Development of a Web-Based Oxygenation Dashboard for Preterm Neonates: A Quality Improvement Initiative.

BACKGROUND: Preterm neonates are extensively monitored to require strict oxygen target attainment for optimal outcomes. In daily practice, detailed oxygenation data are hardly used and crucial patterns may be missed due to the snapshot presentations and subjective observations. This study aimed to develop a web-based dashboard with both detailed and summarized oxygenation data in real-time and to test its feasibility to support clinical decision making. METHODS: Data from pulse oximeters and ventilators were synchronized and stored to enable real-time and retrospective trend visualizations in a web-based viewer. The dashboard was designed based on interviews with clinicians. A preliminary version was evaluated during daily clinical rounds. The routine evaluation of the respiratory condition of neonates (gestational age < 32 weeks) with respiratory support at the NICU was compared to an assessment with the assistance of the dashboard. RESULTS: The web-based dashboard included data on the oxygen saturation (SpO2), fraction of inspired oxygen (FiO2), SpO2/FiO2 ratio, and area < 80% and > 95% SpO2 curve during time intervals that could be varied. The distribution of SpO2 values was visualized as histograms. In 65% of the patient evaluations (n = 86) the level of hypoxia was assessed differently with the use of the dashboard. In 75% of the patients the dashboard was judged to provide added value for the clinicians in supporting clinical decisions. CONCLUSIONS: A web-based customized oxygenation dashboard for preterm neonates at the NICU was developed and found feasible during evaluation. More clear and objective information was found supportive for clinicians during the daily rounds in tailoring treatment strategies.

Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction.

Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR)-regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and negative elongation factor (NELF)-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (Amax) and the steroid concentration required for half-maximal induction (EC50) of GR-mediated gene expression by acting at a minimum of three different kinetically defined steps. The action at two of these steps is dependent on BRD4 concentration, whereas the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR-controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components: GR < Cdk9 < BRD4 ≤ induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side effects of steroid treatments.

Simulation fails to replicate stress in trainees performing a technical procedure in the clinical environment.

INTRODUCTION: Simulation-based training (SBT) has become an increasingly important method by which doctors learn. Stress has an impact upon learning, performance, technical, and non-technical skills. However, there are currently no studies that compare stress in the clinical and simulated environment. We aimed to compare objective (heart rate variability, HRV) and subjective (state trait anxiety inventory, STAI) measures of stress theatre with a simulated environment. METHODS: HRV recordings were obtained from eight anesthetic trainees performing an uncomplicated rapid sequence induction at pre-determined procedural steps using a wireless Polar RS800CX monitor © in an emergency theatre setting. This was repeated in the simulated environment. Participants completed an STAI before and after the procedure. RESULTS: Eight trainees completed the study. The theatre environment caused an increase in objective stress vs baseline (p = .004). There was no significant difference between average objective stress levels across all time points (p = .20) between environments. However, there was a significant interaction between the variables of objective stress and environment (p = .045). There was no significant difference in subjective stress (p = .27) between environments. DISCUSSION: Simulation was unable to accurately replicate the stress of the technical procedure. This is the first study that compares the stress during SBT with the theatre environment and has implications for the assessment of simulated environments for use in examinations, rating of technical and non-technical skills, and stress management training.

Kinetically-defined component actions in gene repression.

Gene repression by transcription factors, and glucocorticoid receptors (GR) in particular, is a critical, but poorly understood, physiological response. Among the many unresolved questions is the difference between GR regulated induction and repression, and whether transcription cofactor action is the same in both. Because activity classifications based on changes in gene product level are mechanistically uninformative, we present a theory for gene repression in which the mechanisms of factor action are defined kinetically and are consistent for both gene repression and induction. The theory is generally applicable and amenable to predictions if the dose-response curve for gene repression is non-cooperative with a unit Hill coefficient, which is observed for GR-regulated repression of AP1LUC reporter induction by phorbol myristate acetate. The theory predicts the mechanism of GR and cofactors, and where they act with respect to each other, based on how each cofactor alters the plots of various kinetic parameters vs. cofactor. We show that the kinetically-defined mechanism of action of each of four factors (reporter gene, p160 coactivator TIF2, and two pharmaceuticals [NU6027 and phenanthroline]) is the same in GR-regulated repression and induction. What differs is the position of GR action. This insight should simplify clinical efforts to differentially modulate factor actions in gene induction vs. gene repression.

A kinase-independent activity of Cdk9 modulates glucocorticoid receptor-mediated gene induction.

A gene induction competition assay has recently uncovered new inhibitory activities of two transcriptional cofactors, NELF-A and NELF-B, in glucocorticoid-regulated transactivation. NELF-A and -B are also components of the NELF complex, which participates in RNA polymerase II pausing shortly after the initiation of gene transcription. We therefore asked if cofactors (Cdk9 and ELL) best known to affect paused polymerase could reverse the effects of NELF-A and -B. Unexpectedly, Cdk9 and ELL augmented, rather than prevented, the effects of NELF-A and -B. Furthermore, Cdk9 actions are not blocked either by Ckd9 inhibitors (DRB or flavopiridol) or by two Cdk9 mutants defective in kinase activity. The mode and site of action of NELF-A and -B mutants with an altered NELF domain are similarly affected by wild-type and kinase-dead Cdk9. We conclude that Cdk9 is a new modulator of GR action, that Ckd9 and ELL have novel activities in GR-regulated gene expression, that NELF-A and -B can act separately from the NELF complex, and that Cdk9 possesses activities that are independent of Cdk9 kinase activity. Finally, the competition assay has succeeded in ordering the site of action of several cofactors of GR transactivation. Extension of this methodology should be helpful in determining the site and mode of action of numerous additional cofactors and in reducing unwanted side effects.

PA1 protein, a new competitive decelerator acting at more than one step to impede glucocorticoid receptor-mediated transactivation.

Numerous cofactors modulate the gene regulatory activity of glucocorticoid receptors (GRs) by affecting one or more of the following three major transcriptional properties: the maximal activity of agonists (A(max)), the potency of agonists (EC(50)), and the partial agonist activity of antisteroids (PAA). Here, we report that the recently described nuclear protein, Pax2 transactivation domain interaction protein (PTIP)-associated protein 1 (PA1), is a new inhibitor of GR transactivation. PA1 suppresses A(max), increases the EC(50), and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream from where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also represses GR induction, increases the EC(50), and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms as follows: PA1 appears to increase GR dissociation from and reduce GR transactivation at the IGFBP1 promoter regions but blocks GR binding to the IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene.

A conserved protein motif is required for full modulatory activity of negative elongation factor subunits NELF-A and NELF-B in modifying glucocorticoid receptor-regulated gene induction properties.

NELF-B is a BRCA1-interacting protein and subunit (with NELF-A, -C/D, and -E) of the human negative elongation factor (NELF) complex, which participates in RNA polymerase II pausing shortly after transcription initiation, especially for synchronized gene expression. We now report new activities of NELF-B and other NELF complex subunits, which are to attenuate glucocorticoid receptor (GR)-mediated gene induction, reduce the partial agonist activity of an antagonist, and increase the EC50 of an agonist during nonsynchronized expression of exogenous and endogenous reporters. Stable knockdown of endogenous NELF-B has the opposite effects on an exogenous gene. The GR ligand-binding domain suffices for these biological responses. ChIP assays reveal that NELF-B diminishes GR recruitment to promoter regions of two endogenous genes. Using a new competition assay, NELF-A and NELF-B are each shown to act independently as competitive decelerators at two steps after the site of GR action and before or at the site of reporter gene activity. A common motif in each NELF was identified that is required for full activity of both NELF-A and NELF-B. These studies allow us to position the actions of two new modulators of GR-regulated transactivation, NELF-A and NELF-B, relative to other factors in the overall gene induction sequence.

Disruption of Ttll5/stamp gene (tubulin tyrosine ligase-like protein 5/SRC-1 and TIF2-associated modulatory protein gene) in male mice causes sperm malformation and infertility.

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp(tm/tm)) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp(tm/tm) sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp(tm/tm) males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.

The use of mode of action information in risk assessment: quantitative key events/dose-response framework for modeling the dose-response for key events.

The HESI RISK21 project formed the Dose-Response/Mode-of-Action Subteam to develop strategies for using all available data (in vitro, in vivo, and in silico) to advance the next-generation of chemical risk assessments. A goal of the Subteam is to enhance the existing Mode of Action/Human Relevance Framework and Key Events/Dose Response Framework (KEDRF) to make the best use of quantitative dose-response and timing information for Key Events (KEs). The resulting Quantitative Key Events/Dose-Response Framework (Q-KEDRF) provides a structured quantitative approach for systematic examination of the dose-response and timing of KEs resulting from a dose of a bioactive agent that causes a potential adverse outcome. Two concepts are described as aids to increasing the understanding of mode of action-Associative Events and Modulating Factors. These concepts are illustrated in two case studies; 1) cholinesterase inhibition by the pesticide chlorpyrifos, which illustrates the necessity of considering quantitative dose-response information when assessing the effect of a Modulating Factor, that is, enzyme polymorphisms in humans, and 2) estrogen-induced uterotrophic responses in rodents, which demonstrate how quantitative dose-response modeling for KE, the understanding of temporal relationships between KEs and a counterfactual examination of hypothesized KEs can determine whether they are Associative Events or true KEs.

Identification of location and kinetically defined mechanism of cofactors and reporter genes in the cascade of steroid-regulated transactivation.

A currently obscure area of steroid hormone action is where the component factors, including receptor and reporter gene, act. The DNA binding of factors can be precisely defined, but the location and timing of factor binding and action are usually not equivalent. These questions are addressed for several factors (e.g. glucocorticoid receptor (GR), reporter, TIF2, NCoR, NELF-A, sSMRT, and STAMP) using our recently developed competition assay. This assay reveals both the kinetically defined mechanism of factor action and where the above factors act relative to both each other and the equilibrium equivalent to the rate-limiting step, which we call the concentration limiting step (CLS). The utility of this competition assay would be greatly increased if the position of the CLS is invariant and if the factor acting at the CLS is known. Here we report that the exogenous GREtkLUC reporter acts at the CLS as an accelerator for gene induction by GRs in U2OS cells. This mechanism of reporter function at the CLS persists with different reporters, factors, receptors, and cell types. We, therefore, propose that the reporter gene always acts at the CLS during gene induction and constitutes a landmark around which one can order the actions of all other factors. Current data suggest that how and where GR and the short form of SMRT act is also constant. These results validate a novel and rational methodology for identifying distally acting factors that would be attractive targets for pharmaceutical intervention in the treatment of diseases involving GR-regulated genes.

Binding of the N-terminal region of coactivator TIF2 to the intrinsically disordered AF1 domain of the glucocorticoid receptor is accompanied by conformational reorganizations.

Control of gene transcription by glucocorticoid receptors (GRs) is important for many physiological processes. Like other steroid hormone receptors, the regulation of target genes by GR is mediated by two transactivation domains: activation function 1 (AF1) in the N-terminal domain and AF2 in the C-terminal ligand-binding domain (LBD). Full receptor activity requires both AF1 and -2 plus assorted coregulatory proteins. Crystal structures of the ligand-bound LBD have provided insight regarding how AF2 interacts with specific coactivators. However, despite its being the major activation domain of GRs, knowledge of AF1 structure/function has languished. This is mainly because of the highly disorganized structure of the GR N-terminal domain. This lack of AF1 structure is shared by all members of the steroid/nuclear receptor superfamily for which it has been examined and AF1 is thought to allow productive interactions with assorted cofactors via protein-induced changes in secondary/tertiary structures. To date, there are no reports of a classical coactivator altering the secondary/tertiary structure of the GR AF1 domain. Earlier, we reported an N-terminal fragment of the p160 coactivator TIF2, called TIF2.0, that binds the GR N-terminal domain and alters GR transcriptional activity. We therefore proposed that TIF2.0 binding to AF1 changes both its conformation and transcriptional activity. We now report that TIF2.0 interacts with the GR AF1 domain to increase the amount of α-helical structure in the complex. Furthermore, TIF2 coactivator activity is observed in the absence of the GR LBD in a manner that requires the AF1 domain. This contrasts with previous models where TIF2 receptor interaction domains binding to GR LBD somehow alter AF1 conformation. Our results establish for the first time that coactivators can modify the structure of the AF1 domain directly via the binding of a second region of the coactivator and suggest a molecular explanation for how coactivators increase the transcriptional activity of GR-agonist complexes.

Clinical evaluation of propofol as sedative for endotracheal intubation in neonates.

AIM: To determine the effects of propofol for endotracheal intubation in neonates in daily clinical practice. METHODS: We prospectively studied the pharmacodynamic effects of intravenous propofol administration in neonates who needed endotracheal intubation at the neonatal intensive care unit. RESULTS: Propofol was used for 62 intubations in neonates with postmenstrual ages ranging from 24 + 3 weeks to 44 + 5 weeks and bodyweights ranging from 520 to 4380 g. A 2 mg/kg bodyweight propofol starting dose was sufficient in 37% of patients; additional propofol was needed less often on the first postnatal day. The mean amount of propofol used was 3.3 (±1.2) mg/kg. The success rate of intubation depended on the experience of the physician and was related to the total administered amount of propofol. Hypotension occurred in 39% of patients and occurred more often at the first postnatal day. In 15% of procedures, propofol mono therapy was insufficient. CONCLUSION: This study shows that high doses of propofol are needed to reach effective sedation in neonates for intubation, with hypotension as a side effect in a considerable percentage of patients. Further research in newborn patients needs to identify optimal propofol doses and risk factors for hypotension.

A theoretical framework for gene induction and experimental comparisons.

Ligand-mediated gene induction by steroid receptors is a multistep process characterized by a dose-response curve for gene product that follows a first-order Hill equation. This behavior has classically been explained by steroid binding to receptor being the rate-limiting step. However, this predicts a constant potency of gene induction (EC(50)) for a given receptor-steroid complex, which is challenged by the findings that various cofactors/reagents can alter this parameter in a gene-specific manner. These properties put strong constraints on the mechanisms of gene induction and raise two questions: How can a first-order Hill dose-response curve (FHDC) arise from a multistep reaction sequence, and how do cofactors modify potency? Here we introduce a theoretical framework in which a sequence of steps yields an FHDC for the final product as a function of the initial agonist concentration. An exact determination of all constants is not required to describe the final FHDC. The theory predicts mechanisms for cofactor/reagent effects on gene-induction potency and maximal activity and it assigns a relative order to cofactors in the sequence of steps. The theory is supported by several observations from glucocorticoid receptor-mediated gene induction. It identifies the mechanism and matches the measured dose-response curves for different concentrations of the combination of cofactor Ubc9 and receptor. It also predicts that an FHDC cannot involve the DNA binding of preformed receptor dimers, which is validated experimentally. The theory is general and can be applied to any biochemical reaction that shows an FHDC.

Quantification of stress exposure in very preterm infants: Development of the NeO-stress score.

BACKGROUND: Stress during treatment at the Neonatal Intensive Care Unit (NICU) has long-term negative consequences on preterm infants' development. AIMS: We developed an instrument suited to validly determine the cumulative stress exposure for preterm infants in a NICU. STUDY DESIGN: This survey study made use of two consecutive questionnaires. SUBJECTS: NICU nurses and physicians from the nine NICUs in the Netherlands. OUTCOME MEASURES: First, respondents rated the relevance of 77 items encompassing potentially stressful procedures, commented on their comprehensibility and the comprehensiveness of the list. We calculated the content validity per item (CVI-I) and included only the relevant items in a second questionnaire in which the participants rated the stressfulness from 0 (not stressful) to 10 (extremely stressful). A stressfulness index - representing the median score - was calculated for each included item. RESULTS: Based on the CVI-I of the 77 items, step 1 resulted in 38 items considered relevant to quantify stress in preterm infants during the first 28 days of life. This list of 38 items exists of 34 items with a CVI-I if 0.78 or higher, one of these items was split into two items, and three items were added to improve comprehensiveness. The stressfulness index ranged from five to nine. CONCLUSIONS: The NeO-stress score consists of stressful items including their severity index and was developed to determine cumulative stress exposure of preterm infants. Evaluating the cross-cultural validity, correlating it to behavioural and biological stress responses, and evaluating its ability to predict preterm infants at risk for the negative effects following stress might expand the possibilities for this instrument.

Cognitive Functioning in Survivors of Hematopoietic Stem Cell Transplantation Compared With a Matched General Population Sample-The Maastricht Observational Study of Late Effects After Stem Cell trAnsplantation Study.

Although cognitive problems can recover over time, a subgroup of hematopoietic stem cell transplantation (HCT) survivors experience persistent cognitive problems in the long term. Despite these implications, studies assessing cognitive functioning in HCT survivors are limited. The aim of the present study was (1) to quantify the prevalence of cognitive impairment in patients treated with HCT who survived at least 2 years and to compare these with a matched reference group representing the general population; (2) to identify potential determinants of cognitive functioning within the HCT survivor group. Within the single-center Maastricht Observational study of late effects after Stem cell trAnsplantation, cognitive performance was assessed by a neuropsychological test battery divided into 3 cognitive domains: memory, information processing speed, and executive function and attention. An overall cognition score was calculated as the average of the domain scores. A total of 115 HCT survivors were group-matched on a 1:4 ratio to the reference group by age, sex, and level of education. Regression analyses adjusted for different sets of covariates including demographic and health- and lifestyle-related factors were used to test for differences in cognition between HCT survivors and the reference group resembling the general population. A limited set of clinical characteristics (diagnosis, type of transplant, time since treatment, conditioning regimen with total body irradiation and age at time of transplantation) were assessed as potential determinants of neurocognitive dysfunction among HCT survivors. Cognitive impairment was defined as scores in the cognitive domains < -1.5 standard deviation (SD) from what can be expected based on someone's age, sex, and education. The mean age at time of transplantation was 50.2 (SD ± 11.2) years, and the mean number of years after transplant was 8.7 (SD ± 5.7) years. The majority of HCT survivors were treated with autologous HCT (n = 73 [64%]). The prevalence of cognitive dysfunction was 34.8% in HCT survivors and 21.3% in the reference group (p = .002.) When adjusted for age, sex, and level of education, HCT survivors had a worse overall cognition score (b = -0.35; 95% confidence interval [CI], -0.55 to -0.16; p < .001), translating into 9.0 years of higher cognitive age. Analyses of specific cognitive domain scores showed that HCT survivors scored worse on memory (b = -0.43; 95% CI, -0.73 to -0.13; p = .005), information processing speed (b = -0.33; 95% CI, -0.55 to -0.11; p = .003), and executive function and attention (b = -0.29; 95% CI, -.55 to -.03; p = .031) than the reference group. The odds of cognitive impairment were on average 2.4 times higher among HCT survivors than the reference group (odd ratio = 2.44; 95% CI, 1.47-4.07; p = .001). Within the HCT survivor group none of the tested clinical determinants of cognitive impairment were significantly associated with cognition. This cohort study showed evidence for worse cognitive functioning in HCT survivors encompassing all three cognitive domains, respectively memory, information processing speed, and executive and attention compared to a reference group that represents the general population translating into nine years of faster cognitive ageing in HCT survivors than can be expected based on their chronological age. It is important to increase awareness for signs of neurocognitive dysfunction after HCT in clinicians and HCT survivors.

Ligand binding domain mutations of the glucocorticoid receptor selectively modify the effects with, but not binding of, cofactors.

We previously reported that several point mutations in the ligand binding domain (LBD) of glucocorticoid receptors (GRs) marginally affect the binding affinity of the synthetic glucocorticoids dexamethasone (Dex) and deacylcortivazol (DAC). However, these mutations dramatically alter the efficacy (A(max)) and potency (EC(50)) of agonists, along with the partial agonist activity (PAA) of the antisteroid Dex-mesylate (DM), for gene induction and repression in a steroid-dependent manner. This was proposed to result, in part, from altered protein-protein interactions in the complex of GR with the coactivator TIF2 despite normal TIF2 binding. To explore the generality of this phenomenon, we now ask whether these mutations also affect the transactivation properties, but not binding, of other GR-bound factors. We find that an elevated concentration of GR, to probe unidentified cofactors, or of the comodulator Ubc9 does not reverse the effects of GR LBD mutations that increase the EC(50) and lower the PAA with the GREtkLUC reporter in both CV-1 and U2OS cells. This behavior is more dramatic with Ubc9 and the isolated GR LBD fused to the GAL4 DNA binding domain, despite normal binding of Ubc9 to the mutant GRs. Similar effects, albeit gene, steroid, and transcriptional property-specific, are seen with full-length GRs and three endogenous genes in U2OS cells. Thus, changes in simple steady-state binding capacities of mutant receptors for factors cannot account for the modified transcriptional properties. In all cases, the nuclear translocation of Dex- and DAC-bound wild-type and mutant receptors is the same. These results are consistent with the earlier results with TIF2 and support the hypothesis that small changes in the GR LBD can alter the activities of the bound cofactor without modifying cofactor binding. We propose that this separation of binding and the modulation of transactivation parameters occurs for a wide variety of GR-associated cofactors.

Susceptibility of clinical Mycobacterium tuberculosis isolates to a potentially less toxic derivate of linezolid, PNU-100480.

Susceptibility of clinical Mycobacterium tuberculosis isolates to PNU-100480 and linezolid was evaluated by the MGIT 960 system. The isolates had various susceptibilities to isoniazid (INH), rifampin, ethambutol, and streptomycin. The mean MIC for PNU-100480 was 3.2 times lower than that for linezolid. Therefore, PNU-100480 is a promising candidate to be developed further as an adjunct in the treatment of multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB).

STAMP alters the growth of transformed and ovarian cancer cells.

BACKGROUND: Steroid receptors play major roles in the development, differentiation, and homeostasis of normal and malignant tissue. STAMP is a novel coregulator that not only enhances the ability of p160 coactivator family members TIF2 and SRC-1 to increase gene induction by many of the classical steroid receptors but also modulates the potency (or EC50) of agonists and the partial agonist activity of antisteroids. These modulatory activities of STAMP are not limited to gene induction but are also observed for receptor-mediated gene repression. However, a physiological role for STAMP remains unclear. METHODS: The growth rate of HEK293 cells stably transfected with STAMP plasmid and overexpressing STAMP protein is found to be decreased. We therefore asked whether different STAMP levels might also contribute to the abnormal growth rates of cancer cells. Panels of different stage human cancers were screened for altered levels of STAMP mRNA. Those cancers with the greatest apparent changes in STAMP mRNA were pursued in cultured cancer cell lines. RESULTS: Higher levels of STAMP are shown to have the physiologically relevant function of reducing the growth of HEK293 cells but, unexpectedly, in a steroid-independent manner. STAMP expression was examined in eight human cancer panels. More extensive studies of ovarian cancers suggested the presence of higher levels of STAMP mRNA. Lowering STAMP mRNA levels with siRNAs alters the proliferation of several ovarian cancer tissue culture lines in a cell line-specific manner. This cell line-specific effect of STAMP is not unique and is also seen for the conventional effects of STAMP on glucocorticoid receptor-regulated gene transactivation. CONCLUSIONS: This study indicates that a physiological function of STAMP in several settings is to modify cell growth rates in a manner that can be independent of steroid hormones. Studies with eleven tissue culture cell lines of ovarian cancer revealed a cell line-dependent effect of reduced STAMP mRNA on cell growth rates. This cell-line dependency is also seen for STAMP effects on glucocorticoid receptor-mediated transactivation. These preliminary findings suggest that further studies of STAMP in ovarian cancer may yield insight into ovarian cancer proliferation and may be useful in the development of biomarker panels.

STAMP, a novel predicted factor assisting TIF2 actions in glucocorticoid receptor-mediated induction and repression.

The coactivator TIF2 was predicted to interact with an unknown factor to modify both the relative inhibition in glucocorticoid receptor (GR)-mediated gene repression and several parameters of agonists and antisteroids in GR-regulated induction. Here, we describe the isolation and characterization of the predicted factor as a new 1,277-amino-acid endogenous protein (STAMP). STAMP associates with coactivators (TIF2 and SRC-1) and is selective for a subset of the steroid/nuclear receptors including GRs. Transfected STAMP increases the effects of TIF2 in GR-mediated repression and induction. Conversely, the levels of both induction and repression of endogenous genes are reduced when STAMP small interfering RNAs are used to lower the level of endogenous STAMP. Endogenous STAMP colocalizes with GR in intact cells and is recruited to the promoters of endogenous GR-induced and -repressed genes. We suggest that STAMP is an important new, downstream component of GR action in both gene activation and gene repression.

What goes on behind closed doors: physiological versus pharmacological steroid hormone actions.

Steroid-hormone-activated receptor proteins are among the best-understood class of factors for altering gene transcription in cells. Steroid receptors are of major importance in maintaining normal human physiology by responding to circulating concentrations of steroid in the nM range. Nonetheless, most studies of steroid receptor action have been conducted using the supra-physiological conditions of saturating concentrations (> or =100 nM) of potent synthetic steroid agonists. Here we summarize the recent developments arising from experiments using two clinically relevant conditions: subsaturating concentrations of agonist (to mimic the circulating concentrations in mammals) and saturating concentrations of antagonists (which are employed in endocrine therapies to block the actions of endogenous steroids). These studies have revealed new facets of steroid hormone action that could not be uncovered by conventional experiments with saturating concentrations of agonist steroids, such as a plethora of factors/conditions for the differential control of gene expression by physiological levels of steroid, a rational approach for examining the gene-specific variations in partial agonist activity of antisteroids, and a dissociation of steroid potency and efficacy that implies the existence of separate, and possibly novel, mechanistic steps and cofactors.