peroxidase activity in the mouse uterus. 2,2′,3′,4′,5′- and 2,2′,3′,4′,6′-Pentachloro-4-biphenylol induced CAT activity in MCF-7 cells transiently transfected with the Vit-CAT plasmid; the remaining congeners did not induce CAT activity but exhibited antiestrogenic activity in MCF-7 cells cotreated with 10−9E2 plus 10−5mhydroxy-PCBs. Complementary structure–estrogenicity relationships were observed utilizing the HeLa cell luciferase induction and MCF-7 cell proliferation assays. The placement of the 2- or 3-chloro groups in the phenolic ring had minimal effects on estrogenic activity, whereas 2,4,6-trichloro- and 2,3,4,6-tetrachloro substitution in the chlorophenyl ring (B, D, F, and H) were required for this response. Substitution in the phenolic ring was also not important for structure–antiestrogenicity relationships, and the most active compounds (A, C, E, and G) contained 2′,3′,4′,5′- and 2′,3′,5′,6′-tetrachlorophenyl groups. Thus, structure–estrogenicity/antiestrogenicity relationships for this series of hydroxy-PCBs were complex and response-specific.

Two populations of the estrogen receptor separated and characterized using aqueous two-phase partitioning.

Two populations of the rat uterine estrogen receptor (ER) were separated and characterized using aqueous two-phase partitioning. Countercurrent distribution of rat uterine cytosolic ER allowed rapid and efficient separation of two populations, one population partitioned preferentially into the top phase (T, K(obs) = 3-6) and the other into the bottom phase (B, K(obs) = 0.01-0.03). The majority of unoccupied cytosolic ER is in the T population. Upon estrogen binding and/or heating to 30 degrees C in vitro the T population is converted to the B population. The transition from T to B does not exclusively involve loss of heat shock protein 90 and does not alter the ligand binding ability of the steroid binding domain. Using the human ER steroid binding domain overproduced in Escherichia coli and the steroid binding domain generated by partial trypsinization of the rat uterine ER, we demonstrate that the characteristic distinguishing T and B populations is not localized to this domain alone but may be associated with the amino terminal half of the ER (the A/B and DNA binding domains). The T to B transition of the ER also occurs in human MCF-7 breast cancer cells upon treatment with estrogen at 37 degrees C.

Response of the primate secretory endometrium to subchronic hypercortisolemia.

To assess the impact of subchronic and moderate hypercortisolism on the secretory endometrium of the cynomolgus monkey. Osmotic pumps containing hydrocortisone phosphate (HP) were implanted subcutaneously in each monkey on the first day of the menstrual cycle; each monkey also received pumps containing saline in another cycle. Blood was obtained three times per week and urine was collected daily for hormone analyses. Endometriectomy was performed 13 +/- 1 days after the serum estradiol (E2) peak in each study cycle. Infusion of HP elevated serum cortisol levels by an average of 70%. Mean serum progesterone (P) levels were decreased by 50% during the secretory phase of HP-treatment cycles by comparison with self-control cycles (P < .01); as a result, the mean endometrial glycogen concentration was reduced by 30% (P < .05) and the activity of 17 beta-hydroxysteroid dehydrogenase was decreased by 70% (P < .05). Serum E2 levels were not consistently elevated by HP treatment, but cytosolic estrogen receptor levels of the endometrium were decreased by 50% (P < .01), indicating increased estrogenic stimulation. Histologic development of the secretory endometrium was retarded, but the length of the secretory phase was not affected by the treatment. A moderate elevation of serum cortisol levels over one menstrual cycle consistently produced a reduction in serum P and a hypoprogestogenic-hyperestrogenic response of the secretory endometrium in the cynomolgus monkey.

A new cell line from human undifferentiated carcinoma of the ovary: establishment and characterization.

A cell line designated CUMO-2 has been established from an undifferentiated ovarian carcinoma. The s.c. injection of cells into nude mice gave rise to fast-growing tumors, while the i.p. route induced a peritoneal carcinomatosis with ascites. Histopathologically, the transplanted s.c. tumors closely resembled the original tumor, but tumors developed in the peritoneal cavity were highly anaplastic. The epithelial nature of the cells was confirmed by ultrastructural analysis. Sequential cytogenetic analyses on early and late passages revealed highly aneuploid tumor cells with consistent structural aberrations of chromosomes 1, 3, 8 and 11. CUMO-2 cells were found to produce CA 125 in vitro and in vivo. Cytosol estrogen receptor (ER) was found but progesterone receptor (PR) was not measured. HLA typing indicated the presence of DR8 and DQw4. A gonadotropin-releasing hormone (Gn-RH) analog inhibited cell growth and Gn-RH receptor mRNA was detected by reverse transcription/polymerase chain reaction in this cell line. Administration of transforming growth factor beta 1 inhibited both cell growth and c-myc mRNA expression. This cell line demonstrated a conformational band shift in exon 7 of the p53 gene. It was a frameshift mutation.

Decrease of Hormone Binding Capacity of Estrogen Receptor by Calcium

We have adressed the question as to whether calcium may modify the [3H]estradiol ([3H]E2) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues.Ca++ (0.1–10 mM) always produced an important loss of [3H]E2 binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [125I]Z-17 α-(2-iodovinyl)-11β-chloromethyl estradiol-17β (an compound with very high selectivity for ER) as well as [125I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca++- induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [3H]E2 binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca++ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca++ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.

Antiestrogenic Activity of Hydroxylated Polychlorinated Biphenyl Congeners Identified in Human Serum

Several hydroxylated polychlorinated biphenyls (PCBs) identified in human serum have been synthesized and these include 2,2′,3,4′,5,5′-hexachloro-4-biphenylol; 2,3,3′,4′,5-pentachloro-4-biphenylol; 2′,3,3′,4′,5-pentachloro-4-biphenylol; 2,2′,3,3′,4′,5-hexachloro-4-biphenylol; 2,2′,3,3′,4′,5,5′-heptachloro-4-biphenylol; 2,2′,3,4′,5,5′,6-heptachloro-4-biphenylol; and 2,2′,3′,4,4′,5,5′-heptachloro-3-biphenylol. The hydroxy-PCBs exhibited minimal binding to the rat uterine cytosolic estrogen receptor (ER) and did not induce proliferation of estrogen-responsive MCF-7 human breast cancer cells at concentrations ranging from 10−5to 10−8m. The estrogenic activity of these compounds was further investigated utilizing two estrogen-responsivein vitrobioassays, namely, (i) HeLa cells stably transfected with a Gal4:human ER chimera and a 17-mer-regulated luciferase reporter gene, and (ii) MCF-7 cells transiently transfected with a full-length human ER expression plasmid and a plasmid containing an estrogen-responsive vitellogenin A2 promoter linked to a chloramphenicol acetyl transferase (CAT) reporter gene. None of the hydroxy-PCBs significantly induced luciferase activity in the stably transfected HeLa cells or CAT activity in MCF-7 cells at concentrations as high as 10−5m. The antiestrogenic effects of the hydroxy-PCBs were also investigated using the same bioassays in which the cells were cotreated with 17β-estradiol plus the hydroxy-PCBs. All of the hydroxy-PCB congeners inhibited one or more estrogenic response, and one congener, 2,2′,3,4′,5,5′,6-heptachloro-4-biphenylol, inhibited 17β-estradiol-induced cell proliferation and CAT activity in MCF-7 cells and luciferase activity in HeLa cells.

Long-term Prognostic Impact of Immunohistochemical Estrogen Receptor Determinations Compared with Biochemical Receptor Determination in Primary Breast Cancer

The estrogen receptor content of primary breast cancer is predictive for the outcome and response to hormonal manipulation ( I ) . Most data on the prognostic implication of estrogen receptor determinations have been generated with biochemical receptor determinations utilising the dextran-coated charcoal (DCC) method and more recently the RIA assay (2). During the last decade immunohistochemical (IH) receptor determinations have gained in popularity due to their lower costs and the lower requirements for sample size as well as the possibility to perform the determination on archival tumour material. A large number of publications have demonstrated a good correlation between IH and biochemical receptor determinations, but also clearly discrepant findings have been reported (for a review see (2)). Few studies have assessed the clinical utility of IH. A number of studies have shown that the predictive value of IH receptor determinations for endocrine treatment response are at least as good as those of biochemical assays for estrogen receptor binding (2). Only a few studies have compared the prognostic value of IH and biochemical estrogen receptor determinations (3-5). The purpose of the present investigation was to study the correlation between IH and DCC estrogen receptor determination as well as their prognostic value for recurrence and death. Material und Methods. A total of 142 consecutive cases of invasive breast cancer analysed at the Department of Pathology, University of Helsinki, during the years 1987-1988, were included in the study. The distribution of TNM-classes and the proportion of estrogen receptor positivity are shown in the Table. In 9 cases the determination was performed on a local recurrence. These patients are excluded from the analysis on the prognostic value of receptor determinations. One patient with previous metastatic contralateral breast cancer was excluded from analysis of survival and disease-free survival. Other patients with synchronous or metachronous bilateral breast cancer (n = 18) were included in all the analyses. In 5 cases TN-status was not known, all were cases with receptor determinations from a local recurrence. The turnour samples were sent to the laboratory on ice and arrived unfixed to the histological laboratory for diagnosis and estrogen receptor (ER) staining. Immunohistochemical staining for ER was performed on frozen sections using the ER-ICA kit (Abbott Laboratories, Chicago, IL, USA). The sections were fixed and stained for ER following the manufacturer’s directions, counterstained lightly in Mayer’s hematoxylin and mounted. The percentage of positively stained turnour cell nuclei was estimated by counting 5001 000 morphologically malignant cells in multiple random fields in every sample. The turnours were divided into four categories according to the number of positive nuclei found: 0-9% = negative, 10-39% = weakly positive (+), 40-69% moderately positive (+ +) and 70% or more = strongly positive ( + + +). Only nuclear immunohistochemical staining was regarded as positive. A sample of each tumour was taken by the surgeon, immediately frozen in liquid nitrogen and analysed at the Department of Clinical Chemistry for cytosol ER by the dextran-coated charcoal method (6) . Cause-specific disease-free survival (DFS) and cause-specific overall survival (0s) were estimated by the Kaplan-Meier method, and the statistical significance of differences tested by the log-rank test. For analysis of both 0s and DFS only breast cancer-related deaths were classified as events, i.e., patients dying from other causes without breast cancer recurrences were classified as censored at the time of death. Follow-up times ranged from 7 to 9 years with a median of 8 years. Fifty-eight patients (42%) had TI tumours, 62 (45%) T2, 12 (9%) T3 and 4 (4%) T4 tumours. Sixty-two patients (45%)) had axillary metastases. Only one patient had distant metastases at diagnosis. Fifty-four patients have died, 13 from causes other than breast cancer without evidence of recurrence. Results. The correlation between IH ER score and ER positivity by DCC assay was studied using a cut-off point of 10 fmol/rng protein (Table). In 44 cases (31%) the result was discrepant. Thirty-three cases (23%) were IH positive and DCC negative, whereas 11 cases (8%) were IH negative and DCC positive. The

CUMC-6, a New Diploid Human Cell Line Derived from a Squamous Carcinoma of the Uterine Cervix

A new cell line, CUMC-6, has been derived from an invasive nonkeratinizing squamous cell carcinoma of the uterine cervix in a 31-year-old patient. It has been maintained in long-term culture for 61 months, and passaged over 300 times. Monolayer-cultured cells were polygonal in shape, showing a pavement-like arrangement and a tendency to pile up without contact inhibition. The epithelial nature of the cultured CUMC-6 cells was confirmed by transmission electron microscopy which demonstrated the presence of desmosomes and tonofilaments. The subcutaneous injection of cultured cells into nude mice gave rise to fast-growing tumors. The transplanted tumor showed similar histological features, but poor differentiation compared to the original tumor. Cultured CUMC-6 cells produced human chorionic gonadotropin β-subunit (β-HCG) and α-fetoprotein (AFP). Cytosol estrogen and progesterone receptors were not measured in this cell line. The results of isozyme analyses were distinct from the HeLa cell line. Repeated chromosome analysis from passage 6 to 300 revealed that most metaphases of this cell line contained diploid number of chromosomes. The structural abnormality consistently observed in this cell line was the elongation of short arm of chromosome 1. The G- or R-banded pattern of this chromosome suggested inv dup (1) (1pter → 1p34:: 1p21 → 1p34:: 1p34 → 1qter). Human leukocyte antigen (HLA) typing of CUMC-6 cells indicated the presence of DR12 and DQw3. Analysis of the DNA extracted from the CUMC-6 cells showed the presence of human papillomavirus (HPV) type 16 and 18 DNAs. The results of oncogene analyses using Southern blotting technique revealed amplification and rearrangement of oncogene c-mycand no amplification of oncogene L-myc.Using the polymerase chain reaction–single strand conformation polymorphism (PCR–SSCP) technique, we have screened CUMC-6 cells forp53mutation in exons 4 to 9. No mobility shift was observed in this cell line. These results suggest that chromosome 1 abnormality, oncogene alteration, and HPV infection work together in cervical tumorigenesis.

Effect of benomyl and carbendazim on steroid and molecular mechanisms in uterine decidual growth in rats.

The present study examined the influence of two benzimidazole fungicides benomyl and carbendazim (MCB), in dosages of 500 and 1000 mg kg-1 for 5 days, on the capacity of decidual growth in pseudopregnant rats. The aim of the research was to determine whether the antimitotic activity of both chemicals was being mediated by way of hormonal and molecular mechanisms at the uterine level. The results show that both fungicides produced reductions in uterine decidual weight (P < 0.01) and uterine protein content (P < 0.05). However, levels of serum estradiol and progesterone and the binding capacities of cytosol estrogen and progesterone receptors remained essentially unchanged. Subsequently, the antigrowth-antimitotic activity of benomyl and MCB, at 500 and 1000 mg kg-1 dosages, on the decidual uterus is direct, and apparently does not involve steroidal or receptor mechanisms.

Effect of Benomyl and Carbendazim on Steroid and Molecular Mechanisms in Uterine Decidual Growth in Rats

The present study examined the influence of two benzimidazole fungicides benomyl and carbendazim (MCB), in dosages of 500 and 1000 mg kg−1 for 5 days, on the capacity of decidual growth in pseudopregnant rats. The aim of the research was to determine whether the antimitotic activity of both chemicals was being mediated by way of hormonal and molecular mechanisms at the uterine level. The results show that both fungicides produced reductions in uterine decidual weight (P < 0.01) and uterine protein content (P < 0.05). However, levels of serum estradiol and progesterone and the binding capacities of cytosol estrogen and progesterone receptors remained essentially unchanged. Subsequently, the antigrowth-antimitotic activity of benomyl and MCB, at 500 and 1000 mg kg−1 dosages, on the decidual uterus is direct, and apparently does not involve steroidal or receptor mechanisms.

Estrogen Receptors in the Stingray (Dasyatis akajei) Ultimobranchial Gland

On the basis of our previous observation that estrogen stimulates calcitonin secretion by the ultimobranchial gland in the stingray,Dasyatis akajei,experiments were conducted to examine the presence of estrogen receptor (ER) and its mRNA in the gland, employing exchange assay and Northern blot analysis, respectively. The optimal incubation conditions of the exchange assay for cytosolic ER were found to be 25° for 2 hr. Scatchard analysis of cytosolic ER revealed two components with dissociation constants (Kd) of 0.090 ± 0.015 and 17.780 ± 1.910 nMand with a maximum number of binding sites (Bmax) of 62.2 ± 7.9 and 2290 ± 180 fmol/mg protein, respectively. Furthermore, Sephadex G-100 filtration of the cytosol and immunoblot analysis using antiserum against a recombinant rat ER confirmed the presence of two specific binding components for estrogen (41 and 57 kDa). Total RNA extracted from the ultimobranchial gland was subjected to Northern blot analysis using a rat ER cDNA as a probe. Two positive signals were detected at 2.5 and 2.0 kb. Thus, the presence of ER was confirmed for the first time in nonmammalian calcitonin-secreting cells.

Changes in radiation sensitivity and steroid receptor content induced by hormonal agents and ionizing radiation in breast cancer cells in vitro.

Possible influences of tamoxifen and estradiol on in vitro radiation sensitivity and cellular receptor content after irradiation and/or tamoxifen treatment were studied in breast cancer cell lines; estrogen receptor (ER) and progesterone receptor (PgR) positive cell lines MCF-7 and MCF-7/TAM(R)-1 and the ER and PgR negative cell line MDA-MB-231. The tamoxifen resistant MCF-7/TAM(R)-1 cells were more resistant to ionizing radiation than the MCF-7 and MDA-MB-231 cells. Exposure to tamoxifen made the MCF-7 cells more radiation resistant, while estradiol made the MDA-MB-231 cells more radiation sensitive. A radiation dose of 6 Gy reduced the ER content in cytosol in both MCF-7 and MCF-7/TAM(R)-1 cells, but brought no alterations to the PgR content. In MCF-7/TAM(R)-1 cells tamoxifen exposure significantly increased the ER and reduced the PgR content, an effect not observed in the MCF-7 cells. To conclude, the present study indicates that irradiation and tamoxifen may modify the ER and PgR content in cytosol in breast cancer cells. Hormonal treatment may alter the radiation sensitivity, even in ER negative cells, suggesting that hormonal agents may act both via receptor and non-receptor binding mechanisms.