BCOR regulates myeloid cell proliferation and differentiation.

BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes.

NRC-interacting factor directs neurite outgrowth in an activity-dependent manner.

Nuclear hormone receptor coregulator-interacting factor 1 (NIF-1) is a zinc finger nuclear protein that was initially identified to enhance nuclear hormone receptor transcription via its interaction with nuclear hormone receptor coregulator (NRC). NIF-1 may regulate gene transcription either by modulating general transcriptional machinery or remodeling chromatin structure through interactions with specific protein partners. We previously reported that the cytoplasmic/nuclear localization of NIF-1 is regulated by the neuronal Cdk5 activator p35, suggesting potential neuronal functions for NIF-1. The present study reveals that NIF-1 plays critical roles in regulating neuronal morphogenesis at early stages. NIF-1 was prominently expressed in the nuclei of developing rat cortical neurons. Knockdown of NIF-1 expression attenuated both neurite outgrowth in cultured cortical neurons and retinoic acid (RA)-treated Neuro-2a neuroblastoma cells. Furthermore, activity-induced Ca(2+) influx, which is critical for neuronal morphogenesis, stimulated the nuclear localization of NIF-1 in cortical neurons. Suppression of NIF-1 expression reduced the up-regulation of neuronal activity-dependent gene transcription. These findings collectively suggest that NIF-1 directs neuronal morphogenesis during early developmental stages through modulating activity-dependent gene transcription.

Cyclin-dependent kinase 1 expression is inhibited by p16(INK4a) at the post-transcriptional level through the microRNA pathway.

The p16(INK4a) protein regulates cell cycle progression mainly by inhibiting the activity of G1-phase cyclin-dependent kinases (CDKs) 4 and 6, the subsequent retinoblastoma protein (pRb) phosphorylation and E2F transcription factor release. The p16(INK4a) protein can also repress the activity of other transcription factors, such as c-myc, nuclear factor-kappaB and c-Jun/AP1. Here, we report that, in two p16(-/-), pRb(WT) and p53(WT) cell lines (MCF7 and U87), p16(INK4a) overexpression induces a dramatic decrease in CDK1 protein expression. In response to p16(INK4a), the decreased rate of CDK1 protein synthesis, its unchanged protein half-life, unreduced CDK1 mRNA steady-state levels and mRNA half-life allow us to hypothesize that p16(INK4a) could regulate CDK1 expression at the post-transcriptional level. This CDK1 downregulation is mediated by the 3'-untranslated region (3'UTR) of CDK1 mRNA as shown by translational inhibition in luciferase assays and is associated with a modified expression balance of microRNAs (miRNAs) that potentially regulate CDK1, analyzed by TaqMan Human microRNA Array. The p16(INK4a)-induced expression of two miRNAs (miR-410 and miR-650 chosen as an example) in MCF7 cells is confirmed by individual reverse transcription-qPCR. Furthermore, we show the interaction of miR-410 or miR-650 with CDK1-3'UTR by luciferase assays. Endogenous CDK1 expression decreases upon both miRNA overexpression and increases with their simultaneous inhibition. The induction of miR-410, but not miR-650 could be related to the pRb/E2F pathway. These results demonstrate the post-transcriptional inhibition of CDK1 by p16(INK4a). We suggest that p16(INK4a) may regulate gene expression by modifying the functional equilibrium of transcription factors and consequently the expression balance of miRNAs.

Prognostic value of miR-16 expression in childhood acute lymphoblastic leukemia relationships to normal and malignant lymphocyte proliferation.

miR-16, a miRNA involved in cell proliferation and apoptosis regulation, may interfere with either oncogenic or tumor-suppressor pathways and is implicated in leukemogenesis. We then explored its expression in 93 childhood acute lymphoblastic leukemia (ALL) cases. A high miR-16 expression was associated with hyperleukocytosis and poor cytogenetic groups. In the whole group and in B-cell ALLs, disease-free survival (DFS) was significantly shorter for miR-16 above quartile 75. In T-cell ALLs, for both DFS and overall survival, a significant trend was found with a survival shortening from the lowest to the highest miR-16 levels. miR-16 expression neither significantly correlated with normal and malignant lymphocyte proliferation nor varied according to lymphocyte differentiation. The prognostic value of miR-16 in childhood ALL highlighted the complexity of miR-16 functions.

Inhibition of normal lymphocyte proliferation by Indirubin-3'-monoxime: a multifactorial process.

Indirubin-3'-monoxime (IO) is a derivative of Indirubin, compound of a Chinese medicinal recipe used to treat various diseases including leukemia. In this study, we investigated to what extent IO inhibits the growth of normal human lymphocytes. We defined various experimental conditions of peripheral blood lymphocyte treatment: IO introduced (i) on unstimulated lymphocytes, (ii) or on stimulated lymphocytes at the time of phytohemagglutinin stimulation (L1 protocol), (iii) 48 h after the beginning of stimulation (L2 protocol), and (iv) after nocodazole synchronization of stimulated lymphocytes. IO induces a concentration dependent cytotoxic effect yielding a characteristic sub-G1 peak in normal stimulated lymphocytes. Cell death was partly due to necrosis and apoptosis. Normal unstimulated lymphocytes remained insensitive to the cytotoxic effect of 10 microM IO treatment. A cell cycle inhibition was observed after IO treatment, stronger for the L1 than for the L2 protocol, without induction of polyploidy after Nocodazole synchronization. These cellular consequences were associated with a decrease in CDK activity, and with CDK and cyclin gene expression modifications. The inhibition of lymphocyte proliferation by IO indicates that indirubin derivatives may be potent immunosuppressive agents.

Sinus node modification using radiofrequency current in a patient with persistent inappropriate sinus tachycardia.

Radiofrequency catheter modification of the sinus node for persistent inappropriate sinus tachycardia has not been previously reported. This article describes a patient in whom radiofrequency current was used to ablate an incessant automatic tachycardia focus mapped to the region of the sinus node, where a discrete multicomponent electrogram demonstrating earliest atrial activation was recorded. A transient junctional rhythm developed immediately after ablation, with rapid subsequent emergence of a stable rhythm having normal sinus nodal characteristics.

Ventricular septal summit stimulation in atrioventricular nodal reentrant tachycardia.

In all, 18 consecutive patients with atrioventricular nodal reentry tachycardia (AVNRT) underwent right ventricular (RV) stimulation during AVNRT from either the RV apex or summit. Stimulation from the RV apex advanced the tachycardia with the same atrial sequence in 6 of 18 patients (33%), but never conclusively excluded the presence of a low atrial tachycardia. RV summit stimulation resulted in direct stimulation of the low septal right atrium in 6 patients. RV summit stimulation advanced the tachycardia in 4 patients, delayed it in 2 and terminated it in 3 without an atrial electrogram. The latter 2 findings exclude the presence of a low atrial tachycardia. Thus, in patients with AVNRT, application of extrastimuli closer to the putative reentrant site enables greater efficacy in tachycardia resetting and in excluding a low septal atrial tachycardia.

Importance of recording the right bundle branch deflection in the diagnosis of His-Purkinje reentrant tachycardia.

Eight of 120 consecutive patients with inducible sustained ventricular tachycardia who were studied at our institution from September 1, 1988 to January 1, 1991, were found to have reentry within the His-Purkinje System as the mechanism of their tachycardias. Two of the eight patients (25%) required the recording of the right bundle branch potential to elucidate the tachycardia circuits. The electrophysiological findings of these two patients are described. In both instances, the diagnosis of supraventricular tachycardia with aberrancy was excluded. In patient 1, a His-bundle electrogram preceded each QRS complex during tachycardia and the His-to-His interval variation preceded changes in QRS intervals. However, recordings from the right bundle branch allowed for exclusion of bundle branch reentry and evidence was found for reentry restricted to the left fascicles. In patient 2, despite instances of dissociation of the His-bundle deflection from the tachycardia, a right bundle branch potential preceded each QRS and spontaneous changes in the interval between successive activation of the right bundle branch preceded changes in ventricular activation. Catheter ablation of the right bundle branch eliminated the tachycardia. It is concluded that the recording of a right bundle branch potential should be included in electrophysiology study of patients in whom there is suspicion of reentry within the His-Purkinje System. Clinically, recognizing these forms of tachycardias can be important because they can be effectively treated with catheter ablation.

Electrophysiological findings and long-term follow-up of patients with the permanent form of junctional reciprocating tachycardia treated by catheter ablation.

BACKGROUND: The permanent form of junctional reciprocating tachycardia (PJRT) commonly presents as recurrent drug-refractory, narrow-complex tachycardia. We studied the efficacy and safety of catheter ablation in treating these patients. METHODS AND RESULTS: Six patients with the diagnosis of PJRT were treated at our institution with direct-current catheter ablation. The study cohort comprised three men and three women with a mean age of 33.8 +/- 4.5 years. The mean time from onset of symptoms to ablation was 129 +/- 44.7 months. All failed multiple drug therapy (mean number of drugs failed was 5.3 +/- 0.5). The left ventricular ejection fractions were calculated by echocardiography and were greater than 60% in all except two patients, whose ejection fractions were 25% and 32%. Symptom duration was significantly longer in those with depressed ejection fraction compared with normal patients (258 versus 64.5 months, p less than 0.01). Electrophysiological findings revealed evidence of an atrioventricular reciprocating tachycardia involving retrograde decremental conduction over an accessory pathway localized to the posteroseptal area. Five patients received two direct-current shocks (250 +/- 16.7 J per shock) via paired electrodes from a catheter positioned just outside the coronary sinus os to a patch placed between the scapulae or on the anterior chest wall. One patient received a single direct-current shock of 300 J. The only complication was the development of complete atrioventricular block in one patient. This patient had previously undergone permanent pacemaker insertion for the sick sinus syndrome. The mean hospital stay after ablation was 2.2 days. Mean peak creatinine phosphokinase after ablation was 352 +/- 58.1 units/l and the MB fraction was 12 +/- 2%. Follow-up echocardiograms or gated nuclear studies showed improvement of ejection fraction in the two patients who presented with depressed ejection fractions. After a mean follow-up of 35.8 +/- 10.3 months, all patients remained free of tachycardia without antiarrhythmic drugs. CONCLUSIONS: We conclude that catheter ablation by using direct current energy appears to be an effective treatment in patients with PJRT.

Patients with supraventricular tachycardia presenting with aborted sudden death: incidence, mechanism and long-term follow-up.

A total of 13 (4.5%) of 290 patients with aborted sudden death had either documented (7; 54%) or strong presumptive evidence of supraventricular tachycardia that deteriorated into ventricular fibrillation. Six (46%) of the 13 had an accessory conduction pathway and either atrial fibrillation (5 patients) or paroxysmal atrioventricular (AV) reentrant tachycardia (1 patient) that deteriorated into ventricular fibrillation. Three patients with AV node reentrant tachycardia and four with atrial fibrillation and enhanced AV node conduction presented with supraventricular arrhythmias that deteriorated into ventricular fibrillation. Patients were treated with medical, surgical or catheter ablative procedures designed to prevent recurrences of supraventricular arrhythmias. Four patients received an implanted automatic defibrillator, but none had an appropriate device discharge. Over a follow-up period of 41.6 +/- 33.6 months, 12 patients are alive without symptomatic arrhythmias. One patient died because of severe chronic lung disease and heart failure. Supraventricular tachycardia was the cause of aborted sudden death in approximately 5% of patients referred for evaluation of sudden cardiac death. Treatment directed at prevention of supraventricular tachycardia was associated with an excellent prognosis. Current treatment techniques appear to obviate the need for automatic defibrillator therapy in these patients.

Radiofrequency catheter ablation for treatment of bundle branch reentrant ventricular tachycardia: results and long-term follow-up.

Seven of 120 consecutive patients with inducible sustained ventricular tachycardia (from September 1, 1988 to January 1, 1991) had bundle branch reentrant tachycardia and underwent percutaneous radiofrequency ablation of the right bundle branch. The seven patients had been unsuccessfully treated with a mean of 3 +/- 1 drugs. Four patients presented with syncope and three with aborted sudden death. The baseline electrocardiogram revealed a left bundle branch block pattern in three patients and an intraventricular conduction defect in four. The baseline HV interval was prolonged in each case (79 +/- 2 ms). With use of programmed ventricular extrastimuli, sustained bundle branch reentrant tachycardia was inducible in all patients at a mean cycle length of 283 +/- 17 ms (range 230 to 350). Bundle branch reentrant tachycardia characteristics included atrioventricular dissociation, a His deflection that preceded each QRS complex and spontaneous His to His variation that preceded changes in ventricular tachycardia cycle length. A quadripolar catheter was positioned across the tricuspid valve with the distal electrode tip of the catheter near the right bundle branch. One to three applications of continuous unmodulated radiofrequency current at 300 kHz between the distal electrode and a large posterior skin patch resulted in complete right bundle branch block in all patients, after which none had inducible bundle branch reentrant tachycardia on restudy. On restudy, three of the seven patients had ventricular tachycardia of myocardial origin (not bundle branch reentry). One patient required no therapy; drug or defibrillator therapy was used in the others.(ABSTRACT TRUNCATED AT 250 WORDS)

Implantable cardioverter defibrillator proarrhythmia: case report and review of the literature.

A 31-year-old man who received an automatic cardioverter defibrillator subsequently underwent exercise testing. During exercise, a sinus tachycardia resulted above his device detect rate prompting two shocks, the second of which produced an unstable polymorphous ventricular tachycardia. In this article, we review the literature on automatic cardioverter defibrillator-induced ventricular tachyarrhythmias as well as the management of exercise testing in patients with these devices.

Pacemaker syndrome in a patient with DDD pacemaker for long QT syndrome.

A patient with long QT syndrome was treated with beta blockers and had a permanent DDD pacemaker implanted. The lower rate was set to 85 beats/min because this provided the best shortening of QT interval at the lowest paced heart rate. The atrioventricular (AV) delay was programmed to 250 msec to allow native AV conduction. Patient returned complaining of symptoms suggestive of pacemaker syndrome. ECG during one of these episodes showed AV sequential pacing. Doppler echocardiography of hepatic vein flow suggested atrial contraction against a closed tricuspid valve. Endocardial electrogram telemetry demonstrated ventriculoatrial (VA) conduction with the retrograde atrial electrogram falling within the atrial refractory period and thus was not sensed. The following atrial stimulus did not capture because of the atrial refractoriness. Ventricular pacing proceeded after the programmed AV delay. Reprogramming the AV delay to 200 msec restored AV synchrony by allowing the atrial stimulus to capture by placing it outside of the refractory period of the atrium. No further symptoms reported during six months of follow-up.

Effect of platelet release products on cytosolic calcium in cardiac myocytes.

The effect of platelet release products on cytosolic calcium [( Ca++]i) was examined by monitoring the fluorescence of chick embryonic heart cells loaded with the fluorescent calcium indicator indo-1 AM. Cell free filtrate of platelet release products was obtained from rabbit platelets activated with thrombin or collagen. This filtrate caused a rapid increase in both systolic and diastolic [Ca++]i in a dose-dependent manner. The effect was not blocked by pretreating the platelets with aspirin or a thromboxane synthetase inhibitor. It was not mimicked by a thromboxane analog, or by several substances known to be released from platelets including ADP, serotonin, or platelet activating factor. Apyrase or ATP-gamma S had no effect on the activity. The responsible product was heat-sensitive, trypsin-sensitive, and partitioned into the aqueous phase of a chloroform suspension. It has a low molecular weight (less than 3kD) and is sensitive to 2-mercaptoethanol. Protease inhibitor appears to prolong the activity. These results suggest that trypsin-sensitive peptide(s) released from activated platelets can increase [Ca++]i in cardiac cells.

Effect of thrombin on calcium homeostasis in chick embryonic heart cells. Receptor-operated calcium entry with inositol trisphosphate and a pertussis toxin-sensitive G protein as second messengers.

UNLABELLED: Thrombin increases intracellular calcium ([Ca++]i) in several cell types and causes a positive inotropic effect in the heart. We examined the mechanism of the thrombin-induced [Ca++]i increase in chick embryonic heart cells loaded with the fluorescent calcium indicator, indo-1. Thrombin (1 U/ml) increased both systolic and diastolic [Ca++]i from 617 +/- 62 and 324 +/- 46 to 1041 +/- 93 and 587 +/- 38 nM, respectively. An initial rapid [Ca++]i increase was followed by a more sustained increase. There were associated increases in contraction strength, beat frequency, and action potential duration. The [Ca++]i increase was not blocked by tetrodotoxin or verapamil, but was blocked by pretreatment with pertussis toxin (100 ng/ml). The thrombin-induced [Ca++]i increase was partly due to intracellular calcium release, since it persisted after removal of external calcium. The [Ca++]i increase in zero calcium was more transitory than in normal calcium and was potentiated by 10 mM Li+. Thrombin also induced influx of calcium across the surface membrane, which could be monitored using Mn++ ions, which quench indo-1 fluorescence when they enter the cell. Thrombin-induced Mn++ entry was insensitive to verapamil, but was blocked by 2 mM Ni++. Thrombin increased inositol trisphosphates by 180% at 90 s and this effect was also blocked by pretreatment with pertussis toxin. CONCLUSION: thrombin promotes calcium entry and release in embryonic heart cells even when action potentials are inhibited. Both modes of [Ca++]i increase may be coupled to the receptor by pertussis toxin-sensitive G proteins.

Inactivation of Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase by an enzyme-generated ethoxyacetylenic ketone in the presence of a thiol.

Replacement of the 21-methyl group of 20 beta-hydroxypregn-4-en-3-one with an ethoxyacetylene group yields a compound that is an excellent substrate (pH 7.4, Km = 2.3 microM, Vmax = 4.6 nmol min-1 micrograms-1) for the Streptomyces hydrogenans NAD(H)-dependent 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53). The enzyme-generated ethoxyacetylenic ketone product is a potent inactivator of the enzyme. Gel filtration chromatography of enzyme inactivated with radiolabeled steroid demonstrates that covalent modification of the enzyme has occurred. Both NAD and NADH retard the rate of inactivation, suggesting that only free enzyme is susceptible to covalent modification. Consequently, enzymatically formed ethoxyacetylenic ketone does not react with the enzyme while it is part of the ternary complex. Moreover, the kinetically preferred release of this reactive ketone prior to NADH release assures that enzyme inactivation occurs only when released ketone subsequently encounters free enzyme. Kinetic analysis of inactivations carried out with chemically prepared ethoxyacetylenic ketone and enzyme at pH 7.4 and 9.2 yields bimolecular rate constants for the inactivation process of 1.15 X 10(4) L mol-1 s-1 and 6.94 X 10(4) L mol-1 s-1, respectively. This bimolecular reaction is faster than the bimolecular reaction of the ethoxyacetylenic ketone with either glutathione, mercaptoethanol, or dithiothreitol. Thus, complete inactivation by ketone generated from 5 microM alcohol and 5 microM NAD occurs in 30 min at pH 7.4 in the presence of 1 mM glutathione.