Study protocol for a randomized controlled trial of the NEIVATECH virtual reality system to improve visual function in children with anisometropic amblyopia.

BACKGROUND: Interest in developing alternative methods for the treatment of amblyopia has long been a topic of interest among clinicians and researchers, as prescribed occlusion and penalization therapies do not always provide an effective response and are associated with a high risk of recurrence and non-compliance. Here, we present the protocol of a small-scale RCT to evaluate the safety and clinical efficacy of a novel VR-based system designed to provide binocular vision training to children with anisometropic amblyopia. METHODS: We aim to recruit a total of 60 children with anisometropic amblyopia aged 5-17 years with no previous treatment for amblyopia other than refractive correction from the pediatric ophthalmology units of the University Clinical Hospital of Valladolid and the Vithas Medimar International Hospital of Alicante. Children who meet the eligibility criteria and consent to participate will be randomly assigned to a three-month intervention group of 18 half-hour in-office therapy sessions with the NEIVATECH system (group A) or to a parallel group receiving 2 hours of conventional patching per day at home for the same period of time (group B). Assessments of visual function will be carried out before the intervention and at 1, 2 and 3 months, with changes in distance BCVA being the primary outcome measure to be considered. Patient safety, compliance, satisfaction and acceptance to treatment will also be assessed after therapy as other valuable outcome measures. In addition, a rsfMRI scan will be performed on a subgroup of 5 patients from each group at the pre-intervention visit and at the post-intervention visit to test the effects of both therapies on neural plasticity in the visual cortex. DISCUSSION: The NEIVATECH system has been conceived as a serious game designed to provide binocular vision training to anisometropic amblyopic children by complementing the concepts of perceptual learning, dichoptic training and gamification in an immersive VR environment. We hope that this novel approach may lead to greater improvements in vision performance than those provided so far by conventional patching in anisometropic amblyopic children. TRIAL REGISTRATION: This protocol was registered with ClinicalTrials.gov ( NCT04819386 ) on 29 March 2021.

Profile of patients who visit medical internists for an osteoporosis assessment: The OSTEOMED registry. / Perfil de los pacientes que acuden al médico internista para valoración de osteoporosis: registro OSTEOMED.

BACKGROUND AND OBJECTIVES: Osteoporosis is considered a generalised skeletal disorder in which there is impaired bone resistance, which predisposes the individual to a greater risk of fracture. The aim of this cross-sectional study was to collect and present data on the main clinical characteristics of patients who consult medical internists in Spain. Understanding these characteristics can help in implementing action plans to improve these patients' care more effectively and efficiently. MATERIAL AND METHODS: Through an analysis of the Osteoporosis in Internal Medicine (OSTEOMED) registry, this study presents the main clinical characteristics of patients with osteoporosis who attended internal medicine consultations in 23 Spanish hospital centres between 2012 and 2017. We analysed the reasons for the consultations, the densitometric values, the presence of comorbidities, the prescribed treatment and other lifestyle-related factors. RESULTS: In total, 2024 patients with osteoporosis were assessed (89.87% women, 10.13% men). The patients' mean age was 64.1±12.1 years (women, 64.7±11.5 years; men, 61.2±14.2 years). There was no significant difference between the sexes in their history of recent falls (9.1% and 6.7%); however, there were significant differences in the daily intake of calcium from milk products (553.8±332.6mg for women vs. 450.2±303.3mg for men; P<.001) and in the secondary causes of osteoporosis (13% of men vs. 6.5% of women; P<.001). In the sample, there were 404 fractures (20%), with a notable number of confirmed vertebral fractures (17.2%, 35.6% in men vs. 15.2% in women; P<.001). A large portion of the patients did not undergo the indicated treatment and presented low levels of physical activity and sun exposure. A significant percentage of the patients presented associated comorbidities, the most common of which were hypertension (32%) and dyslipidaemia (28%). CONCLUSIONS: These results define the profile of patients with osteoporosis who attend internal medicine consultations in Spain. The results also show the multisystemic character of this condition, which, along with its high prevalence, determine that the specific internal medicine consultations dedicated to managing the condition are the appropriate place for caring for these patients.

Atomic layer deposited oxide films as protective interface layers for integrated graphene transfer.

The transfer of chemical vapour deposited graphene from its parent growth catalyst has become a bottleneck for many of its emerging applications. The sacrificial polymer layers that are typically deposited onto graphene for mechanical support during transfer are challenging to remove completely and hence leave graphene and subsequent device interfaces contaminated. Here, we report on the use of atomic layer deposited (ALD) oxide films as protective interface and support layers during graphene transfer. The method avoids any direct contact of the graphene with polymers and through the use of thicker ALD layers (≥100 nm), polymers can be eliminated from the transfer-process altogether. The ALD film can be kept as a functional device layer, facilitating integrated device manufacturing. We demonstrate back-gated field effect devices based on single-layer graphene transferred with a protective Al2O3 film onto SiO2 that show significantly reduced charge trap and residual carrier densities. We critically discuss the advantages and challenges of processing graphene/ALD bilayer structures.

Profile of patients who consult with internists for an osteoporosis assessment: The OSTEOMED registry.

BACKGROUND AND OBJECTIVES: Osteoporosis is considered a generalised skeletal disorder in which there is impaired bone resistance, which predisposes the individual to a greater risk of fracture. The aim of this cross-sectional study was to collect and present data on the main clinical characteristics of patients who consult medical internists in Spain. Understanding these characteristics can help in implementing action plans to improve these patients' care more effectively and efficiently. MATERIAL AND METHODS: Through an analysis of the Osteoporosis in Internal Medicine (OSTEOMED) registry, this study presents the main clinical characteristics of patients with osteoporosis who attended internal medicine consultations in 23 Spanish hospital centres between 2012 and 2017. We analysed the reasons for the consultations, the densitometric values, the presence of comorbidities, the prescribed treatment and other lifestyle-related factors. RESULTS: In total, 2024 patients with osteoporosis were assessed (89.87% women, 10.13% men). The patients' mean age was 64.1±12.1 years (women, 64.7±11.5 years; men, 61.2±14.2 years). There was no significant difference between the sexes in their history of recent falls (9.1% and 6.7%); however, there were significant differences in the daily intake of calcium from milk products (553.8±332.6mg for women vs. 450.2±303.3mg for men; p<.001) and in the secondary causes of osteoporosis (13% of men vs. 6.5% of women; p<.001). In the sample, there were 404 fractures (20%), with a notable number of confirmed vertebral fractures (17.2%, 35.6% in men vs. 15.2% in women; p<.001). A large portion of the patients did not undergo the indicated treatment and presented low levels of physical activity and sun exposure. A significant percentage of the patients presented associated comorbidities, the most common of which were hypertension (32%) and dyslipidaemia (28%). CONCLUSIONS: These results define the profile of patients with osteoporosis who attend internal medicine consultations in Spain. The results also show the multisystemic character of this condition, which, along with its high prevalence, determine that the specific internal medicine consultations dedicated to managing the condition are the appropriate place for caring for these patients.

[Anesthesia in interventional neuroradiology]. / Anestesia en neurorradiología intervencionista.

Interventional neuroradiology procedures have become increasingly complex, requiring planning and coordination. Key roles are played by the anesthesiologist and the radiologist, as well as by technicians and nurses. This review aims to analyze the anesthesiologist's part in managing these procedures, from the start of the intervention through the immediate postoperative period. First concerns are to assure patient safety during transfer, maintain the airway, keep the patient immobile and hemodynamically stable, and manage anticoagulant and antiplatelet treatments. Rapid awakening must also be assured so that the patient's neurologic status can be assessed in situ. The anesthesiologist should treat any neurologic complications that develop and that might lead to emergency situations during the procedures.

TG101209, a small molecule JAK2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated JAK2V617F and MPLW515L/K mutations.

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling, which is implicated in MPD pathogenesis. We developed TG101209, an orally bioavailable small molecule that potently inhibits JAK2 (IC(50)=6 nM), FLT3 (IC(50)=25 nM) and RET (IC(50)=17 nM) kinases, with significantly less activity against other tyrosine kinases including JAK3 (IC(50)=169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC(50) of approximately 200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209-induced cell cycle arrest and apoptosis, and inhibited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore, TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations.

[Ten years' experience with vagus nerve stimulation in a paediatric population]. / Diez años de experiencia con el estimulador del nervio vago en una poblacion pediatrica.

INTRODUCTION: Fifty million people are affected by epilepsy. Up to 30% are not controlled with the aid of antiepileptic drugs. The vagus nerve stimulator (VNS) is a therapeutic alternative that must be taken into account. AIMS: To determine the effect of the VNS in a cohort of paediatric patients with refractory epilepsy. PATIENTS AND METHODS: A retrospective study of children with a VNS implanted between 2008 and 2017 in a tertiary hospital. Epidemiological, aetiological, clinical and electrophysiological data, along with VNS parameters were analysed. RESULTS: The study included 35 patients, with a mean age when the VNS was implanted of 12.84 years (range: 3.1-18.7 years) and a mean time between onset of epilepsy and implantation of 7.2 years (range: 1.3-17.7 years). The causation was structural in 62.9% of cases. The most frequent epileptic conditions were: Lennox-Gastaut syndrome and focal epilepsy, with a predominance of tonic seizures (57.1%). The video electroencephalogram showed multifocal anomalies (54%) and a pattern of epileptic encephalopathies (34.3%). Intellectual disability was associated in 94% of the cases. The mean of previous antiepileptic drugs was 9.6 ± 3 (range: 4-16). 43% responded to treatment (>= 50% reduction in number of seizures), with a mean reduction of 67.3%, which improved with higher ages of onset of epilepsy. Three patients were seizure-free (8.5%). The number of seizures decreased by 33% at six months and by 47.4% at 24 months. There was also a notable degree of cognitive (57%) and behavioural improvement (53%). In 28% of cases there were some side effects, but in general they were mild. CONCLUSIONS: The VNS is a valid option in refractory epilepsy, with improvements not only in terms of seizures but also regarding cognitive-behavioural aspects, this being very important for the paediatric population.

TITLE: Diez años de experiencia con el estimulador del nervio vago en una poblacion pediatrica.Introduccion. La epilepsia afecta a 50 millones de personas. Hasta un 30% no se controla con farmacos antiepilepticos. El estimulador del nervio vago (ENV) constituye una alternativa terapeutica que hay que valorar. Objetivo. Determinar el efecto del ENV en una cohorte pediatrica con epilepsia refractaria. Pacientes y metodos. Estudio retrospectivo de niños con ENV implantado entre 2008 y 2017 en un hospital terciario. Se han analizado datos epidemiologicos, etiologicos, clinicos, electrofisiologicos y parametros del ENV. Resultados. Se incluyo a 35 pacientes, con una mediana de edad de implantacion de 12,84 años (rango: 3,1-18,7 años) y una mediana de evolucion entre el inicio de la epilepsia y la implantacion de 7,2 años (rango: 1,3-17,7 años). La etiologia fue estructural en el 62,9% de los casos. Cuadros epilepticos mas frecuentes: sindrome de Lennox-Gastaut y epilepsia focal, con predominio de las crisis tonicas (57,1%). El videoelectroencefalograma mostro anomalias multifocales (54%) y un patron de encefalopatia epileptica (34,3%). El 94% asociaba discapacidad intelectual. La media de farmacos antiepilepticos previos fue de 9,6 ± 3 (rango: 4-16). El 43% fueron respondedores (>= 50% reduccion de crisis), con una media de reduccion del 67,3%, mejor cuanto mayor era la edad de inicio de la epilepsia. Tres pacientes quedaron libres de crisis (8,5%). La reduccion de crisis fue del 33% a los 6 meses y del 47,4% a los 24 meses. Mejoria cognitiva (57%) y conductual (53%). El 28% tuvo efectos secundarios, generalmente leves. Conclusiones. El ENV es una opcion valida en la epilepsia refractaria con mejoria no solo de las crisis, sino tambien cognitiva y conductual, con la importancia que ello tiene para la poblacion pediatrica.

Consumption of a high-fat diet alters estrogen receptor content, protein kinase C activity, and mammary gland morphology in virgin and pregnant mice and female offspring.

Previous studies have shown that a diet high in polyunsaturated fatty acids increases mammary tumor incidence in adult and pregnant mice and rats and in the female offspring. The present study investigated whether a high-fat diet alters the number of estrogen receptor (ER) binding sites and protein kinase C (PKC) activity in the mammary gland of these animals. In the female offspring, the effects of maternal exposure to a high-fat diet during pregnancy on development of the mammary epithelial tree were studied also. BALB/c mice were kept on a diet containing either 43% (high-fat) or 16% (low-fat) calories from corn oil, which consists mostly of n-6 polyunsaturated fatty acids, for 1 month. In adult female mice, a 6-fold increase in the number of ER binding sites and 2-fold increase in PKC activity were found in the mammary glands of the high-fat mice when compared with the low-fat mice. In pregnant mice, a high-fat diet increased ER binding sites by 61% and PKC activity by 51%. In contrast to adult and pregnant mice, females exposed to a high-fat diet only in utero through their pregnant mother exhibited a significantly reduced number of mammary ER binding sites by age 45 days (78% decrease) and a reduction in PKC activity by ages 30 and 100 days (44 and 20% decrease, respectively). The mammary epithelial tree of the high-fat offspring contained more terminal end buds and was less differentiated than that of the low-fat offspring. These findings show that consumption of a high-fat diet increases ER and PKC in the adult and pregnant mouse mammary gland, perhaps contributing to the fat-induced promotion of mammary tumorigenesis. In contrast, reduced ER and PKC following a high-fat exposure in utero may be associated with increased susceptibility to carcinogenesis, possibly due to an increased number of terminal end buds that are the sites of neoplastic transformation in the mammary gland.

Activation of estrogen receptor-alpha by the heavy metal cadmium.

Previous studies from this laboratory have shown that the heavy metal cadmium (Cd) mimics the effects of estradiol in estrogen-responsive breast cancer cell lines. To understand the mechanism by which cadmium activates estrogen receptor-alpha (ER-alpha), the ability of cadmium to bind to and activate wild-type and various mutants of ER-alpha was examined. When tested in transient cotransfection assays in COS-1 cells, cadmium concentrations as low as 10(-11) M activated ER-alpha. Scatchard analysis employing either purified human recombinant ER-alpha or extracts from ER-containing MCF-7 cells demonstrated that l09Cd binds to the ER with an equilibrium dissociation constant of approximately 4 to 5 x 10(-10) M. Cadmium also blocks the binding of estradiol to ER-alpha in a noncompetitive manner (K(i) = 2.96 x 10(-10) M), suggesting that the heavy metal interacts with the hormone-binding domain of the receptor. To study the role of the hormone-binding domain in cadmium activation, COS-1 cells were transiently cotransfected with GAL-ER, a chimeric receptor containing the DNA-binding domain of the transcription factor GAL4 and the hormone-binding domain of ER-alpha, and a GAL4-responsive reporter gene. Treatment of the transfected cells with either 10(-6) M cadmium or 10(-9) M estradiol resulted in a 4-fold increase in reporter gene activity. The effect of cadmium on the chimeric receptor was blocked by the antiestrogen, ICI-164,384, suggesting that cadmium activates ER-alpha through an interaction with the hormone-binding domain of the receptor. Transfection and binding assays with ER-alpha mutants identified C381, C447, E523, H524, and D538 as possible interaction sites of cadmium with the hormone-binding domain of ER-alpha.

Posttranscriptional regulation of albumin gene expression in Xenopus liver: evidence for an estrogen receptor-dependent mechanism.

We have previously shown that estrogen administration to male Xenopus laevis results in the posttranscriptional suppression of serum albumin mRNA concurrent with the transcriptional activation of the genes for the yolk protein precursor vitellogenin. To determine whether the posttranscriptional regulation of albumin gene expression is mediated through a mechanism involving the high affinity estrogen receptor protein or through a receptor-independent mechanism involving a middle affinity cytoplasmic estrogen-binding protein we examined the effects of the competitive estrogen receptor antagonist 4-hydroxytamoxifen. Administration of 4-hydroxytamoxifen 24 h before estradiol completely blocked both the suppression of albumin mRNA and the transcriptional activation of the vitellogenin genes. Albumin gene transcription remained constitutive under all treatment regimens. Competitive binding experiments demonstrated that 4-hydroxytamoxifen has an affinity for the estrogen receptor similar to that of estradiol. However, 4-hydroxytamoxifen displays little or no interaction with the middle affinity cytoplasmic estrogen-binding protein. These data indicate that the estrogen receptor occupies a key role in the posttranscriptional regulation of albumin mRNA.

Role of an estrogen receptor-dependent mechanism in the regulation of estrogen receptor mRNA in MCF-7 cells.

We have previously demonstrated that regulation of estrogen receptor (ER) expression in MCF-7 breast cancer cells is a complex process involving transcriptional and posttranscriptional regulation by estradiol. Treatment of MCF-7 cells with estradiol results in the down-regulation of receptor expression; posttranscriptional suppression of receptor mRNA appears to be the predominant mechanism. To determine whether posttranscriptional regulation of ER gene expression is mediated by an ER-dependent mechanism independent of protein synthesis, we have used the competitive estrogen antagonist, 4-hydroxytamoxifen, and the inhibitor of protein synthesis, cycloheximide, to study regulation of ER mRNA by estradiol. 4-Hydroxytamoxifen had no effect on the steady-state level of receptor mRNA and effectively blocked the suppression of ER mRNA by estradiol. The metabolic inhibitor, cycloheximide, was unable to prevent the estrogen induced decrease in ER mRNA. These data provide evidence that the posttranscriptional suppression of ER expression through estradiol is mediated through the ER independent of protein synthesis. A study of the effects of estradiol on the steady-state levels of nuclear and cytoplasmic receptor mRNA suggest that posttranscriptional suppression is a nuclear event.

Regulation of the estrogen receptor in MCF-7 cells by estradiol.

The role of estradiol in the regulation of its cognate receptor in MCF-7 cells was investigated in this study. After treatment with 10(-9) M estradiol, the level of receptor protein was measured using an enzymeimmunoassay. By 6 h, the receptor protein declined by about 60% from a level of approximately 3.6 to 1.2 fmol/micrograms DNA. The level of receptor remained suppressed for 24-48 h. Similar results were obtained with an estrogen receptor (ER) binding assay. The steady state level of ER mRNA was determined by an RNase protection assay. Estrogen treatment resulted in a maximum suppression of mRNA by 6 h. Receptor mRNA remained depressed for 48 h. Transcription run on experiments demonstrated a transient decrease of about 90% in ER transcription after 1 h. By 3-6 h transcription increased approximately 2-fold and remained elevated for at least 48 h. These data suggest that estrogen down-regulates ER mRNA by inhibition of ER gene transcription at early times and by a posttranscriptional effect on receptor mRNA at later times.

Symptomatic venous thromboembolism in cancer patients treated with chemotherapy: an underestimated phenomenon.

BACKGROUND: The exact incidence of venous thromboembolism (VTE) in cancer patients is unknown, partly because of confounding factors. Prophylactic treatment is warranted in surgical patients with cancer because of a high incidence of VTE. We performed a retrospective study to evaluate if the same applies for cancer patients treated with chemotherapy. METHODS: The medical records of 206 consecutive patients with malignancy, treated with chemotherapy, were identified. The kind of malignancy and chemotherapeutic treatment were recorded, as was the date of treatment. The records were reviewed for other risk factors for VTE, and were searched for proved deep venous thrombosis or pulmonary embolism. RESULTS: Of those 206 patients, 15 (7.3%) had proved VTE during or within 3 months after chemotherapeutic treatment. The annual incidence was 10.9%. The incidence of VTE was specifically high in the 39 patients treated with a combination of fluorouracil and leucovorin calcium because of colorectal cancer (6 [15%] of the patients were affected). The occurrence of VTE in the latter group of patients was not influenced by factors such as surgery, central venous catheters, or tumor load. CONCLUSIONS: The annual incidence of VTE in patients treated with chemotherapy was high, specifically in patients with colorectal cancer treated with fluorouracil-leucovorin. If these observations are confirmed, trials to evaluate the use of prophylactic anticoagulant treatment should be conducted.

Effects of arsenite on estrogen receptor-alpha expression and activity in MCF-7 breast cancer cells.

To determine whether arsenite has estrogen-like activities, the effects of this compound on estrogen receptor-alpha (ERalpha) and other estrogen-regulated genes were measured in the human breast cancer cell line MCF-7. Treatment of cells with 1 microM arsenite resulted in a 60% decrease in the amount of ERalpha and in a parallel decrease of 40% in ERalpha messenger RNA. Progesterone receptor concentration increased 22-fold after arsenite treatment. pS2 messenger RNA also increased 2. 1-fold after treatment. The induction of progesterone receptor and pS2 was blocked by the antiestrogen ICI-182,780. In transient cotransfection experiments of wild-type ERalpha and an estrogen response element-reporter construct, arsenite stimulated chloramphenicol acetyltransferase (CAT) activity. In growth assays, arsenite significantly stimulated the proliferation of MCF-7 cells compared with cells grown in estrogen-depleted medium. Addition of an antiestrogen blocked growth stimulation by arsenite. In binding assays, arsenite blocked the binding of estradiol to ERalpha (Ki = 5 +/- 0.5 nM; n = 3), suggesting that the compound interacts with the hormone-binding domain of the receptor. To determine whether interaction of arsenite with the hormone-binding domain results in receptor activation, COS-1 cells were transiently cotransfected with the chimeric receptors GAL-ER, which contains the hormone-binding domain of ERalpha and the DNA-binding domain of the transcription factor GAL4, and a GAL4-responsive CAT reporter gene. Treatment of cells with estradiol or arsenite resulted in a 4-fold increase in CAT activity. The effects of arsenite on the chimeric receptor were blocked by the antiestrogen, suggesting that arsenite activates ERalpha through an interaction with the hormone-binding domain of the receptor. Transfection assays with ERalpha mutants identified C381, C447, H524, and N532 as interaction sites of arsenite with the hormone-binding domain.

Differential induction of hepatic estrogen receptor and vitellogenin gene transcription in Xenopus laevis.

Low levels of estrogen receptor (200-500 per cell) are present in the liver of hormonally naive male Xenopus. However, administration of estradiol results in a rapid 2- to 5-fold increase in cellular estrogen receptor content concurrent with the de novo transcriptional activation of the genes for the yolk protein precursor vitellogenin. Studies on Xenopus embryogenesis suggest that estrogen receptor induction is required for the activation of vitellogenin transcription. The purpose of the present study was to examine the mechanism of estrogen receptor induction in male Xenopus liver. The experimental protocol used 4-hydroxytamoxifen, an antiestrogen with a high affinity for the estrogen receptor, to inhibit the effects of estradiol. Changes in estrogen receptor content were then determined through the use of an exchange assay. 4-Hydroxytamoxifen alone suppressed the level of estrogen receptor from 800 sites per cell in hormonally naive animals to 250 sites per cell. Administration of estradiol 24 h after the antiestrogen resulted in the induction of estrogen receptor to a level equivalent to that found in control animals (800 sites per cell). However, under the same conditions, estradiol was unable to overcome the antiestrogen inhibition of vitellogenin gene transcription. Although 4-hydroxytamoxifen displayed a high affinity for the hepatic estrogen receptor, it did not inhibit the binding of estradiol to a middle affinity cytoplasmic estrogen-binding protein. These results suggest that different mechanisms are involved in the induction of estrogen receptor and vitellogenin gene transcription.

Regulation of estrogen receptor concentration and activity by an erbB/HER ligand in breast carcinoma cell lines.

Expression of the erbB-2 oncogene in breast cancer patients correlates with poor prognosis and failure of hormonal therapy. In this study, the effects of a putative erbB/HER ligand, gp30, on estrogen receptor (ER) concentration and activity was investigated in the estrogen receptor positive human breast cancer cells, BT474 and MCF-7, which express either high or low levels of erbB-2 and erbB-4, respectively. Treatment of cells with gp30 resulted in a decrease in the steady-state level of estrogen receptor protein by approximately 70-80%. The effect of gp30 on the concentration of ER was independent of serum in the media and was not inhibited by an epidermal growth factor receptor blocking antibody. In addition to the effect on ER protein, gp30 decreased the steady-state level of ER messenger RNA. Transcription run on experiments demonstrated that the decrease in ER expression was mediated by a decrease in ER gene transcription. The effect of gp30 on estrogen receptor activity was also investigated in this study. Treatment of cells with gp30 blocked estradiol induction of progesterone receptor. Inhibition was observed at the level of progesterone receptor protein, messenger RNA, and gene transcription. gp30 also blocked estradiol induction of pS2 gene transcription. In addition to its effects on progesterone receptor and pS2, gp30 blocked activation of an estrogen response element in a transient transfection assay and inhibited ER binding to its response element in a DNA mobility shift assay, suggesting a direct effect on the estrogen receptor. The effects of gp30 on estrogen receptor concentration and activity were independent of the level of erbB-2 and erbB-4 in the cell. These data show that gp30 regulates the concentration of ER and modulates ER activity.

The role of transforming growth factor-beta in the regulation of estrogen receptor expression in the MCF-7 breast cancer cell line.

The role of transforming growth factor-beta1 (TGFbeta1) in the regulation of estrogen receptor (ER) expression in MCF-7 cells was investigated. After treatment of the cells with 100 pM TGFbeta1, ER protein declined by about 30% at 6 h from a concentration of 413.5 fmol/mg protein in control cells to 289.5 fmol/mg protein in treated cells. The concentration of receptor remained suppressed for 24 h. Scatchard analysis demonstrated that the decrease in ER protein corresponded to a decrease in estradiol-binding sites, with no effect on the binding affinity of the ER. The dissociation constant of the estradiol-ER complex was 0.117 nM in TGFbeta1-treated cells compared to 0.155 nM in control cells. Treatment with TGFbeta1 did not influence the half-life of the ER. In TGFbeta1-treated cells, as well as in control cells, the half-life of the receptor was approximately 4 h. In contrast to the effect on ER concentration, TGFbeta1 treatment resulted in a greater decrease in the steady state level of ER messenger RNA (approximately 75%) at 6 h. By 24 h, a small recovery in the amount of messenger RNA was observed. Transcription run-on experiments demonstrated a decrease of approximately 70% in the level of ER gene transcription at 3 h. Transient transfections using an ER promoter-chloramphenicol acetyltransferase construct demonstrated that after TGFbeta1 treatment, chloramphenicol acetyltransferase activity decreased by 50%, suggesting that TGFbeta1 inhibition of the ER gene transcription is mediated through the ER promoter. Although treatment with TGFbeta1 decreased the ER concentration, the growth factor had no effect on the activity of ER, as measured by its effects on estradiol induction of progesterone receptor and pS2, suggesting that TGFbeta1 does not inhibit proliferation of MCF-7 cells by blocking ER activity.

A role for Akt in mediating the estrogenic functions of epidermal growth factor and insulin-like growth factor I.

This study examines whether the serine/threonine protein kinase, Akt, is involved in the cross-talk between epidermal growth factor (EGF) and insulin-related growth factor I (IGF-I) receptors and ER-alpha. Treatment of MCF-7 cells with either EGF or IGF-I resulted in a rapid phosphorylation of Akt and a 14- to 16-fold increase in Akt activity, respectively. Akt activation was blocked by inhibitors of phosphatidylinositol 3-kinase, but not by an inhibitor of the ribosomal protein kinase p70S6K. Stable transfection of cells with a dominant negative Akt mutant blocked the effects of EGF and IGF-I on ER-alpha expression and activity, whereas stable transfection of cells with a constitutively active Akt mutant mimicked the effects of EGF and IGF-I. In the latter cells, there was a decrease in the amount of ER-alpha protein and messenger RNA (70-80%) and an increase in the amount of progesterone receptor protein, messenger RNA (4- to 9- and by 3.5- to 7-fold, respectively) and pS2 (3- to 5-fold). Coexpression of wild-type ER-alpha and the dominant negative Akt mutant in COS-1 cells also blocked the growth factor-stimulated activation of ER-alpha, but coexpression of the wild-type receptor with the constitutively active Akt mutant increased ER-alpha activity. Receptor activation was blocked by an antiestrogen. Studies using mutants of ER-alpha demonstrated that Akt increased estrogen receptor activity through the amino-terminal activation function-1 (AF-1). Serines S104 S106, S118, and S167 appear to play a role in the activation of ER-alpha by Akt.

Regulation of retinoblastoma gene expression in hormone-dependent breast cancer.

Studies have shown an increased risk for breast cancer in the mothers of children suffering from retinoblastoma and osteosarcoma, suggesting a role for the retinoblastoma susceptibility (Rb) gene product in breast cancer. We now show that estradiol decreases the expression of Rb at the level of protein and messenger RNA (mRNA) in estrogen-dependent breast cancer cell lines. Treatment of MCF-7 cells with 10(-9) M estradiol for 48 h resulted in a 70% decrease in the level of Rb protein. Ribonuclease protection assays showed a 50% decrease in the steady state levels of Rb mRNA by 12 h and a 70% decrease in Rb mRNA by 24 h. Treatment with estradiol had no effect on the rate of Rb gene transcription or on Rb mRNA stability, but resulted in an increase in the steady state level of Rb mRNA in the nucleus. The effect of estradiol was inhibited by 10(-7) M 4-hydroxytamoxifen. In the absence of estradiol, the antiestrogens 4-hydroxytamoxifen and ICI 164,384 increased Rb mRNA by 50% over that in estrogen-depleted conditions. Estradiol regulation of Rb mRNA also occurred in other estrogen-dependent breast cancer cell lines. Insulin-like growth factor I, insulin, progestins, and epidermal growth factor had no effect on Rb expression. In summary, these results show that estradiol specifically regulates the expression of the Rb susceptibility gene product in hormone-dependent breast cancer by a posttranscriptional mechanism that occurs in the nucleus. The results from this study suggest that the negative regulation of Rb expression by estradiol, rather than Rb loss or mutation, may play an important role in breast carcinogenesis.

Iron chelation therapy in sickle cell disease.

Iron chelation therapy with deferoxamine enhances iron excretion and removes excessive tissue iron in regularly transfused patients with sickle cell disease. Long-term studies of deferoxamine in other hemoglobinopathies demonstrate that regular chelation therapy also reduces iron-related organ damage and mortality. Careful design of chelation regimens and attention to compliance are critical elements of successful therapy. The role of new chelators in sickle cell disease is currently under Investigation.