Trescowthick Research Laboratories

Melbourne, Australia

Trescowthick Research Laboratories

Melbourne, Australia
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Chan C.J.,Trescowthick Research Laboratories | Chan C.J.,Monash University | Martinet L.,QIMR Berghofer Medical Research Institute | Gilfillan S.,University of Washington | And 13 more authors.
Nature Immunology | Year: 2014

CD96, CD226 (DNAM-1) and TIGIT belong to an emerging family of receptors that interact with nectin and nectin-like proteins. CD226 activates natural killer (NK) cell-mediated cytotoxicity, whereas TIGIT reportedly counterbalances CD226. In contrast, the role of CD96, which shares the ligand CD155 with CD226 and TIGIT, has remained unclear. In this study we found that CD96 competed with CD226 for CD155 binding and limited NK cell function by direct inhibition. As a result, Cd96 -/- mice displayed hyperinflammatory responses to the bacterial product lipopolysaccharide (LPS) and resistance to carcinogenesis and experimental lung metastases. Our data provide the first description, to our knowledge, of the ability of CD96 to negatively control cytokine responses by NK cells. Blocking CD96 may have applications in pathologies in which NK cells are important. © 2014 Nature America, Inc.


Chan C.J.,Trescowthick Research Laboratories | Chan C.J.,University of Melbourne | Chan C.J.,Monash University | Smyth M.J.,Trescowthick Research Laboratories | And 6 more authors.
Cell Death and Differentiation | Year: 2014

Protection against cellular stress from various sources, such as nutritional, physical, pathogenic, or oncogenic, results in the induction of both intrinsic and extrinsic cellular protection mechanisms that collectively limit the damage these insults inflict on the host. The major extrinsic protection mechanism against cellular stress is the immune system. Indeed, it has been well described that cells that are stressed due to association with viral infection or early malignant transformation can be directly sensed by the immune system, particularly natural killer (NK) cells. Although the ability of NK cells to directly recognize and respond to stressed cells is well appreciated, the mechanisms and the breadth of cell-intrinsic responses that are intimately linked with their activation are only beginning to be uncovered. This review will provide a brief introduction to NK cells and the relevant receptors and ligands involved in direct responses to cellular stress. This will be followed by an in-depth discussion surrounding the various intrinsic responses to stress that can naturally engage NK cells, and how therapeutic agents may induce specific activation of NK cells and other innate immune cells by activating cellular responses to stress. © 2014 Macmillan Publishers Limited All rights reserved.


Sampurno S.,Trescowthick Research Laboratories | Sampurno S.,University of Melbourne | Cross R.,Trescowthick Research Laboratories | Cross R.,University of Melbourne | And 8 more authors.
Growth Factors | Year: 2015

Skin integrity requires an ongoing replacement and repair orchestrated by several cell types. We previously investigated the architecture of the skin of avian myeloblastosis viral oncogene homolog (Myb) knock-out (KO) embryos and wound repair in Myb+/- mice revealing a need for Myb in the skin, attributed to fibroblast-dependent production of collagen type 1. Here, using targeted Myb deletion in keratin-14 (K14) positive cells we reveal further Myb-specific defects in epidermal cell proliferation, thickness and ultrastructural morphology. This was associated with a severe deficit in collagen type 1 production, reminiscent of that observed in patients with ichthyosis vulgaris and Ehlers-Danlos syndrome. Since collagen type 1 is a product of fibroblasts, the collagen defect observed was unexpected and appears to be directed by the loss of Myb with significantly reduced tumor growth factor beta 1 (Tgfβ-1) expression by primary keratinocytes. Our findings support a specific role for Myb in K14+ epithelial cells in the preservation of adult skin integrity and function. © 2015 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.


Lobachevsky P.,Trescowthick Research Laboratories | Smith J.,Trescowthick Research Laboratories | Denoyer D.,Trescowthick Research Laboratories | Skene C.,University of Melbourne | And 5 more authors.
International Journal of Radiation Biology | Year: 2012

Purpose: The objective of the study was to conjugate the DNA binding ligand para-[125I]-iodoHoechst to octreotate, and to explore the tumour targeting potential of this conjugate in the octreotate-somatostatin receptor system. Methods: We synthesized a Hoechst analogue containing a tri-butylstannyl group in the para position of phenyl ring, conjugated it to the N-terminal amino group of octreotate and prepared 125I-labelled conjugate by iododestannylation. We used the somatostatin receptor (SSTR2) over-expressing cell line A427-7 derived from its parent A427 human non-small cell lung carcinoma cell line to investigate SSTR2 affinity and receptor-mediated internalisation of the conjugate, and the mouse A427-7 tumour xenograft model for in vivo biodistribution studies of the radiolabelled conjugate. Results: A method was developed for convenient preparation of high specific activity radioiodinated conjugate which retains affinity for somatostatin receptors and is internalised into A427-7 SSTR2 over-expressing cells via a receptor-mediated mechanism. The conjugate accumulates in mouse A427-7 tumour xenografts following intravenous administration. Conclusions: A dual targeting strategy for Auger endoradiotherapy, in which a DNA ligand is used to target the Auger decay to DNA, in conjunction with receptor-mediated targeting to specific receptors, using a labelled DNA ligand/peptide conjugate, has been demonstrated for the octreotate-somatostatin receptor system. © 2012 Informa UK, Ltd.


Astle M.V.,Trescowthick Research Laboratories | Hannan K.M.,Trescowthick Research Laboratories | Ng P.Y.,Trescowthick Research Laboratories | Lee R.S.,Trescowthick Research Laboratories | And 12 more authors.
Oncogene | Year: 2012

The phosphatidylinositol 3-kinase (PI3K)/AKT and RAS oncogenic signalling modules are frequently mutated in sporadic human cancer. Although each of these pathways has been shown to play critical roles in driving tumour growth and proliferation, their activation in normal human cells can also promote cell senescence. Although the mechanisms mediating RAS-induced senescence have been well characterised, those controlling PI3K/AKT-induced senescence are poorly understood. Here we show that PI3K/AKT pathway activation in response to phosphatase and tensin homolog (PTEN) knockdown, mutant PI3K, catalytic, α polypeptide (PIK3CA) or activated AKT expression, promotes accumulation of p53 and p21, increases cell size and induces senescence-associated Β-galactosidase activity. We demonstrate that AKT-induced senescence is p53-dependent and is characterised by mTORC1-dependent regulation of p53 translation and stabilisation of p53 protein following nucleolar localisation and inactivation of MDM2. The underlying mechanisms of RAS and AKT-induced senescence appear to be distinct, demonstrating that different mediators of senescence may be deregulated during transformation by specific oncogenes. Unlike RAS, AKT promotes rapid proliferative arrest in the absence of a hyperproliferative phase or DNA damage, indicating that inactivation of the senescence response is critical at the early stages of PI3K/AKT-driven tumourigenesis. Furthermore, our data imply that chronic activation of AKT signalling provides selective pressure for the loss of p53 function, consistent with observations that PTEN or PIK3CA mutations are significantly associated with p53 mutation in a number of human tumour types. Importantly, the demonstration that mTORC1 is an essential mediator of AKT-induced senescence raises the possibility that targeting mTORC1 in tumours with activated PI3K/AKT signalling may exert unexpected detrimental effects due to inactivation of a senescence brake on potential cancer-initiating cells. © 2012 Macmillan Publishers Limited All rights reserved.


Lobachevsky P.N.,Trescowthick Research Laboratories | Vasireddy R.S.,Trescowthick Research Laboratories | Broadhurst S.,Trescowthick Research Laboratories | Sprung C.N.,Trescowthick Research Laboratories | And 8 more authors.
International Journal of Radiation Biology | Year: 2011

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage. © 2011 Informa UK, Ltd.


Ivashkevich A.N.,Trescowthick Research Laboratories | Martin O.A.,U.S. National Cancer Institute | Smith A.J.,Trescowthick Research Laboratories | Redon C.E.,U.S. National Cancer Institute | And 3 more authors.
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis | Year: 2011

The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches.We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens. © 2011 Elsevier B.V.


PubMed | Trescowthick Research Laboratories
Type: Journal Article | Journal: Oncogene | Year: 2012

The phosphatidylinositol 3-kinase (PI3K)/AKT and RAS oncogenic signalling modules are frequently mutated in sporadic human cancer. Although each of these pathways has been shown to play critical roles in driving tumour growth and proliferation, their activation in normal human cells can also promote cell senescence. Although the mechanisms mediating RAS-induced senescence have been well characterised, those controlling PI3K/AKT-induced senescence are poorly understood. Here we show that PI3K/AKT pathway activation in response to phosphatase and tensin homolog (PTEN) knockdown, mutant PI3K, catalytic, polypeptide (PIK3CA) or activated AKT expression, promotes accumulation of p53 and p21, increases cell size and induces senescence-associated -galactosidase activity. We demonstrate that AKT-induced senescence is p53-dependent and is characterised by mTORC1-dependent regulation of p53 translation and stabilisation of p53 protein following nucleolar localisation and inactivation of MDM2. The underlying mechanisms of RAS and AKT-induced senescence appear to be distinct, demonstrating that different mediators of senescence may be deregulated during transformation by specific oncogenes. Unlike RAS, AKT promotes rapid proliferative arrest in the absence of a hyperproliferative phase or DNA damage, indicating that inactivation of the senescence response is critical at the early stages of PI3K/AKT-driven tumourigenesis. Furthermore, our data imply that chronic activation of AKT signalling provides selective pressure for the loss of p53 function, consistent with observations that PTEN or PIK3CA mutations are significantly associated with p53 mutation in a number of human tumour types. Importantly, the demonstration that mTORC1 is an essential mediator of AKT-induced senescence raises the possibility that targeting mTORC1 in tumours with activated PI3K/AKT signalling may exert unexpected detrimental effects due to inactivation of a senescence brake on potential cancer-initiating cells.


PubMed | Trescowthick Research Laboratories
Type: Journal Article | Journal: Growth factors (Chur, Switzerland) | Year: 2015

Skin integrity requires an ongoing replacement and repair orchestrated by several cell types. We previously investigated the architecture of the skin of avian myeloblastosis viral oncogene homolog (Myb) knock-out (KO) embryos and wound repair in Myb(+/)(-) mice revealing a need for Myb in the skin, attributed to fibroblast-dependent production of collagen type 1. Here, using targeted Myb deletion in keratin-14 (K14) positive cells we reveal further Myb-specific defects in epidermal cell proliferation, thickness and ultrastructural morphology. This was associated with a severe deficit in collagen type 1 production, reminiscent of that observed in patients with ichthyosis vulgaris and Ehlers-Danlos syndrome. Since collagen type 1 is a product of fibroblasts, the collagen defect observed was unexpected and appears to be directed by the loss of Myb with significantly reduced tumor growth factor beta 1 (Tgf-1) expression by primary keratinocytes. Our findings support a specific role for Myb in K14+ epithelial cells in the preservation of adult skin integrity and function.


Karagiannis T.C.,Trescowthick Research Laboratories | El-Osta A.,BakerIDI Heart and Diabetes Institute
Cellular and Molecular Life Sciences | Year: 2010

The central dogma in radiation biology is that nuclear DNA is the critical target with respect to radiosensitivity. In accordance with the theoretical expectations, and in the absence of a conclusive model, the general consensus in the field has been to view chromatin as a homogeneous template for DNA damage and repair. This paradigm has been called into question by recent findings indicating a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin. Here, we have extended those studies and provide evidence that γH2AX foci form preferentially in actively transcribing euchromatin following γ-irradiation. © 2009 Birkhäuser Verlag, Basel/Switzerland.

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