Anti-malarial drugs: Mechanisms underlying their proarrhythmic effects.

Malaria remains the leading cause of parasitic death in the world. Artemisinin resistance is an emerging threat indicating an imminent need for novel combination therapy. Given the key role of mass drug administration, it is pivotal that the safety of anti-malarial drugs is investigated thoroughly prior to widespread use. Cardiotoxicity, most prominently arrhythmic risk, has been a concern for anti-malarial drugs. We clarify the likely underlying mechanisms by which anti-malarial drugs predispose to arrhythmias. These relate to disruption of (1) action potential upstroke due to effects on the sodium currents, (2) action potential repolarisation due to effects on the potassium currents, (3) cellular calcium homeostasis, (4) mitochondrial function and reactive oxygen species production and (5) cardiac fibrosis. Together, these alterations promote arrhythmic triggers and substrates. Understanding these mechanisms is essential to assess the safety of these drugs, stratify patients based on arrhythmic risk and guide future anti-malarial drug development.

Transcriptional profiles of genes related to electrophysiological function in Scn5a+/- murine hearts.

The Scn5a gene encodes the major pore-forming Nav 1.5 (α) subunit, of the voltage-gated Na+ channel in cardiomyocytes. The key role of Nav 1.5 in action potential initiation and propagation in both atria and ventricles predisposes organisms lacking Scn5a or carrying Scn5a mutations to cardiac arrhythmogenesis. Loss-of-function Nav 1.5 genetic abnormalities account for many cases of the human arrhythmic disorder Brugada syndrome (BrS) and related conduction disorders. A murine model with a heterozygous Scn5a deletion recapitulates many electrophysiological phenotypes of BrS. This study examines the relationships between its Scn5a+/- genotype, resulting transcriptional changes, and the consequent phenotypic presentations of BrS. Of 62 selected protein-coding genes related to cardiomyocyte electrophysiological or homeostatic function, concentrations of mRNA transcribed from 15 differed significantly from wild type (WT). Despite halving apparent ventricular Scn5a transcription heterozygous deletion did not significantly downregulate its atrial expression, raising possibilities of atria-specific feedback mechanisms. Most of the remaining 14 genes whose expression differed significantly between WT and Scn5a+/- animals involved Ca2+ homeostasis specifically in atrial tissue, with no overlap with any ventricular changes. All statistically significant changes in expression were upregulations in the atria and downregulations in the ventricles. This investigation demonstrates the value of future experiments exploring for and clarifying links between transcriptional control of Scn5a and of genes whose protein products coordinate Ca2+ regulation and examining their possible roles in BrS.

Molecular basis of ventricular arrhythmogenicity in a Pgc-1α deficient murine model.

Mitochondrial dysfunction underlying metabolic disorders such as obesity and diabetes mellitus is strongly associated with cardiac arrhythmias. Murine Pgc-1α-/- hearts replicate disrupted mitochondrial function and model the associated pro-arrhythmic electrophysiological abnormalities. Quantitative PCR, western blotting and histological analysis were used to investigate the molecular basis of the electrophysiological changes associated with mitochondrial dysfunction. qPCR analysis implicated downregulation of genes related to Na+-K+ ATPase activity (Atp1b1), surface Ca2+ entry (Cacna1c), action potential repolarisation (Kcnn1), autonomic function (Adra1d, Adcy4, Pde4d, Prkar2a), and morphological properties (Myh6, Tbx3) in murine Pgc-1α-/- ventricles. Western blotting revealed reduced NaV1.5 but normal Cx43 expression. Histological analysis revealed increased tissue fibrosis in the Pgc-1α-/- ventricles. These present findings identify altered transcription amongst a strategically selected set of genes established as encoding proteins involved in cardiac electrophysiological activation and therefore potentially involved in alterations in ventricular activation and Ca2+ homeostasis in arrhythmic substrate associated with Pgc-1α deficiency. They complement and complete previous studies examining such expression characteristics in the atria and ventricles of Pgc-1 deficient murine hearts.

Circulating microRNA as a Biomarker for Coronary Artery Disease.

Coronary artery disease (CAD) is the leading cause of sudden cardiac death in adults, and new methods of predicting disease and risk-stratifying patients will help guide intervention in order to reduce this burden. Current CAD detection involves multiple modalities, but the consideration of other biomarkers will help improve reliability. The aim of this narrative review is to help researchers and clinicians appreciate the growing relevance of miRNA in CAD and its potential as a biomarker, and also to suggest useful miRNA that may be targets for future study. We sourced information from several databases, namely PubMed, Scopus, and Google Scholar, when collating evidentiary information. MicroRNAs (miRNA) are short, noncoding RNAs that are relevant in cardiovascular physiology and pathophysiology, playing roles in cardiac hypertrophy, maintenance of vascular tone, and responses to vascular injury. CAD is associated with changes in miRNA expression profiles, and so are its risk factors, such as abnormal lipid metabolism and inflammation. Thus, they may potentially be biomarkers of CAD. Nevertheless, there are limitations in using miRNA. These include cost and the presence of several confounding factors that may affect miRNA profiles. Furthermore, there is difficulty in the normalisation of miRNA values between published studies, due to pre-analytical variations in samples.

Protein expression profiles in murine ventricles modeling catecholaminergic polymorphic ventricular tachycardia: effects of genotype and sex.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is associated with mutations in the cardiac ryanodine receptor (RyR2). These result in stress-induced ventricular arrhythmic episodes, with clinical symptoms and prognosis reported more severe in male than female patients. Murine homozygotic RyR2-P2328S (RyR2S/S ) hearts replicate the proarrhythmic CPVT phenotype of abnormal sarcoplasmic reticular Ca2+ leak and disrupted Ca2+ homeostasis. In addition, RyR2S/S hearts show decreased myocardial action potential conduction velocities (CV), all features implicated in arrhythmic trigger and substrate. The present studies explored for independent and interacting effects of RyR2S/S genotype and sex on expression levels of molecular determinants of Ca2+ homeostasis (CASQ2, FKBP12, SERCA2a, NCX1, and CaV 1.2) and CV (NaV 1.5, Connexin (Cx)-43, phosphorylated-Cx43, and TGF-ß1) in mice. Expression levels of Ca2+ homeostasis proteins were not altered, hence implicating abnormal RyR2 function alone in disrupted cytosolic Ca2+ homeostasis. Furthermore, altered NaV 1.5, phosphorylated Cx43, and TGF-ß1 expression were not implicated in the development of slowed CV. By contrast, decreased Cx43 expression correlated with slowed CV, in female, but not male, RyR2S/S mice. The CV changes may reflect acute actions of the increased cytosolic Ca2+ on NaV 1.5 and Cx43 function.

Molecular basis of arrhythmic substrate in ageing murine peroxisome proliferator-activated receptor γ co-activator deficient hearts modelling mitochondrial dysfunction.

INTRODUCTION: Ageing and chronic metabolic disorders are associated with mitochondrial dysfunction and cardiac pro-arrhythmic phenotypes which were recently attributed to slowed atrial and ventricular action potential (AP) conduction in peroxisome proliferator-activated receptor γ co-activator deficient (Pgc-1ß-/-) mice. METHODS: We compared expression levels of voltage-gated Na+ channel (NaV1.5) and gap junction channels, Connexins 40 and 43 (Cx40 and Cx43) in the hearts of young and old, and wild-type (WT) and Pgc-1ß-/- mice. This employed Western blotting (WB) for NaV1.5, Cx40 and Cx43 in atrial/ventricular tissue lysates, and immunofluorescence (IF) from Cx43 was explored in tissue sections. Results were analysed using two-way analysis of variance (ANOVA) for independent/interacting effects of age and genotype. RESULTS: In atria, increased age and Pgc-1ß-/- genotype each independently decreased both Cx40 and Cx43 expression without interacting effects. In IF experiments, both age and Pgc-1ß deletion independently reduced Cx43 expression. In ventricles, age and genotype exerted interacting effects in WB studies of NaV1.5 expression. Young Pgc-1ß-/- then showed greater NaV1.5 expression than young WT ventricles. However, neither age nor Pgc-1ß deletion affected Cx43 expression, independently or through interacting effects in both WB and IF studies. CONCLUSION: Similar pro-arrhythmic atrial/ventricular phenotypes arise in aged/Pgc-1ß-/- from differing contributions of altered protein expression and functional effects that may arise from multiple acute mechanisms.

Preclinical validation of 3-phosphoinositide-dependent protein kinase 1 inhibition in pancreatic cancer.

BACKGROUND: The very aggressive nature and low survival rate of pancreatic ductal adenocarcinoma (PDAC) dictates the necessity to find novel efficacious therapies. Recent evidence suggests that phosphoinositide 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase 1 (PDK1) are key effectors of oncogenic KRAS in PDAC. Herein, we report the role and mechanism of action of PDK1, a protein kinase of the AGC family, in PDAC. METHODS: PDAC cell lines were treated with selective PDK1 inhibitors or transfected with specific PDK1-targeting siRNAs. In vitro and in vivo assays were performed to investigate the functional role of PDK1 in PDAC. Specifically, anchorage-dependent and anchorage-independent growth was assessed in PDAC cells upon inhibition or downregulation of PDK1. Detailed investigation of the effect of PDK1 inhibition/downregulation on specific signalling pathways was also performed by Western blotting analysis. A xenograft tumour mouse model was used to determine the effect of pharmacological inhibition of PDK1 on PDAC cells growth in vivo. RESULTS: Treatment with specific inhibitors of PDK1 impaired anchorage-dependent and anchorage-independent growth of pancreatic cancer cell lines, as well as pancreatic tumour growth in a xenograft model. Mechanistically, inhibition or downregulation of PDK1 resulted in reduced activation of the serum/glucocorticoid regulated kinase family member 3 and subsequent reduced phosphorylation of its target N-Myc downstream regulated 1. Additionally, we found that combination of sub-optimal concentrations of inhibitors selective for PDK1 and the class IB PI3K isoform p110γ inhibits pancreatic cancer cell growth and colonies formation more potently than each single treatment. CONCLUSIONS: Our data indicate that PDK1 is a suitable target for therapeutic intervention in PDAC and support the clinical development of PDK1 inhibitors for PDAC.

Atrial Transcriptional Profiles of Molecular Targets Mediating Electrophysiological Function in Aging and Pgc-1ß Deficient Murine Hearts.

BACKGROUND: Deficiencies in the transcriptional co-activator, peroxisome proliferative activated receptor, gamma, coactivator-1ß are implicated in deficient mitochondrial function. The latter accompanies clinical conditions including aging, physical inactivity, obesity, and diabetes. Recent electrophysiological studies reported that Pgc-1ß-/- mice recapitulate clinical age-dependent atrial pro-arrhythmic phenotypes. They implicated impaired chronotropic responses to adrenergic challenge, compromised action potential (AP) generation and conduction despite normal AP recovery timecourses and background resting potentials, altered intracellular Ca2+ homeostasis, and fibrotic change in the observed arrhythmogenicity. OBJECTIVE: We explored the extent to which these age-dependent physiological changes correlated with alterations in gene transcription in murine Pgc-1ß-/- atria. METHODS AND RESULTS: RNA isolated from murine atrial tissue samples from young (12-16 weeks) and aged (>52 weeks of age), wild type (WT) and Pgc-1ß-/- mice were studied by pre-probed quantitative PCR array cards. We examined genes encoding sixty ion channels and other strategic atrial electrophysiological proteins. Pgc-1ß-/- genotype independently reduced gene transcription underlying Na+-K+-ATPase, sarcoplasmic reticular Ca2+-ATPase, background K+ channel and cholinergic receptor function. Age independently decreased Na+-K+-ATPase and fibrotic markers. Both factors interacted to alter Hcn4 channel activity underlying atrial automaticity. However, neither factor, whether independently or interactively, affected transcription of cardiac Na+, voltage-dependent K+ channels, surface or intracellular Ca2+ channels. Nor were gap junction channels, ß-adrenergic receptors or transforming growth factor-ß affected. CONCLUSION: These findings limit the possible roles of gene transcriptional changes in previously reported age-dependent pro-arrhythmic electrophysiologial changes observed in Pgc-1ß-/- atria to an altered Ca2+-ATPase (Atp2a2) expression. This directly parallels previously reported arrhythmic mechanism associated with p21-activated kinase type 1 deficiency. This could add to contributions from the direct physiological outcomes of mitochondrial dysfunction, whether through reactive oxygen species (ROS) production or altered Ca2+ homeostasis.

Ageing in Pgc-1ß-/- mice modelling mitochondrial dysfunction induces differential expression of a range of genes regulating ventricular electrophysiology.

Mice deficient in mitochondrial promoter peroxisome proliferator activated receptor-γ co-activator-1ß (Pgc-1ß-/- ) is a valuable model for metabolic diseases and has been found to present with several pathologies including ventricular arrhythmia. In the present study, our aim was to shed light on the molecular mechanisms behind the observed arrhythmic substrate by studying how the expression of selected genes critical for cardiac function differs in wild-type (WT) compared with Pgc-1ß knockout mice and young compared with aged mice. We found that a clear majority of genes are down-regulated in the Pgc-1ß-/- ventricular tissue compared with the WT. Although most individual genes are not significantly differentially expressed, a pattern is apparent when the genes are grouped according to their functional properties. Genes encoding proteins relating to ATPase activity, potassium ion channels relating to repolarisation and resting membrane potential, and genes encoding proteins in the cAMP pathway are found to be significantly down-regulated in the Pgc-1ß deficient mice. On the contrary, the pacemaker channel genes Hcn3 and Hcn4 are up-regulated in subsets of the Pgc-1ß deficient tissue. Furthermore, we found that with age, especially in the Pgc-1ß-/- genotype, most genes are up-regulated including genes relating to the resting membrane potential, calcium homeostasis, the cAMP pathway, and most of the tested adrenoceptors. In conclusion, we here demonstrate how a complex pattern of many modest changes at gene level may explain major functional differences of the action potential related to ageing and mitochondrial dysfunction.

Gene and Protein Expression Profile of Selected Molecular Targets Mediating Electrophysiological Function in Pgc-1α Deficient Murine Atria.

Increases in the prevalence of obesity, insulin resistance, and metabolic syndrome has led to the increase of atrial fibrillation (AF) cases in the developed world. These AF risk factors are associated with mitochondrial dysfunction, previously modelled using peroxisome proliferator activated receptor-γ (PPARγ) coactivator-1 (Pgc-1)-deficient murine cardiac models. We explored gene and protein expression profiles of selected molecular targets related to electrophysiological function in murine Pgc-1α-/- atria. qPCR analysis surveyed genes related to Na⁺-K⁺-ATPase, K⁺ conductance, hyperpolarisation-activated cyclic nucleotide-gated (Hcn), Na⁺ channels, Ca2+ channels, and indicators for adrenergic and cholinergic receptor modulation. Western blot analysis for molecular targets specific to conduction velocity (Nav1.5 channel and gap junctions) was performed. Transcription profiles revealed downregulation of molecules related to Na⁺-K⁺-ATPase transport, Hcn-dependent pacemaker function, Na⁺ channel-dependent action potential activation and propagation, Ca2+ current generation, calsequestrin-2 dependent Ca2+ homeostasis, and adrenergic α1D dependent protection from hypertrophic change. Nav1.5 channel protein expression but not gap junction expression was reduced in Pgc-1α-/- atria compared to WT. Nav1.5 reduction reflects corresponding reduction in its gene expression profile. These changes, as well as the underlying Pgc-1α-/- alteration, suggest potential pharmacological targets directed towards either upstream PGC-1 signalling mechanisms or downstream ion channel changes.

Arrhythmogenic mechanisms of obstructive sleep apnea in heart failure patients.

Heart failure (HF) affects 23 million people worldwide and results in 300000 annual deaths. It is associated with many comorbidities, such as obstructive sleep apnea (OSA), and risk factors for both conditions overlap. Eleven percent of HF patients have OSA and 7.7% of OSA patients have left ventricular ejection fraction <50% with arrhythmias being a significant comorbidity in HF and OSA patients. Forty percent of HF patients develop atrial fibrillation (AF) and 30%-50% of deaths from cardiac causes in HF patients are from sudden cardiac death. OSA is prevalent in 32%-49% of patients with AF and there is a dose-dependent relationship between OSA severity and resistance to anti-arrhythmic therapies. HF and OSA lead to various downstream arrhythmogenic mechanisms, including metabolic derangement, remodeling, inflammation, and autonomic imbalance. (1) Metabolic derangement and production of reactive oxidative species increase late Na+ currents, decrease outward K+ currents and downregulate connexin-43 and cell-cell coupling. (2) remodeling also features downregulated K+ currents in addition to decreased Na+/K+ ATPase currents, altered Ca2+ homeostasis, and increased density of If current. (3) Chronic inflammation leads to downregulation of both Nav1.5 channels and K+ channels, altered Ca2+ homeostasis and reduced cellular coupling from alterations of connexin expression. (4) Autonomic imbalance causes arrhythmias by evoking triggered activity through increased Ca2+ transients and reduction of excitation wavefront wavelength. Thus, consideration of these multiple pathophysiological pathways (1-4) will enable the development of novel therapeutic strategies that can be targeted against arrhythmias in the context of complex disease, such as the comorbidities of HF and OSA.

Effects of ageing on pro-arrhythmic ventricular phenotypes in incrementally paced murine Pgc-1ß -/- hearts.

A range of chronic clinical conditions accompany cardiomyocyte energetic dysfunction and constitute independent risk factors for cardiac arrhythmia. We investigated pro-arrhythmic and arrhythmic phenotypes in energetically deficient C57BL mice with genetic ablation of the mitochondrial promoter peroxisome proliferator-activated receptor-γ coactivator-1ß (Pgc-1ß), a known model of ventricular arrhythmia. Pro-arrhythmic and cellular action potential (AP) characteristics were compared in intact Langendorff-perfused hearts from young (12-16 week) and aged (> 52 week), wild-type (WT) and Pgc-1ß -/- mice. Simultaneous electrocardiographic and intracellular microelectrode recordings were made through successive trains of 100 regular stimuli at progressively incremented heart rates. Aged Pgc-1ß -/- hearts displayed an increased incidence of arrhythmia compared to other groups. Young and aged Pgc-1ß -/- hearts showed higher incidences of alternans in both AP activation (maximum AP upshoot velocity (dV/dt)max and latency), recovery (action potential duration (APD90) and resting membrane potential (RMP) characteristics compared to WT hearts. This was particularly apparent at lower pacing frequencies. These findings accompanied reduced (dV/dt)max and increased AP latency values in the Pgc-1ß -/- hearts. APs observed prior to termination of the protocol showed lower (dV/dt)max and longer AP latencies, but indistinguishable APD90 and RMPs in arrhythmic compared to those in non-arrhythmic hearts. APD restitution analysis showed that Pgc-1ß -/- and WT hearts showed similar limiting gradients. However, Pgc-1ß -/- hearts had shortened plateau AP wavelengths, particularly in aged Pgc-1ß -/- hearts. Pgc-1ß -/- hearts therefore show pro-arrhythmic instabilities attributable to altered AP conduction and activation rather than recovery characteristics.

Caffeine and the analog CGS 15943 inhibit cancer cell growth by targeting the phosphoinositide 3-kinase/Akt pathway.

Caffeine is a naturally occurring methylxanthine that acts as a non-selective adenosine receptor antagonist. Epidemiological studies demonstrated habitual coffee drinking to be significantly associated with liver cancer survival. We aimed to investigate the effects of caffeine and its analog CGS 15943 on hepatocellular carcinoma (HCC) and pancreatic cancer adenocarcinoma (PDAC). We demonstrate that caffeine and CGS 15943 block proliferation in HCC and PDAC cell lines by inhibiting the PI3K/Akt pathway. Importantly a kinase profiling assay reveals that CGS 15943 targets specifically the catalytic subunit of the class IB PI3K isoform (p110γ). These data give mechanistic insight into the action of caffeine and its analogs and they identify these compounds as promising lead compounds to develop drugs that can specifically target this PI3K isoform whose key role in cancer progression is emerging.

PI3K class IB controls the cell cycle checkpoint promoting cell proliferation in hepatocellular carcinoma.

Alterations of the cell cycle checkpoint frequently occur during hepatocarcinogenesis. Dysregulation of the phosphatidylinositol-3-kinases (PI3K) signaling pathway is believed to exert a potential oncogenic effect in hepatocellular carcinoma (HCC), ultimately promoting tumor cell proliferation. However, the impact of PI3K on cell cycle regulation remains unclear. We used a combined loss- and gain-of-function approach to address the involvement of p110γ in HCC cell proliferation, apoptosis and the cell cycle. We also investigated the correlation between p110γ and Ki-67 in 24 HCC patients. Finally, we analyzed the expression levels of p110γ and cell cycle regulators in HCC tissues. We found that PI3K class IB, but not class IA, is required for HCC cell proliferation. In particular, we found that knock-down of p110γ inhibits cell proliferation because of an arrest of the cell cycle in the G0-G1 phase. This effect is associated with an altered expression of proteins regulating the cell cycle progression, including p21, and with an increased apoptosis. By contrast, we found that ectopic expression of p110γ promotes HCC cell proliferation. Tissues analysis performed in HCC patients showed a positive correlation between the expression of p110γ and Ki-67, a marker of proliferation, and, even more importantly, that p21 expression is up-regulated in HCC patients with a lower p110γ expression. Our results emphasize the role of p110γ as a promoter of HCC proliferation and unveil an important cell cycle regulation function of this molecule.

Targeting phosphoinositide 3-kinase pathways in pancreatic cancer--from molecular signalling to clinical trials.

Pancreatic cancer has one of the poorest prognoses among all cancers partly because of its silent nature and tendency for late discovery but also because of its persistent resistance to chemotherapy. At present there are very limited treatment alternatives for pancreatic cancer, hence the need to develop novel and more efficient drugs. It is well known that mutations in K-Ras oncogene accumulate early in the disease progression and occur in almost all of pancreatic ductal adenocarcinomas (PDAC). A key downstream target of the Ras family is phosphoinositide 3-kinase (PI3K), the enzyme responsible for generation of 3-phosphorylated phosphoinositides and activation of Akt (Protein Kinase B/Akt). The PI3K/Akt pathway is involved in inhibition of apoptosis and stimulation of cell proliferation and it is has been estimated that at least 50% of all cancer types are related to deregulation of this signalling pathway. In this review we will discuss how the PI3K/Akt/mTOR signaling network is altered in pancreatic cancer and further give an overview of preclinical and clinical studies where this pathway has been targeted.

Class II phosphoinositide 3-kinase regulates exocytosis of insulin granules in pancreatic beta cells.

Phosphoinositide 3-kinases (PI3Ks) are critical regulators of pancreatic ß cell mass and survival, whereas their involvement in insulin secretion is more controversial. Furthermore, of the different PI3Ks, the class II isoforms were detected in ß cells, although their role is still not well understood. Here we show that down-regulation of the class II PI3K isoform PI3K-C2α specifically impairs insulin granule exocytosis in rat insulinoma cells without affecting insulin content, the number of insulin granules at the plasma membrane, or the expression levels of key proteins involved in insulin secretion. Proteolysis of synaptosomal-associated protein of 25 kDa, a process involved in insulin granule exocytosis, is impaired in cells lacking PI3K-C2α. Finally, our data suggest that the mRNA for PI3K-C2α may be down-regulated in islets of Langerhans from type 2 diabetic compared with non-diabetic individuals. Our results reveal a critical role for PI3K-C2α in ß cells and suggest that down-regulation of PI3K-C2α may be a feature of type 2 diabetes.

Key role of phosphoinositide 3-kinase class IB in pancreatic cancer.

PURPOSE: Phosphoinositide 3-kinase (PI3K) signaling is well established as important in cancer. To date most studies have been focused on the PI3K/p110α isoform, which has been found to be mutated in several different cancers. The aim of our study was to determine which specific PI3K isoforms are involved in pancreatic ductal adenocarcinoma (PDAC) and investigate the effects of these isoforms on proliferation, survival, and induction of Akt activation in pancreatic cancer cells. EXPERIMENTAL DESIGN: The expression of all PI3K isoforms and downstream targets was analyzed by immunohistochemistry in human pancreatic cancer tissue and normal counterparts. Isoform selective inhibitors and short interfering RNA (siRNA) were employed to investigate the effects of the different PI3Ks on proliferation, survival, and intracellular signaling in PDAC cell lines. RESULTS: Immunohistochemical screening revealed high specific expression of the PI3K/p110γ isoform. Scoring indicated that 72% of the PDAC tissue stained positive for PI3K/p110γ, whereas no stain was detected in normal pancreatic ducts. Proliferation analyses after selective inhibition and siRNA downregulation of PI3K/p110γ showed that PI3K/p110γ, but not other PI3K isoforms, was required for cell proliferation. Overexpression of PI3K/p110γ indeed increased cell numbers and mediated activation of Akt in PDAC cell lines. Moreover, PI3K/p110γ was required for Akt activation via lysophosphatidic acid receptors. CONCLUSIONS: These data represent the first identification of a tumor-specific accumulation of the PI3K isoform p110γ in human cancer. Further, our results signify a critical role for PI3K/p110γ in pancreatic cancer, and we hypothesize that PI3K/p110γ overexpression is a key event in the disease progression.

c-Kit--a hematopoietic cell essential receptor tyrosine kinase.

The receptor tyrosine kinase c-Kit is expressed in hematopoietic stem and progenitor cells and in several non-hematopoietic tissues. In the hematopoietic system, c-Kit is critical for proliferation, survival and differentiation. During recent years exploration of the signalling pathways downstream of this receptor has yielded significant new insights in the field. In this review, we will summarise the c-Kit background, structure, downstream signalling and medical significance with particular focus on its role in hematopoietic progenitor cells and mast cells.

Haematopoietic progenitor cells utilise conventional PKC to suppress PKB/Akt activity in response to c-Kit stimulation.

Receptor tyrosine kinase (RTK) c-Kit signalling is crucial for the proliferation, survival and differentiation of haematopoietic stem cells (HSCs). To further understand the mechanisms underlying these events we explored how the downstream mediators interact. The present study investigated the function of conventional protein kinase Cs (c-PKC) in c-Kit mediated signalling pathways in HSC-like cell lines. This analysis supported earlier findings, that steel factor (SF) activates c-PKC, extracellular signal-regulated kinase (Erk) and protein kinase B (PKB). The present results were consistent with an important role of c-PKC in the positive activation of Erk and for proliferation. Further, it was observed that c-PKC negatively regulated PKB activity upon SF stimulation, indicating that c-PKC acts as a suppressor of c-Kit signalling. Finally, these observations were extended to show that c-PKC mediated the phosphorylation of the endogenous c-Kit receptor on serine 746, resulting in decreased overall tyrosine phosphorylation of c-Kit upon SF stimulation. This report showed that this specific feedback mechanism of c-PKC mediated phosphorylation of the c-Kit receptor has consequences for both proliferation and survival of HSC-like cell lines.

Activation of the MAP kinase pathway by c-Kit is PI-3 kinase dependent in hematopoietic progenitor/stem cell lines.

The Steel factor (SF) and its receptor c-Kit play a critical role for various cell types at different levels in the hematopoietic hierarchy. Whether similar or distinct signaling pathways are used upon c-Kit activation in different cell types within the hematopoietic hierarchy is not known. To study c-Kit signaling pathways in the hematopoietic system we have compared c-Kit downstream signaling events in SF-dependent hematopoietic stem cell (HSC)-like cell lines to those of mast cells. Both Erk and protein kinase B (PKB)/Akt are activated by ligand-induced activation of the c-Kit receptor in the HSC-like cell lines. Surprisingly, phosphoinositide-3 (PI-3) kinase inhibitors block not only PKB/Akt activation but also activation of Raf and Erk. SF-induced activation of Ras is not affected by inhibition of PI-3 kinase. In mast cells and other more committed hematopoietic precursors, the activation of Erk by SF is not PI-3 kinase dependent. Our results suggest that a molecular signaling switch occurs during differentiation in the hematopoietic system whereby immature hematopoietic progenitor/stem cells use a PI-3 kinase-sensitive pathway in the activation of both Erk and PKB/Akt, which is then switched upon differentiation to the more commonly described PI-3 kinase-independent mitogen-activated protein (MAP) kinase pathway.