St Vincents Institute Of Medical Research
St Vincents Institute Of Medical Research
Walia M.K.,St Vincents Institute Of Medical Research |
Ho P.M.W.,St Vincents Institute Of Medical Research |
Taylor S.,St Vincents Institute Of Medical Research |
Ng A.J.M.,St Vincents Institute Of Medical Research |
And 10 more authors.
eLife | Year: 2016
Mutations in the P53 pathway are a hallmark of human cancer. The identification of pathways upon which p53-deficient cells depend could reveal therapeutic targets that may spare normal cells with intact p53. In contrast to P53 point mutations in other cancer, complete loss of P53 is a frequent event in osteosarcoma (OS), the most common cancer of bone. The consequences of p53 loss for osteoblastic cells and OS development are poorly understood. Here we use murine OS models to demonstrate that elevated Pthlh (Pthrp), cAMP levels and signalling via CREB1 are characteristic of both p53-deficient osteoblasts and OS. Normal osteoblasts survive depletion of both PTHrP and CREB1. In contrast, p53-deficient osteoblasts and OS depend upon continuous activation of this pathway and undergo proliferation arrest and apoptosis in the absence of PTHrP or CREB1. Our results identify the PTHrP-cAMP-CREB1 axis as an attractive pathway for therapeutic inhibition in OS. © Walia et al.
Johanson T.M.,St Vincents Institute Of Medical Research |
Johanson T.M.,Walter and Eliza Hall Institute of Medical Research |
Johanson T.M.,University of Melbourne |
Skinner J.P.J.,St Vincents Institute Of Medical Research |
And 7 more authors.
International Journal of Hematology | Year: 2014
The immune system is composed of a diverse range of cell types, each with a distinct function. It can be broadly divided into the lymphoid (T, B, NK, etc.) and myeloid (monocyte, granulocyte, etc.) arms. Lymphopoiesis, the development and differentiation of lymphoid lineages, has been studied extensively for decades. For example, the influence of extracellular signals, signaling pathways and transcription factors has already been well documented. However, the importance of microRNAs has been highlighted by a surge of studies in recent years. In this review, we will discuss what is currently known about the role of microRNAs in lymphopoiesis, from the hematopoietic stem cell through to the differentiation of mature lymphocytes including thymic development, helper and regulatory T cells, fate determination of B cells and dendritic cells. © 2014, The Japanese Society of Hematology.
Ng A.J.M.,St Vincents Institute Of Medical Research |
Ng A.J.M.,University of Melbourne |
Walia M.K.,St Vincents Institute Of Medical Research |
Smeets M.F.,St Vincents Institute Of Medical Research |
And 12 more authors.
PLoS Genetics | Year: 2015
RECQL4 mutations are associated with Rothmund Thomson Syndrome (RTS), RAPADILINO Syndrome and Baller-Gerold Syndrome. These patients display a range of benign skeletal abnormalities such as low bone mass. In addition, RTS patients have a highly increased incidence of osteosarcoma (OS). The role of RECQL4 in normal adult bone development and homeostasis is largely uncharacterized and how mutation of RECQL4 contributes to OS susceptibility is not known. We hypothesised that Recql4 was required for normal skeletal development and both benign and malignant osteoblast function, which we have tested in the mouse. Recql4 deletion in vivo at the osteoblastic progenitor stage of differentiation resulted in mice with shorter bones and reduced bone volume, assessed at 9 weeks of age. This was associated with an osteoblast intrinsic decrease in mineral apposition rate and bone formation rate in the Recql4-deficient cohorts. Deletion of Recql4 in mature osteoblasts/osteocytes in vivo, however, did not cause a detectable phenotype. Acute deletion of Recql4 in primary osteoblasts or shRNA knockdown in an osteoblastic cell line caused failed proliferation, accompanied by cell cycle arrest, induction of apoptosis and impaired differentiation. When cohorts of animals were aged long term, the loss of Recql4 alone was not sufficient to initiate OS. We then crossed the Recql4fl/fl allele to a fully penetrant OS model (Osx-Cre p53fl/fl). Unexpectedly, the Osx-Cre p53fl/flRecql4fl/fl (dKO) animals had a significantly increased OS-free survival compared to Osx-Cre p53fl/fl or Osx-Cre p53fl/flRecql4fl/+ (het) animals. The extended survival was explained when the Recql4 status in the tumors that arose was assessed, and in no case was there complete deletion of Recql4 in the dKO OS. These data provide a mechanism for the benign skeletal phenotypes of RECQL4 mutation syndromes. We propose that tumor suppression and osteosarcoma susceptibility are most likely a function of mutant, not null, alleles of RECQL4. © 2015 Ng et al.
Fynch S.,St Vincents Institute Of Medical Research
Immunology and Cell Biology | Year: 2015
In type 1 diabetes, cytotoxic CD8+ T lymphocytes (CTLs) directly interact with pancreatic beta cells through major histocompatibility complex class I. An immune synapse facilitates delivery of cytotoxic granules, comprised mainly of granzymes and perforin. Perforin deficiency protects the majority of non-obese diabetic (NOD) mice from autoimmune diabetes. Intriguingly perforin deficiency does not prevent diabetes in CD8+ T-cell receptor transgenic NOD8.3 mice. We therefore investigated the importance of perforin-dependent killing via CTL-beta cell contact in autoimmune diabetes. Perforin-deficient CTL from NOD mice or from NOD8.3 mice were significantly less efficient at adoptive transfer of autoimmune diabetes into NODRag1-/- mice, confirming that perforin is essential to facilitate beta cell destruction. However, increasing the number of transferred in vitro-activated perforin-deficient 8.3 T cells reversed the phenotype and resulted in diabetes. Perforin-deficient NOD8.3 T cells were present in increased proportion in islets, and proliferated more in response to antigen in vivo indicating that perforin may regulate the activation of CTLs, possibly by controlling cytokine production. This was confirmed when we examined the requirement for direct interaction between beta cells and CD8+ T cells in NOD8.3 mice, in which beta cells specifically lack major histocompatibility complex (MHC) class I through conditional deletion of β2-microglobulin. Although diabetes was significantly reduced, 40% of these mice developed diabetes, indicating that NOD8.3 T cells can kill beta cells in the absence of direct interaction. Our data indicate that although perforin delivery is the main mechanism that CTL use to destroy beta cells, they can employ alternative mechanisms to induce diabetes in a perforin-independent manner.Immunology and Cell Biology advance online publication, 3 November 2015; doi:10.1038/icb.2015.89. © 2015 Australasian Society for Immunology Inc.
Heraud-Farlow J.E.,St Vincents Institute Of Medical Research |
Walkley C.R.,St Vincents Institute Of Medical Research |
Walkley C.R.,University of Melbourne
Journal of Molecular Medicine | Year: 2016
The innate immune system is the first line of the cellular defence against invading pathogens. A critical component of this defence is the capacity to discriminate foreign RNA molecules, which are distinct from most cellular RNAs in structure and/or modifications. However, a series of rare autoimmune/autoinflammatory diseases in humans highlight the propensity for the innate immune sensing system to be activated by endogenous cellular double-stranded RNAs (dsRNAs), underscoring the fine line between distinguishing self from non-self. The RNA editing enzyme ADAR1 has recently emerged as a key regulator that prevents innate immune pathway activation, principally the cytosolic dsRNA sensor MDA5, from inducing interferon in response to double-stranded RNA structures within endogenous RNAs. Adenosine-to-Inosine RNA editing by ADAR1 is proposed to destabilise duplexes formed from inverted repetitive elements within RNAs, which appear to prevent MDA5 from sensing these RNA as virus-like in the cytoplasm. Aberrant activation of these pathways has catastrophic effects at both a cellular and organismal level, contributing to one of the causes of the conditions collectively known as the type I interferonopathies. © 2016 Springer-Verlag Berlin Heidelberg
Bouter Y.,University of Gottingen |
Noguerola J.S.L.,University of Gottingen |
Tucholla P.,University of Gottingen |
Crespi G.A.N.,St Vincents Institute Of Medical Research |
And 6 more authors.
Acta Neuropathologica | Year: 2015
Solanezumab and Crenezumab are two humanized antibodies targeting Amyloid-β (Aβ) which are currently tested in multiple clinical trials for the prevention of Alzheimer’s disease. However, there is a scientific discussion ongoing about the target engagement of these antibodies. Here, we report the immunohistochemical staining profiles of biosimilar antibodies of Solanezumab, Crenezumab and Bapineuzumab in human formalin-fixed, paraffin-embedded tissue and human fresh frozen tissue. Furthermore, we performed a direct comparative immunohistochemistry analysis of the biosimilar versions of the humanized antibodies in different mouse models including 5XFAD, Tg4-42, TBA42, APP/PS1KI, 3xTg. The staining pattern with these humanized antibodies revealed a surprisingly similar profile. All three antibodies detected plaques, cerebral amyloid angiopathy and intraneuronal Aβ in a similar fashion. Remarkably, Solanezumab showed a strong binding affinity to plaques. We also reaffirmed that Bapineuzumab does not recognize N-truncated or modified Aβ, while Solanezumab and Crenezumab do detect N-terminally modified Aβ peptides Aβ4–42 and pyroglutamate Aβ3–42. In addition, we compared the results with the staining pattern of the mouse NT4X antibody that recognizes specifically Aβ4–42 and pyroglutamate Aβ3–42, but not full-length Aβ1–42. In contrast to the biosimilar antibodies of Solanezumab, Crenezumab and Bapineuzumab, the murine NT4X antibody shows a unique target engagement. NT4X does barely cross-react with amyloid plaques in human tissue. It does, however, detect cerebral amyloid angiopathy in human tissue. In Alzheimer mouse models, NT4X detects intraneuronal Aβ and plaques comparable to the humanized antibodies. In conclusion, the biosimilar antibodies Solanezumab, Crenezumab and Bapineuzumab strongly react with amyloid plaques, which are in contrast to the NT4X antibody that hardly recognizes plaques in human tissue. Therefore, NT4X is the first of a new class of therapeutic antibodies. © 2015, Springer-Verlag Berlin Heidelberg.
Shah M.H.,St Vincents Institute Of Medical Research |
Liu G.-S.,University of Melbourne |
Liu G.-S.,Obrien Institute |
Thompson E.W.,St Vincents Institute Of Medical Research |
And 4 more authors.
Breast Cancer Research and Treatment | Year: 2015
Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2) have been implicated in development and progression of breast cancer. In the present study, we have evaluated the effects of the superoxide dismutase (SOD) mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 on superoxide and H2O2 formation as well as proliferation, adhesion, and migration of MCF-7 and MDA-MB-231 cells. Superoxide and H2O2 production was examined using dihydroethidium and Amplex red assays, respectively. Cell viability and adhesion were measured using a tetrazolium-based MTT assay. Cell proliferation was determined using trypan blue assay. Cell cycle progression was analyzed using flow cytometry. Clonal expansion of a single cell was performed using a colony formation assay. Cell migration was measured using transwell migration assay. Dual luciferase assay was used to determine NF-κB reporter activity. EUK 134 effectively reduced both superoxide and H2O2, whereas MnTmPyP removed superoxide but enhanced H2O2 formation. EUK 134 effectively attenuated viability, proliferation, clonal expansion, adhesion, and migration of MCF-7 and MDA-MB-231 cells. In contrast, MnTmPyP only reduced clonal expansion of MCF-7 and MDA-MB-231 cells but had no effect on adhesion and cell cycle progression. Tumor necrosis factor-alpha-induced NF-κB activity was reduced by EUK 134, whereas MnTmPyP enhanced this activity. These data indicate that the SOD mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 exert differential effects on breast cancer cell growth. Inhibition of H2O2 signaling using EUK 134-like compound might be a promising approach to breast cancer therapy. © 2015, Springer Science+Business Media New York.
Joglekar M.V.,University of Sydney |
Trivedi P.M.,St Vincents Institute Of Medical Research |
Trivedi P.M.,University of Melbourne |
Kay T.W.,St Vincents Institute Of Medical Research |
And 8 more authors.
Apoptosis | Year: 2016
Cell death via FAS/CD95 can occur either by activation of caspases alone (extrinsic) or by activation of mitochondrial death signalling (intrinsic) depending on the cell type. The BH3-only protein BID is activated in the BCL-2-regulated or mitochondrial apoptosis pathway and acts as a switch between the extrinsic and intrinsic cell death pathways. We have previously demonstrated that islets from BID-deficient mice are protected from FAS ligand-mediated apoptosis in vitro. However, it is not yet known if BID plays a similar role in human beta cell death. We therefore aimed to test the role of BID in human islet cell apoptosis immediately after isolation from human cadaver donors, as well as after de-differentiation in vitro. Freshly isolated human islets or 10–12 day cultured human islet cells exhibited BID transcript knockdown after BID siRNA transfection, however they were not protected from FAS ligand-mediated cell death in vitro as determined by DNA fragmentation analysis using flow cytometry. On the other hand, the same cells transfected with siRNA for FAS-associated via death domain (FADD), a molecule in the extrinsic cell death pathway upstream of BID, showed significant reduction in cell death. De-differentiated islets (human islet-derived progenitor cells) also demonstrated similar results with no difference in cell death after BID knockdown as compared to scramble siRNA transfections. Our results indicate that BID-independent pathways are responsible for FAS-dependent human islet cell death. These results are different from those observed in mouse islets and therefore demonstrate potentially alternate pathways of FAS ligand-induced cell death in human and mouse islet cells. © 2016 Springer Science+Business Media New York
Zhao Y.,St Vincents Institute Of Medical Research |
Scott N.A.,St Vincents Institute Of Medical Research |
Scott N.A.,University of Melbourne |
Fynch S.,St Vincents Institute Of Medical Research |
And 13 more authors.
Diabetologia | Year: 2015
Methods: We altered expression levels of critical cell death proteins in mouse islets and tested their ability to survive CD4+ T cell-mediated attack using an in vivo graft model.Aims/hypothesis: Type 1 diabetes results from T cell-mediated destruction of pancreatic beta cells. The mechanisms of beta cell destruction in vivo, however, remain unclear. We aimed to test the relative roles of the main cell death pathways: apoptosis, necrosis and necroptosis, in beta cell death in the development of CD4+ T cell-mediated autoimmune diabetes.Results: Loss of the B cell leukaemia/lymphoma 2 (BCL-2) homology domain 3-only proteins BIM, PUMA or BID did not protect beta cells from this death. Overexpression of the anti-apoptotic protein BCL-2 or combined deficiency of the pro-apoptotic multi-BCL2 homology domain proteins BAX and BAK also failed to prevent beta cell destruction. Furthermore, loss of function of the death receptor Fas or its essential downstream signalling molecule Fas-associated death domain (FADD) in islets was also not protective. Using electron microscopy we observed that dying beta cells showed features of necrosis. However, islets deficient in receptor-interacting serine/threonine protein kinase 3 (RIPK3), a critical initiator of necroptosis, were still normally susceptible to CD4+ T cell-mediated destruction. Remarkably, simultaneous inhibition of apoptosis and necroptosis by combining loss of RIPK3 and overexpression of BCL-2 in islets did not protect them against immune attack either.Conclusions/interpretation: Collectively, our data indicate that beta cells die by necrosis in autoimmune diabetes and that the programmed cell death pathways apoptosis and necroptosis are both dispensable for this process. © 2014, Springer-Verlag Berlin Heidelberg.
Kuiper M.J.,University of Melbourne |
Morton C.J.,St Vincents Institute Of Medical Research |
Abraham S.E.,University of Melbourne |
Gray-Weale A.,University of Melbourne
eLife | Year: 2015
Antifreeze proteins (AFPs) protect certain cold-adapted organisms from freezing to death by selectively adsorbing to internal ice crystals and inhibiting ice propagation. The molecular details of AFP adsorption-inhibition is uncertain but is proposed to involve the Gibbs–Thomson effect. Here we show by using unbiased molecular dynamics simulations a protein structure-function mechanism for the spruce budworm Choristoneura fumiferana AFP, including stereo-specific binding and consequential melting and freezing inhibition. The protein binds indirectly to the prism ice face through a linear array of ordered water molecules that are structurally distinct from the ice. Mutation of the ice binding surface disrupts water-ordering and abolishes activity. The adsorption is virtually irreversible, and we confirm the ice growth inhibition is consistent with the Gibbs–Thomson law. © 2015, eLife Sciences Publications Ltd. All rights reserved.