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Introduction: Current AHA/ACC guidelines on the management of ST-elevation myocardial infarction (STEMI) suggest that an ECG is indicated within 10minutes of arrival for patients arriving to the Emergency Department (ED) with symptoms concerning for STEMI. In response, there has been a creep towards performing ECGs more frequently in triage. The objectives of this study were to quantify the number of triage ECGs performed at our institution, assess the proportion of ECGs performed within current hospital guidelines, and evaluate the rate of STEMI detection in triage ECGs., Methods: A retrospective chart review of all emergency department patients presenting over a period of 8days who had a triage ECG performed. Cases of bradycardia or tachycardia were excluded. Data collection included patient demographics, presenting complaint, cardiac risk factors, troponin values, and final diagnosis. Summary statistics are reported in a descriptive manner., Results: During the study period, 538 patients had a triage ECG for possible STEMI with no STEMI identified and 16 NSTEMI diagnoses (confirmed as positive troponins following ED assessment). Sixty-three (11.7%) patients did not meet internal criteria for a triage ECG. A NSTEMI ED diagnosis was identified in 3% of patients who met internal triage ECG criteria and 1.6% who did not meet criteria (p=0.29). A cost analysis was performed using an average of 50 STEMI cases diagnosed in our ED per given year. Current institutional ECG billing rates for ECGs performed and interpreted is $125 per ECG, providing an estimated triage ECG charge to detect one STEMI at $54,295., Discussion: This retrospective study of 538 triage ECG's performed over an 8day period identified no STEMIs and 16 NSTEMIs. A very large number of ECGs were done at triage overall and included patients who do not meet our own hospital criteria. Given the extremely low yield and high associated charges, current guidelines for triage ECG for identifying a possible STEMI should be reviewed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: To derive distinct clusters of septic emergency department (ED) patients based on their presenting noninvasive hemodynamic (HD) measurements and to determine if any clinical parameters could identify these groups., Methods: Prospective, observational, convenience study of individuals with confirmed systemic infection. Presenting, pretreatment noninvasive HD parameters were compiled using Nexfin (Bmeye/Edwards LifeSciences) from 127 cases. Based on normalized parameters, k-means clustering was performed to identify a set of variables providing the greatest level of intercluster discrimination and intracluster cohesion., Results: Our best HD clustering model used 2 parameters: the cardiac index (CI [L/min per square meter]) and systemic vascular resistance index (SVRI [dynes·s/cm 5 per square meter]). Using this model, 3 different patient clusters were identified. Cluster 1 had high CI with normal SVRI (CI, 4.03 ± 0.61; SVRI, 1655.20 ± 348.08); cluster 2 low CI with increased vascular tone (CI, 2.50 ± 0.50; SVRI, 2600.83 ± 576.81); and cluster 3 very low CI with markedly elevated SVRI (CI, 1.37 ± 0.81; SVRI, 5951.49 ± 1480.16). Cluster 1 patients had the lowest 30-day overall mortality. Among clinically relevant variables available during the initial patient evaluation in the ED age, heart rate and temperature were significantly different across the 3 clusters., Conclusions: Emergency department patients with confirmed sepsis had 3 distinct cluster groupings based on their presenting noninvasively derived CI and SVRI. Further clinical studies evaluating the effect of early cluster-specific therapeutic interventions are needed to determine if there are outcome benefits of ED HD phenotyping in these patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: The Global Burden of Disease Study 2013 (GBD 2013) aims to bring together all available epidemiological data using a coherent measurement framework, standardised estimation methods, and transparent data sources to enable comparisons of health loss over time and across causes, age-sex groups, and countries. The GBD can be used to generate summary measures such as disability-adjusted life-years (DALYs) and healthy life expectancy (HALE) that make possible comparative assessments of broad epidemiological patterns across countries and time. These summary measures can also be used to quantify the component of variation in epidemiology that is related to sociodemographic development., Methods: We used the published GBD 2013 data for age-specific mortality, years of life lost due to premature mortality (YLLs), and years lived with disability (YLDs) to calculate DALYs and HALE for 1990, 1995, 2000, 2005, 2010, and 2013 for 188 countries. We calculated HALE using the Sullivan method; 95% uncertainty intervals (UIs) represent uncertainty in age-specific death rates and YLDs per person for each country, age, sex, and year. We estimated DALYs for 306 causes for each country as the sum of YLLs and YLDs; 95% UIs represent uncertainty in YLL and YLD rates. We quantified patterns of the epidemiological transition with a composite indicator of sociodemographic status, which we constructed from income per person, average years of schooling after age 15 years, and the total fertility rate and mean age of the population. We applied hierarchical regression to DALY rates by cause across countries to decompose variance related to the sociodemographic status variable, country, and time., Findings: Worldwide, from 1990 to 2013, life expectancy at birth rose by 6·2 years (95% UI 5·6-6·6), from 65·3 years (65·0-65·6) in 1990 to 71·5 years (71·0-71·9) in 2013, HALE at birth rose by 5·4 years (4·9-5·8), from 56·9 years (54·5-59·1) to 62·3 years (59·7-64·8), total DALYs fell by 3·6% (0·3-7·4), and age-standardised DALY rates per 100 000 people fell by 26·7% (24·6-29·1). For communicable, maternal, neonatal, and nutritional disorders, global DALY numbers, crude rates, and age-standardised rates have all declined between 1990 and 2013, whereas for non-communicable diseases, global DALYs have been increasing, DALY rates have remained nearly constant, and age-standardised DALY rates declined during the same period. From 2005 to 2013, the number of DALYs increased for most specific non-communicable diseases, including cardiovascular diseases and neoplasms, in addition to dengue, food-borne trematodes, and leishmaniasis; DALYs decreased for nearly all other causes. By 2013, the five leading causes of DALYs were ischaemic heart disease, lower respiratory infections, cerebrovascular disease, low back and neck pain, and road injuries. Sociodemographic status explained more than 50% of the variance between countries and over time for diarrhoea, lower respiratory infections, and other common infectious diseases; maternal disorders; neonatal disorders; nutritional deficiencies; other communicable, maternal, neonatal, and nutritional diseases; musculoskeletal disorders; and other non-communicable diseases. However, sociodemographic status explained less than 10% of the variance in DALY rates for cardiovascular diseases; chronic respiratory diseases; cirrhosis; diabetes, urogenital, blood, and endocrine diseases; unintentional injuries; and self-harm and interpersonal violence. Predictably, increased sociodemographic status was associated with a shift in burden from YLLs to YLDs, driven by declines in YLLs and increases in YLDs from musculoskeletal disorders, neurological disorders, and mental and substance use disorders. In most country-specific estimates, the increase in life expectancy was greater than that in HALE. Leading causes of DALYs are highly variable across countries., Interpretation: Global health is improving. Population growth and ageing have driven up numbers of DALYs, but crude rates have remained relatively constant, showing that progress in health does not mean fewer demands on health systems. The notion of an epidemiological transition--in which increasing sociodemographic status brings structured change in disease burden--is useful, but there is tremendous variation in burden of disease that is not associated with sociodemographic status. This further underscores the need for country-specific assessments of DALYs and HALE to appropriately inform health policy decisions and attendant actions., Funding: Bill & Melinda Gates Foundation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Emergency physicians (EPs) estimate the underlying hemodynamics of acutely ill patients and use them to help both diagnose and formulate a treatment plan. This trial compared the EP clinically derived estimates of cardiac output (CO) and systemic vascular resistance (SVR) to those measured noninvasively., Methods: Forty acutely ill emergency department patients with a broad range of diagnosis and blood pressure (BP) and pulse were monitored for 2 hours using novel noninvasive finger cuff technology (Nexfin; BMEYE, Amsterdam, The Netherlands). The Nexfin device provides continuous BP monitoring and, from the resulting pulse pressure waveform, calculates beat-to-beat CO and SVR. At baseline assessment and after 2 hours of testing and therapy, treating EPs were asked to estimate the CO and SVR (low, normal, or high), and these were compared with Nexfin measurements., Results: Twenty-five men and 15 women were enrolled with a mean age of 62.2 years (SD, 12.6 years). Eighteen had acute shortness of breath; 11, with probable stroke syndrome; 3, with suspected sepsis; and 8, with a systolic BP greater than 180 or less than 100 mm Hg. Concordance tables showed that there was very little agreement (κ values) between either the compared initial CO (-0.0873) and SVR (-0.0645) or the 2-hour values (-0.0645 and -0.1949, respectively)., Conclusions: Emergency physicians cannot accurately estimate the underlying hemodynamic profiles of acutely ill patients when compared with more objective measurements. This inaccuracy may have important clinical ramifications. Further study is needed to determine how to use these measured continuous CO and SVR monitoring values., (Copyright © 2012 Elsevier Inc. All rights reserved.)

S1P (sphingosine 1-phosphate) is a signalling molecule involved in a host of cellular and physiological functions, most notably cell survival and migration. S1P, which signals via a set of five G-protein-coupled receptors (S1P1-S1P5), is formed by the action of two SphKs (sphingosine kinases) from Sph (sphingosine). Interfering RNA strategies and SphK1 (sphingosine kinase type 1)-null (Sphk1-/-) mouse studies implicate SphK1 in multiple signalling cascades, yet there is a paucity of potent and selective SphK1 inhibitors necessary to evaluate the effects of rapid onset inhibition of this enzyme. We have identified a set of submicromolar amidine-based SphK1 inhibitors and report using a pair of these compounds to probe the cellular and physiological functions of SphK1. In so doing, we demonstrate that our inhibitors effectively lower S1P levels in cell-based assays, but we have been unable to correlate SphK1 inhibition with changes in cell survival. However, SphK1 inhibition did diminish EGF (epidermal growth factor)-driven increases in S1P levels and Akt (also known as protein kinase B)/ERK (extracellular-signal-regulated kinase) phosphorylation. Finally, administration of the SphK1 inhibitor to wild-type, but not Sphk1-/-, mice resulted in a rapid decrease in blood S1P levels indicating that circulating S1P is rapidly turned over.

Objective: Continuous invasive blood pressure (BP) and heart rate (HR) monitoring in the emergency department (ED) is valuable in managing critically ill patients. Novel noninvasive finger cuff technology allows this same uninterrupted monitoring for almost any individual. This exploratory study compares ED noninvasive continuous to intermittent measurements of these variables., Methods: A variety of acutely ill ED patients (n = 40) with broad ranges of BP and HR underwent simultaneous monitoring using interval standard automated ED devices and continuous finger cuff technology (Nexfin; Bmeye, Amsterdam, The Netherlands) over a period of 2 hours. At baseline and at 15-minute intervals, simultaneous measurements for BP and HR were recorded and compared., Results: There were 25 men and 15 women enrolled with a mean age of 62.2 years (SD, 12.6). Eighteen had acute dyspnea, 11 with probable stroke syndrome, 3 with suspected sepsis, and 8 with a systolic BP greater than 180 or less than 100 mm Hg. Blood pressure and HR values (n = 344) simultaneously measured by automated ED equipment and the Nexfin finger cuff device were compared. The Pearson correlation coefficients were high, and the Bland-Altman plots showed good agreement between the 2 determinations., Conclusion: Continuous BP and HR monitoring measured by the Nexfin finger cuff device in this trial showed reasonable agreement when compared with the intermittent values obtained by automated ED equipment. However, theoretically, noninvasive and continuous monitoring of the BP and HR might better reflect underlying hemodynamics than these same measurements obtained intermittently and, thus, could be important in patient management. More study is needed to determine the optimal method of monitoring these parameters., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Sphingosine 1-phosphate (S1P) is a bioactive lipid that has been identified as an accelerant of cancer progression. The sphingosine kinases (SphKs) are the sole producers of S1P, and thus, SphK inhibitors may prove effective in cancer mitigation and chemosensitization. Of the two SphKs, SphK1 overexpression has been observed in a myriad of cancer cell lines and tissues and has been recognized as the presumptive target over that of the poorly characterized SphK2. Herein, we present the design and synthesis of amidine-based nanomolar SphK1 subtype-selective inhibitors. A homology model of SphK1, trained with this library of amidine inhibitors, was then used to predict the activity of additional, more potent, inhibitors. Lastly, select amidine inhibitors were validated in human leukemia U937 cells, where they significantly reduced endogenous S1P levels at nanomolar concentrations.

Hepatic cholesterol 7alpha-hydroxylase (CYP7A) and sterol 27 hydroxylase activities were measured in fetal, newborn, suckling, and weaned piglets from 76 d into gestation to 49 d of age. Hepatic CYP7A activity was not detected in fetal microsomes, but it increased to 6.8 +/- 2.6 pmol/min x mg(-1) protein in suckling piglets at 21 d of age and to 18.2 +/- 2.5 in weaned piglets at 49 d of age. Hepatic CYP7A activity was not different between 49-d-old piglets weaned at 21 d and piglets suckled for 49 d (18.9 +/- 2.6 and 18.2 +/- 2.5 pmol/min x mg protein, respectively). Fasting for 14 h decreased CYP7A activity by 86% in both suckled and weaned piglets. Cholesterol 7alpha-hydroxylase activity remained decreased for at least 5 h after refeeding. Sterol 27-hydroxylase activity was also undetectable near birth, but was detectable by 21 d of age. Postnatally, sterol 27-hydroxylase activity was not influenced by age or suckling and weaning, as was CYP7A. Sterol 27-hydroxylase was decreased by 80% in piglets deprived of feed compared with piglets given free access. In contrast to CYP7A activity, 27-hydroxylase activity returned within 5 h after refeeding to levels observed in piglets given ad libitum access to feed. Similar to CYP7A enzyme activity, hepatic CYP7A mRNA was not detected in newborn piglets, but increased from 2.7 +/- 1.7 pg mRNA/microg RNA in suckling piglets at 21 d to 13.7 +/- 1.2 in 49-d-old piglets weaned at 21 d. As with enzyme activity, feed deprivation decreased CYP7A mRNA to barely detectable levels (< .5 pg/microg RNA), and which remained decreased for at least 5 h following refeeding (.6 +/- .3 and 2.67 +/- .4 pg mRNA/microg RNA for suckled and weaned piglets, respectively). In piglets allowed free access to feed, CYP7A mRNA concentrations were associated positively (P = .001) with enzyme activity. These results suggest that developmental regulation of CYP7A activity is the result of a pretranslational mechanism.

The HMG-CoA reductase inhibitor, lovastatin, blocks targeting of the Rho and Ras families of small GTPases to their active sites by inhibiting protein prenylation. Control NIH3T3 cells, and those overexpressing human cyclin E protein were treated with lovastatin for 24 h to determine the effects of cyclin E overexpression on lovastatin-induced growth arrest and cell rounding. Lovastatin treatment (10 microM) of control 3T3 cells resulted in growth arrest at G1 accompanied by actin stress fiber disassembly, cell rounding, and decreased active RhoA from the membranous protein fraction. By contrast, in NIH3T3 cells overexpressing cyclin E, lovastatin did not cause loss of RhoA from the membrane (active) protein fraction, actin stress fiber disassembly, cell rounding or growth arrest within 24 h. Analysis of cell cycle proteins showed that 24 h of lovastatin treatment in the control cells caused an elevation in the levels of the cyclin-dependent kinase inhibitor p27(kip1), inhibition of both cyclin E- and cyclin A-dependent kinase activity, and decreased levels of hyperphosphorylated retinoblastoma protein (pRb). By contrast, lovastatin treatment of the cyclin E overexpressors did not suppress either cyclin E- or cyclin A-dependent kinase activity, nor did it alter the level of maximally phosphorylated pRb, despite increased levels of p27(kip1). However, by 72 h, the cyclin E overexpressors rounded up but remained attached to the substratum, indicating a delayed response to lovastatin. In contrast with lovastatin, inactivation of membrane-bound Rho proteins (i.e., GTP-bound RhoA, RhoB, RhoC) with botulinum C3 transferase caused cell rounding and G1 growth arrest in both cell types but did not inhibit cyclin E-dependent histone kinase activity in the cyclin E overexpressors. In addition, 24 h of cycloheximide treatment caused depletion of RhoA from the membrane (active) fraction in neo cells, but in the cells overexpressing cyclin E, RhoA remained in the active (membrane-associated) fraction. Our observations suggest that (1) RhoA activation occurs downstream of cyclin E-dependent kinase activation, and (2) overexpression of cyclin E decreased the turnover rate of active RhoA., (Copyright 1999 Wiley-Liss, Inc.)

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

The ATPase mechanism for a monomeric Drosophila kinesin construct, K341, was determined by pre-steady-state kinetic methods and compared to dimeric kinesin, K401. We directly measured the kinetics of binding mantATP (a fluorescent ATP analog) to the microtubule K341 complex, the dissociation of K341 from the microtubule, and release of phosphate and ADP from K341. Measurements of phosphate release kinetics at low salt concentration show that K341 hydrolyzes 18 molecules of ATP per kinesin monomer prior to release from the microtubule. At a higher salt concentration the amplitude of the pre-steady-state burst of phosphate release was reduced to 8 molecules per kinesin monomer. The maximum rate of dissociation of K341 from the microtubule following the addition of ATP was 22 s-1. The rate of mantADP release from the M.K341.mantADP complex increased as a function of tubulin concentration with a second-order rate constant of 11 microM-1 s-1 for K341 binding to the microtubule and reached a maximum rate of mantADP release of 303 s-1. ADP release kinetics were also determined by monitoring the binding of mantATP to K341.ADP and K401.ADP after mixing with microtubules. We show that monomeric kinesin remains associated with the microtubule through multiple rounds of ATP hydrolysis. This apparent processivity implies that one of the functions of the cooperative interaction between the two kinesin heads in dimeric kinesin is for the reactions occurring on one kinesin head to facilitate the release of the adjacent head from the microtubule.

The processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results show that the release of two molecules of ADP from dimeric kinesin (K401) after the binding of kinesin ADP to the microtubule is a sequential process leading to biphasic kinetics. The maximum rate of release of mantADP from the first motor domain of K401 or monomeric K341 is fast (300 s-1) and independent of added nucleotide. The rate of mantADP release from the second motor domain of K401 is slow in the absence of added nucleotide (0.4 s-1) and reaches a maximum rate of 300 s-1 at saturating concentrations of ATP. High concentrations of ADP stimulate mantADP release from the second head to a maximum rate of 3.8 s-1. The nonhydrolyzable analog AMP-PNP and ATP-gamma S also stimulate ADP release from the second head (maximum rate of 30 s-1), suggesting that ATP hydrolysis is not necessary to stimulate the ADP release. These experiments establish an alternating site mechanism for dimeric kinesin whereby ATP binding to one kinesin active site stimulates the release of ADP from the second site such that the reactions occurring at the active sites of the two monomer units are kept out of phase from each other by interactions between the heads. These results define the steps of the ATPase pathway that lead to the efficient coupling of ATP hydrolysis to force production in a processive reaction whereby force production in forming a tight microtubule complex by one head is coupled to the rate-limiting release of the other head from the microtubule.

Steady-state and pre-steady-state kinetic methods were used to analyze two shorter Drosophila kinesin constructs (K341 and K366) in comparison to K401. K341, K366, and K401 represent the kinesin motor domains containing the N-terminal 341, 366, or 401 amino acids, respectively. K401 is dimeric (Kd = 37 +/- 17 nM) whereas both K366 and K341 are monomeric [Correia et al. (1995) Biochemistry 34, 4898-4907]. Like native kinesin and K401, K341 and K366 demonstrate low ATPase activity in the absence of microtubules (0.03 and 0.01 s-1, respectively), and ADP release is rate-limiting during steady-state turnover. Microtubules activate the steady-state ATPase to 84 s-1 for K341 (K(m),ATP = 100 microM; K0.5,MT = 3.2 microM tubulin) and 64 s-1 for K366 (K(m),ATP = 65 microM; K0.5,MT = 2.5 microM tubulin) in comparison to K401 at 20 s-1 (K(m)ATP = 60 microM; K0.5,MT = 1 microM tubulin). The rapid quench experiments for all three constructs show a burst of product formation during the first turnover, indicating the rate-limiting step for the microtubule-activated ATPase occurs after ATP hydrolysis. The interaction of K341 and K366 with the microtubule was analyzed by electron microscopy. The results show that K341 and K366, like K401, bind to the microtubule with an 8 nm axial periodicity. However, the addition of K366 to microtubules resulted in significant aggregation of microtubules. The pre-steady-state kinetic results show that K341 retains the kinetic and structural properties necessary to compare directly the kinetic properties of monomeric and dimeric kinesins, although the microtubule-activated ATPase is significantly faster for the monomeric constructs, suggesting possible interactions in the dimer which inhibit ATP turnover as part of the coupling to force production.

Bacterial expressed kinesin motor domains hydrolyze ATP and promote microtubule-dependent motility. It has routinely been assumed that motor domain preparations are monomeric on the basis of the presumption that dimerization is mediated by the stalk region. However, experimental verification of the oligomeric state of the kinesin construct is required to interpret the results from single-molecule motility assays as well as presteady-state kinetic experiments. We have measured directly the state of assembly of three conventional kinesin motor domain constructs-K401, K366, and K341, comprising the N-terminal 401, 366, and 341 amino acids, respectively, of the Drosophila kinesin heavy chain-by sedimentation velocity and sedimentation equilibrium methods in an analytical ultracentrifuge. K401 (MW of ADP complex, 45,532) is a predominantly a dimer with a sedimentation coefficient, s020,w, of 5.06 S, but it is able to self-associate by means of a 1-2-4 mechanism into higher oligomers. Molecular weight measurements establish the dissociation constant for dimerization at 37 +/- 17 nM in the presence of ATP. The dissociation constant in the presence of ADP is 35 +/- 26 nM and in the presence of AMPPNP is 42 +/- 28 nM. The construct K366 (MW of ADP complex, 41,404) is a monomer (measured MW, 41,768 +/- 1219) at concentrations below 4 microM K366, with a sedimentation coefficient, s020,w, of 3.25 S. At higher concentrations, there is evidence for a weak association of K366 to a 1-2-4-8 model with a slight preference for octamer formation. The smallest construct, K341 (MW of ADP complex, 38,274), is a monomer (measured MW, 38,191 +/- 734) up to at least 10 microM total K341 concentration with a sedimentation coefficient, s020,w, of 2.9 S. Thus, the dimerization domain either is between amino acid residues 367 and 401 or is strongly affected by the removal of this region. Higher oligomers of K401 form by a mechanism involving dimers of dimers, and suggest that native kinesin may also undergo self-association. These results have important implications for the interpretation of ATP-dependent motility assays.

Treatment of the T47D(A1-2) mammary carcinoma cell line with the nonspecific kinase inhibitor 2-aminopurine (2AP) has an unusual effect on induction of the mouse mammary tumor virus promoter by glucocorticoids. 2AP initially abrogates the dexamethasone-mediated increase, but after about 16-20 h, this effect is reversed, and continued incubation with 2AP potently stimulates the hormone induction. The biphasic kinetics of 2AP action displayed cell specificity. No inhibitory phase was seen in fibroblasts. Cell type specificity is also seen upon treatment of cells with isobutylmethylxanthine (IBMX). Dexamethasone action is inhibited in mammary cells, but potentiated in fibroblasts. Unlike 2AP treatment, IBMX continues to suppress the hormone response through at least 48 h of treatment. IBMX is commonly used as a phosphodiesterase inhibitor, and indeed, it stimulates a cAMP-dependent promoter in both mammary carcinoma cells and fibroblasts. Because elevating cAMP will potentiate dexamethasone-mediated induction in mammary cells, IBMX must be interventing in another pathway that elicits IBMX-dependent suppression of the hormone response. Pharmacological studies suggest that this interaction is not mediated through adenosine receptors, histamine receptors, or imidazoline receptors. The five-member imidazole ring plays a critical role in the suppressive activity; substitutions at the 7 position inhibit suppression. These results give further indication of coupling of steroid receptor action to other cellular signaling pathways. This complex spectrum of interactions may play a central determining role in the tissue specificity of the response to steroid hormones.

The T47D (A1-2) cell line is a human mammary carcinoma-derived cell line that has been engineered to constitutively express comparable levels of both glucocorticoid and progesterone receptors. In addition, these cells possess a stably integrated mouse mammary tumor virus (MMTV) luciferase reporter gene. Because the MMTV promoter is recognized similarly by both receptors, we have used this cell line to examine the transcriptional regulatory mechanisms employed by the two receptors. The stably integrated MMTV luciferase gene is highly inducible by glucocorticoids, whereas it is almost entirely refractory to induction by progestins. In contrast, a transiently transfected MMTV chloroamphenicol acetyl transferase reporter, while much more inducible by glucocorticoids, can be induced significantly by progestins. The differential inducibility of the stably integrated template is reflected in the superior ability of glucocorticoids to initiate alterations in the chromatin structure of the promoter. Concomitant with the changes in nuclease accessibility, glucocorticoids, unlike progestins, recruit transcription factors to the MMTV promoter. These results emphasize a central role for the modulation of the chromatin environment by steroid receptors in defining their capacity to regulate gene expression in vivo.

A continuous spectrophotometric method for assaying ATCase activity has been devised that couples the production of inorganic phosphate from the ATCase-catalyzed reaction to the phosphorolysis reaction catalyzed by purine nucleoside phosphorylase, using a chromophoric nucleotide analogue, methylthioguanosine (MESG). This latter reaction results in a change in extinction coefficient of 11,000 M-1 cm-1 at 360 nm, providing a means for continuous assay of ATCase activity by spectrophotometry in the visible light region. This delta epsilon 360 is sufficiently large to allow continuous determination of reaction rates with micromolar levels of carbamyl-phosphate, a feature not offered by other currently used assay methods. Other currently available ATCase assay methods typically include fixed-time incubations involving [14C]Asp that require multiple chromatographic separations, colorimetry requiring long incubations with corrosive chemicals in the dark, or relatively insensitive continuous approaches involving a pH stat or far uv spectrophotometry. This facile, inexpensive MESG-coupled assay can be routinely applied to studies of ATCase altered by feedback modifiers or by site-specific mutations. Saturation curves for Asp and CP determined by other methods at pH 7 and 8 have been reproduced by the MESG/PNP-coupled approach. The kinetic binding of CP was demonstrated to be non-cooperative at low [Asp], i.e., under conditions at which ATCase was primarily in the T state. Cooperative binding of CP observed under conditions of saturating [Asp] (i.e., with ATCase in the R state) appears to reflect binding of Asp rather than CP.(ABSTRACT TRUNCATED AT 250 WORDS)

In a human breast carcinoma-derived cell line engineered to contain a hormone-responsive luciferase reporter gene, manipulation of cell growth conditions or cellular signal transduction in a variety of ways can enhance or impair glucocorticoid-mediated induction of a target gene. Induction may be enhanced as much as 10-fold or inhibited 90% by different treatments. For example, two different inhibitors of protein phosphatase-1 and -2A potentiated the hormone-dependent induction of luciferase. Activation of protein kinase-A via addition of 8-bromo-cAMP or forskolin also potentiated the hormonal induction, whereas 8-bromo-cGMP was ineffective. In contrast, activating protein kinase-A by inhibiting cAMP turnover with the phosphodiesterase inhibitors isobutylmethylxanthine or Ro20-1724 inhibited the hormone response rather than potentiated it. The inhibitory activity of isobutylmethylxanthine was evident even when activators of protein kinase-A are administered simultaneously. Isobutylmethylxanthine must, therefore, activate a signal transduction pathway in addition to the protein kinase-A pathway. Activation of protein kinase-C potentiated the hormone response in a cell-specific manner. Treatment with epidermal growth factor and imposition of cell stress by heat shock or inhibition of protein synthesis also enhanced the glucocorticoid response. Thus, our results suggest an elaborate coupling of the steroid response pathway with other cellular signal transduction mechanisms that permits an additional layer of control to be imposed on hormone-mediated transcriptional responses. It is proposed that cell-specific phosphorylation events influence steroid receptor interaction with the basal transcription apparatus, thereby altering receptor-mediated induction mechanisms.

We have stably introduced expression vectors for the glucocorticoid receptor and a sensitive, hormone-responsive reporter (mouse mammary tumor virus-luciferase) into a human breast carcinoma-derived cell line. Employing this cell line, we have conducted a detailed examination of the induction of glucocorticoid-regulated genes and the phosphorylation of glucocorticoid receptor following pharmacologic manipulation of cell signaling pathways. The hormone response can be enhanced from 2 to 10-fold by activators of protein kinase A, protein kinase C, and inhibitors of protein phosphatase. Forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (BrcAMP), but not BrcGMP, enhance the hormone effect, yet surprisingly, phosphodiesterase inhibitors, isobutylmethylxanthine and Ro20-1724, strongly inhibit hormone-mediated induction of the reporter gene. These treatments do not alter cellular receptor content, dexamethasone binding, nor hormone-mediated receptor down-regulation. Tryptic peptide analysis of 32P-labeled receptor reveals that neither BrcAMP, isobutylmethylxanthine, nor the tumor promoter and protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate, detectably alter the state of glucocorticoid receptor phosphorylation. The only agent which alters receptor phosphorylation is the protein phosphatase inhibitor okadaic acid, but only at concentrations higher than required for maximum effects on glucocorticoid receptor transactivation. We propose that these effectors do not modify receptor directly but alter its interaction with transcription complexes.

RU486 is a glucocorticoid and progesterone antagonist. In glucocorticoid-responsive fibroblasts, it mediates little or no induction of a truncated, hormone-responsive mouse mammary tumor virus promoter; moreover, it abrogates the induction mediated by the glucocorticoid agonist, dexamethasone. However, when the fibroblasts are treated with activators of protein kinase A, 8-Br-cAMP or forskolin, along with RU486, the steroid now acts as a partial agonist, capable of mediating an induction of hormone-responsive reporter genes. In addition, the ability of RU486 to block the action of the glucocorticoid agonist, dexamethasone, is compromised by concomitant treatment with 8-Br-cAMP. Activators of protein kinase C fail to elicit these phenomena. Induction of gene expression in the presence of 8-Br-cAMP is dependent on the dose of RU486 over a range consistent with a glucocorticoid receptor-mediated mechanism. An antagonist, ZK98 299, which unlike RU486 is not thought to permit receptor binding to DNA, is not activated by 8-Br-cAMP. The elicitation of RU486 agonist activity cannot be attributed solely to idiosyncrasies of the cell line or the promoter. Similar phenomena are observed in another glucocorticoid-responsive fibroblast line. Furthermore, RU486 can induce a minimal promoter bearing two copies of a synthetic receptor target site. However, we have identified at least one promoter toward which RU486 still behaves as an antagonist despite 8-Br-cAMP treatment. These observations suggest that the unmasking of latent agonist activity in a type II antagonist is not an isolated phenomenon and may, therefore, be seen with other receptors and antagonists. The finding that modulation of cellular signal transduction pathways can unmask agonist activity in an otherwise effective steroid antagonist has significant implications for the use of steroid antagonists in the clinical setting and could represent a heretofore unrecognized mechanism for the development of steroid resistance.

The protein kinase A stimulator cAMP can potentiate the ability of progestins to induce the transactivation function of the human progesterone receptor (hPR). We questioned in the present study whether cAMP could functionally cooperate with the progestin antagonist RU486. In T47D human breast cancer cells, RU486 behaves as a pure antagonist with respect to induction of the progesterone-responsive mouse mammary tumor virus chloramphenicol acetyltransferase (MMTV-CAT) reporter gene. It fails to stimulate MMTV-CAT expression and completely inhibits induction by the synthetic progestin R5020. However, when RU486 is combined with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), MMTV-CAT is induced to levels approaching that stimulated by R5020 alone. Also, RU486 in the presence of 8-Br-cAMP is only partially effective in antagonizing R5020 action. The agonist activity exhibited under these conditions appears to be due to RU486 acting through hPR as evidenced by the fact that 8-Br-cAMP alone has no effect on MMTV-CAT, whereas induction by the combination of 8-Br-cAMP and RU486 is dose responsive to RU486 in a saturable manner and can be inhibited by the type I antiprogestin (prevents hPR-DNA binding) ZK98299, which does not exhibit positive functional cooperation with cAMP. Acquisition of agonist activity in the presence of 8-Br-cAMP also extends to the type II antiprogestin (permits hPR-DNA binding) ZK112993. Since RU486 is also a type II antagonist, these results suggest that detection of functional synergism between cAMP and antiprogestins may require binding of the hPR-antagonist complex to DNA. We propose that cross-talk between second messenger and steroid receptor signal transduction pathways may be one mechanism for resistance to steroid antagonists that frequently develops in breast cancer.