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Junkins R.D.,Dalhousie University | Junkins R.D.,Health Center | Junkins R.D.,Beatrice Hunter Cancer Research Institute | Shen A.,Dalhousie University | And 5 more authors.
PLoS ONE | Year: 2013

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection. © 2013 Junkins et al.


Cutler M.J.,Dalhousie University | Cutler M.J.,Beatrice Hunter Cancer Research Institute | Choo E.F.,Genentech
Current Drug Metabolism | Year: 2011

The effectiveness of many anticancer agents is dependent on their disposition to the intracellular space of cancerous tissue. Accumulation of anticancer drugs at their sites of action can be altered by both uptake and efflux transport proteins, however the majority of research on the disposition of anticancer drugs has focused on drug efflux transporters and their ability to confer multidrug resistance. Here we review the roles of uptake transporters of the SLC22A and SLCO families in the context of cancer therapy. The many first-line anticancer drugs that are substrates of organic cation transporters (OCTs) organic cation/carnitine transporters (OCTNs) and organic anion-transporting polypeptides (OATPs) are summarized. In addition, where data is available a comparison of the localization of drug uptake transporters in healthy and cancerous tissues is provided. Expression of drug uptake transporters increases the sensitivity of cancer cell lines to anticancer substrates. Furthermore, early observational studies have suggested a causal link between drug uptake transporter expression and positive outcome in some cancers. Quantification of drug transporters by mass spectrometry will provide an essential technique for generation of expression data during future observational clinical studies. Screening of drug uptake transporter expression in primary tumors may help differentiate between susceptible and resistant cancers prior to therapy. © 2011 Bentham Science Publishers.


Kalousi A.,French Institute of Health and Medical Research | Hoffbeck A.-S.,French Institute of Health and Medical Research | Selemenakis P.N.,National and Kapodistrian University of Athens | Pinder J.,Dalhousie University | And 11 more authors.
Cell Reports | Year: 2015

Cells experience damage from exogenous and endogenous sources that endanger genome stability. Several cellular pathways have evolved to detect DNA damage and mediate its repair. Although many proteins have been implicated in these processes, only recent studies have revealed how they operate in the context of high-ordered chromatin structure. Here, we identify the nuclear oncogene SET (I2PP2A) as a modulator of DNA damage response (DDR) and repair in chromatin surrounding double-strand breaks (DSBs). We demonstrate that depletion of SET increases DDR and survival in the presence of radiomimetic drugs, while overexpression of SET impairs DDR and homologous recombination (HR)-mediated DNA repair. SET interacts with the Kruppel-associated box (KRAB)-associated co-repressor KAP1, and its overexpression results inthe sustained retention of KAP1 and Heterochromatin protein 1 (HP1) on chromatin. Our results are consistent with a model in which SET-mediated chromatin compaction triggers an inhibition of DNA end resection and HR. © 2015 The Authors.


Junkins R.D.,Dalhousie University | Junkins R.D.,Health Center | Junkins R.D.,Beatrice Hunter Cancer Research Institute | McCormick C.,Dalhousie University | And 4 more authors.
Autophagy | Year: 2014

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), a channel that normally transports anions across epithelial cell membranes. The most common manifestation of CF is buildup of mucus in the airways and bacterial colonization of the lower respiratory tract, accompanied by chronic inflammation. Antibiotics are used to control CF-associated opportunistic infections, but lengthy antibiotic treatment risks the emergence of multiple-drug resistant (MDR) strains. New antimicrobial strategies are needed to prevent and treat infections in these high-risk individuals. Autophagy contributes to the control of a variety of microbial infections. For this reason, the recent discovery of functional impairment of autophagy in CF provides a new basis for understanding susceptibility to severe infections. Here, we review the role of autophagy in host defense against CF-associated bacterial and fungal pathogens, and survey pharmacologic approaches to restore normal autophagy function in these individuals. Autophagy restoration therapy may improve pathogen clearance and mitigate lung inflammation in CF airways. © 2014 Landes Bioscience.


Kepkay R.,Dalhousie University | Attwood K.M.,Dalhousie University | Ziv Y.,Tel Aviv University | Shiloh Y.,Tel Aviv University | And 2 more authors.
Cell Cycle | Year: 2011

The promyelocytic leukemia (PML) protein is the main structural component of subnuclear domains termed PML nuclear bodies (PML NBs), which are implicated in tumor suppression by regulating apoptosis, cell senescence and DNA repair. Previously, we demonstrated that ATM kinase can regulate changes in PML NB number in response to DNA doublestrand breaks (DSBs). PML NBs make extensive contacts with chromatin and ATM mediates DNA damage-dependent changes in chromatin structure in part by the phosphorylation of the KRAB-associated protein 1 (KAP1) at S824. We now demonstrate that in the absence of DNA damage, reduced KAP1 expression results in a constitutive increase in PML NB number in both human U2-OS cells and normal human diploid fibroblasts. This increase in PML NB number correlated with decreased nuclear lamina-associated heterochromatin and a 30% reduction in chromatin density as observed by electron microscopy, which is reminiscent of DNA damaged chromatin. These changes in chromatin ultrastructure also correlated with increased histone H4 acetylation, and treatment with the HDAC inhibitor TSA failed to further increase PML NB number. Although PML NB number could be restored by complementation with wild-type KAP1, both the loss of KAP1 or complementation with phospho-mutants of KAP1 inhibited the early increase in PML NB number and reduced the fold induction of PML NBs by 25-30% in response to etoposide-induced DNA DSBs. Together these data implicate KAP1-dependent changes in chromatin structure as one possible mechanism by which ATM may regulate PML NB number in response to DNA damage. © 2011 Landes Bioscience.


Corcoran J.A.,Dalhousie University | Corcoran J.A.,Beatrice Hunter Cancer Research Institute | Johnston B.P.,Dalhousie University | Johnston B.P.,Beatrice Hunter Cancer Research Institute | And 2 more authors.
PLoS Pathogens | Year: 2015

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of several AIDS-related cancers, including the endothelial cell (EC) neoplasm Kaposi's sarcoma (KS). KSHV-infected ECs secrete abundant host-derived pro-inflammatory molecules and angiogenic factors that contribute to tumorigenesis. The precise contributions of viral gene products to this secretory phenotype remain to be elucidated, but there is emerging evidence for post-transcriptional regulation. The Kaposin B (KapB) protein is thought to contribute to the secretory phenotype in infected cells by binding and activating the stress-responsive kinase MK2, thereby selectively blocking decay of AU-rich mRNAs (ARE-mRNAs) encoding pro-inflammatory cytokines and angiogenic factors. Processing bodies (PBs) are cytoplasmic ribonucleoprotein foci in which ARE-mRNAs normally undergo rapid 5′ to 3′ decay. Here, we demonstrate that PB dispersion is a feature of latent KSHV infection, which is dependent on kaposin protein expression. KapB is sufficient to disperse PBs, and KapB-mediated ARE-mRNA stabilization could be partially reversed by treatments that restore PBs. Using a combination of genetic and chemical approaches we provide evidence that KapB-mediated PB dispersion is dependent on activation of a non-canonical Rho-GTPase signaling axis involving MK2, hsp27, p115RhoGEF and RhoA. PB dispersion in latently infected cells is likewise dependent on p115RhoGEF. In addition to PB dispersion, KapB-mediated RhoA activation in primary ECs caused actin stress fiber formation, increased cell motility and angiogenesis; these effects were dependent on the activity of the RhoA substrate kinases ROCK1/2. By contrast, KapB-mediated PB dispersion occurred in a ROCK1/2-independent manner. Taken together, these observations position KapB as a key contributor to viral reprogramming of ECs, capable of eliciting many of the phenotypes characteristic of KS tumor cells, and strongly contributing to the post-transcriptional control of EC gene expression and secretion. © 2015 Corcoran et al.


Corkery D.P.,Dalhousie University | Holly A.C.,Dalhousie University | Lahsaee S.,Dalhousie University | Dellaire G.,Dalhousie University | Dellaire G.,Beatrice Hunter Cancer Research Institute
Nucleus | Year: 2015

Abbreviations: SR, serine arginine; SRPK, serine arginine protein kinase; CLK, CDC-like kinase; PRP4K, pre-mRNA processing factor 4 kinase; snRNP, small nuclear ribonucleic protein. Alternative pre-mRNA splicing in higher eukaryotes enhances transcriptome complexity and proteome diversity. Its regulation is mediated by a complex RNA-protein network that is essential for the maintenance of cellular and tissue homeostasis. Disruptions to this regulatory network underlie a host of human diseases and contribute to cancer development and progression. The splicing kinases are an important family of pre-mRNA splicing regulators, , which includes the CDC-like kinases (CLKs), the SRSF protein kinases (SRPKs) and pre-mRNA splicing 4 kinase (PRP4K/PRPF4B). These splicing kinases regulate pre-mRNA splicing via phosphorylation of spliceosomal components and serine- arginine (SR) proteins, affecting both their nuclear localization within nuclear speckle domains as well as their nucleocytoplasmic shuttling. Here we summarize the emerging evidence that splicing kinases are dysregulated in cancer and play important roles in both tumorigenesis as well as therapeutic response to radiation and chemotherapy. © Dale P Corkery, Alice C Holly, Sara Lahsaee, and Graham Dellaire.


Gebremeskel S.,Dalhousie University | Gebremeskel S.,Beatrice Hunter Cancer Research Institute | Johnston B.,Dalhousie University | Johnston B.,Beatrice Hunter Cancer Research Institute
Oncotarget | Year: 2015

Chemotherapy has historically been thought to induce cancer cell death in an immunogenically silent manner. However, recent studies have demonstrated that therapeutic outcomes with specific chemotherapeutic agents (e.g. anthracyclines) correlate strongly with their ability to induce a process of immunogenic cell death (ICD) in cancer cells. This process generates a series of signals that stimulate the immune system to recognize and clear tumor cells. Extensive studies have revealed that chemotherapy-induced ICD occurs via the exposure/release of calreticulin (CALR), ATP, chemokine (C-X-C motif) ligand 10 (CXCL10) and high mobility group box 1 (HMGB1). This review provides an in-depth look into the concepts and mechanisms underlying CALR exposure, activation of the Toll-like receptor 3/IFN/CXCL10 axis, and the release of ATP and HMGB1 from dying cancer cells. Factors that influence the impact of ICD in clinical studies and the design of therapies combining chemotherapy with immunotherapy are also discussed.


Cann K.L.,Dalhousie University | Cann K.L.,Beatrice Hunter Cancer Research Institute | Dellaire G.,Dalhousie University | Dellaire G.,Beatrice Hunter Cancer Research Institute
Biochemistry and Cell Biology | Year: 2011

Higher order chromatin structure has an impact on all nuclear functions, including the DNA damage response. Over the past several years, it has become increasingly clear that heterochromatin and euchromatin represent separate entities with respect to both damage sensitivity and repair. The chromatin compaction present in heterochromatin helps to protect this DNA from damage; however, when lesions do occur, the compaction restricts the ability of DNA damage response proteins to access the site, as evidenced by its ability to block the expansion of H2AX phosphorylation. As such, DNA damage in heterochromatin is refractory to repair, which requires the surrounding chromatin structure to be decondensed. In the case of DNA double-strand breaks, this relaxation is at least partially mediated by the ATM kinase phosphorylating and inhibiting the function of the transcriptional repressor KAP1. This review will focus on the functions of KAP1 and other proteins involved in the maintenance or restriction of heterochromatin, including HP1 and TIP60, in the DNA damage response. As heterochromatin is important for maintaining genomic stability, cells must maintain a delicate balance between allowing repair factors access to these regions and ensuring that these regions retain their organization to prevent increased DNA damage and chromosomal mutations.


Khaperskyy D.A.,Dalhousie University | Hatchette T.F.,Dalhousie University | Hatchette T.F.,Queen Elizabeth Health science Center | McCormick C.,Dalhousie University | McCormick C.,Beatrice Hunter Cancer Research Institute
FASEB Journal | Year: 2012

An important component of the mammalian stress response is the reprogramming of translation. A variety of stresses trigger abrupt polysome disassembly and the accumulation of stalled translation preinitiation complexes. These complexes nucleate cytoplasmic stress granules (SGs), sites of mRNA triage in which mRNAs from disassembling polysomes are sorted and the fates of individual transcripts are determined. Here, we demonstrate that influenza A virus (IAV) actively suppresses SG formation during infection, thereby allowing translation of viral mRNAs. Complete inhibition of SG formation is dependent on the function of the viral nonstructural protein 1 (NS1); at late times postinfection, cells infected with NS1-mutant viruses formed SGs in a double-stranded RNA-activated protein kinase (PKR)-dependent fashion. In these cells, SG formation correlated with inhibited viral protein synthesis. Together, these experiments demonstrate the antiviral potential of SGs and reveal a viral countermeasure that limits SG formation. © FASEB.

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