CRUK Beatson Institute for Cancer Research

Glasgow, United Kingdom

CRUK Beatson Institute for Cancer Research

Glasgow, United Kingdom
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Renault T.T.,University of Bordeaux 1 | Renault T.T.,CRUK Beatson Institute for Cancer Research | Teijido O.,New York University | Teijido O.,U.S. National Institutes of Health | And 16 more authors.
International Journal of Biochemistry and Cell Biology | Year: 2015

Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release. © 2015 Elsevier Ltd.


Muinonen-Martin A.J.,CRUK Beatson Institute for Cancer Research | Knecht D.A.,University of Connecticut | Veltman D.M.,CRUK Beatson Institute for Cancer Research | Thomason P.A.,CRUK Beatson Institute for Cancer Research | And 2 more authors.
Methods in Molecular Biology | Year: 2013

Direct visualization chambers are considered the gold standard for measuring and analyzing chemotactic responses, because they allow detailed analysis of cellular behavior during the process of chemotaxis. We have previously described the Insall chamber, an improved chamber for measuring cancer cell chemotaxis. Here, we describe in detail how this system can be used to perform two key assays for both fast- and slow-moving mammalian and nonmammalian cell types. This allows for the detailed analysis of chemotactic responses in linear gradients at the levels of both overall cell behavior and subcellular dynamics. © Springer Science+Business Media, LLC 2013.


Itakura A.,Oregon Health And Science University | Aslan J.E.,Oregon Health And Science University | Kusanto B.T.,Oregon Health And Science University | Phillips K.G.,Oregon Health And Science University | And 7 more authors.
PLoS ONE | Year: 2013

Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca2+ signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils. © 2013 Itakura et al.


Lu W.-Y.,Center for Regenerative Medicine | Bird T.G.,Center for Regenerative Medicine | Boulter L.,Institute of Genetics and Molecular Medicine | Tsuchiya A.,Niigata University | And 16 more authors.
Nature Cell Biology | Year: 2015

Hepatocytes and cholangiocytes self-renew following liver injury. Following severe injury hepatocytes are increasingly senescent, but whether hepatic progenitor cells (HPCs) then contribute to liver regeneration is unclear. Here, we describe a mouse model where the E3 ubiquitin ligase Mdm2 is inducibly deleted in more than 98% of hepatocytes, causing apoptosis, necrosis and senescence with nearly all hepatocytes expressing p21. This results in florid HPC activation, which is necessary for survival, followed by complete, functional liver reconstitution. HPCs isolated from genetically normal mice, using cell surface markers, were highly expandable and phenotypically stable in vitro. These HPCs were transplanted into adult mouse livers where hepatocyte Mdm2 was repeatedly deleted, creating a non-competitive repopulation assay. Transplanted HPCs contributed significantly to restoration of liver parenchyma, regenerating hepatocytes and biliary epithelia, highlighting their in vivo lineage potency. HPCs are therefore a potential future alternative to hepatocyte or liver transplantation for liver disease. © 2015 Macmillan Publishers Limited.


Itakura A.,Oregon Health And Science University | Verbout N.G.,Oregon Health And Science University | Phillips K.G.,Oregon Health And Science University | Insall R.H.,CRUK Beatson Institute for Cancer Research | And 4 more authors.
Journal of Leukocyte Biology | Year: 2011

PMN leukocytes are the most abundant leukocytes in the circulation and play an important role in host defense. PMN leukocyte recruitment and inflammatory responses at sites of infection are critical components in innate immunity. Although inflammation and coagulation are known to have bidirectional relationships, little is known about the interaction between PMN leukocytes and coagulation factors. Coagulation FXI participates in the intrinsic coagulation pathway upon its activation, contributing to hemostasis and thrombosis. We have shown previously that FXI-deficient mice have an increased survival and less leukocyte accumulation into the peritoneum in severe polymicrobial peritonitis. This result suggests a role for FXI in leukocyte trafficking and/or function. In this study, we characterized the functional consequences of FXIa binding to PMN leukocytes. FXIa reduced PMN leukocyte chemotaxis triggered by the chemokine, IL-8, or the bacterial-derived peptide, fMLP, perhaps as a result of the loss of directed migration. In summary, our data suggest that FXIa modulates the inflammatory response of PMN leukocytes by altering migration. These studies highlight the interplay between inflammation and coagulation and suggest that FXIa may play a role in innate immunity. © Society for Leukocyte Biology.


Huser C.A.,University of Glasgow | Pringle M.A.,University of Glasgow | Heath V.J.,University of Glasgow | Heath V.J.,Now at Nature Publishing Group | And 7 more authors.
Cell Death and Differentiation | Year: 2010

Transforming growth factor Β (TGFΒ)-stimulated clone-22 domain family member 1 (TSC-22D1) has previously been associated with enhanced apoptosis in several cell systems. In an attempt to identify novel factors that are involved in the control of cell death during mammary gland involution, we found that the mRNA for isoform 2 of TSC-22D1 was highly upregulated 24 h after forced weaning, when a dramatic increase in cell death occurred, closely following the expression of the known inducer of cell death during involution, TGFΒ3. This was paralleled by strongly increased TSC-22D1 isoform 2 protein levels in the luminal epithelium. In contrast, RNA and protein expression levels of the isoform 1 of TSC-22D1 did not change during development. Whereas isoform 2 induced cell death, isoform 1 suppressed TGFΒ-induced cell death and enhanced proliferation in mammary epithelial cell lines. Furthermore, four distinct forms of isoform 2 protein were detected in the mammary gland, of which only a 15-kDa form was associated with early involution. Our data describe novel opposing functions of the two mammalian TSC-22D1 isoforms in cell survival and proliferation, and establish the TSC-22D1 isoform 2 as a potential regulator of cell death during mammary gland involution. © 2010 Macmillan Publishers Limited All rights reserved.


Matthews J.R.,University of Cardiff | Sansom O.J.,CRUK Beatson Institute for Cancer Research | Clarke A.R.,University of Cardiff
Cell Death and Differentiation | Year: 2011

The transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated in human cancers. Interestingly, STAT3 also maintains the pluripotency and self-renewal of murine embryonic stem cells, and several tissue stem cell types. To investigate whether STAT3 also maintains the small-intestine crypt stem cell, we conditionally inactivated a Floxed Stat3 allele (Stat3 fl) in murine small-intestine crypt stem cells. Following Cre recombinase expression, apoptosis increased in Stat3 fl/ experimental crypts relative to Stat3 wt/ controls before declining. Control Stat3 wt/ mice carrying a Flox-STOP LacZ reporter transgene stably expressed LacZ after Cre induction. In contrast, Stat3 fl/ intestine LacZ expression initially increased modestly, before declining to background levels. Quantitative PCRs revealed a similar transient in recombined Stat3 fl allele levels. Long-term bromodeoxyuridine labelling directly demonstrated that functional STAT3 is required for 4 to 6 region label-retaining small-intestine stem cell survival. Rapid clearance of recombined Stat3 fl/ cells involves apoptosis potentially induced by elevated c-Myc in non-recombined cells and involves elevated p53 expression and caspase 3 activation. Intriguingly, Stat3 fl/ intestine recombination triggered dramatically upregulated polycomb transcriptional repressor Bmi1-potentially accelerating recombined crypt repopulation. In summary, STAT3 activity is absolutely required for small-intestine crypt stem cell survival at both the 4 to 6 label-retaining and crypt base columnar cell locations. © 2011 Macmillan Publishers Limited All rights reserved.


Muinonen-Martin A.J.,CRUK Beatson Institute for Cancer Research | Veltman D.M.,CRUK Beatson Institute for Cancer Research | Kalna G.,CRUK Beatson Institute for Cancer Research | Insall R.H.,CRUK Beatson Institute for Cancer Research
PLoS ONE | Year: 2010

There has been a growing appreciation over the last decade that chemotaxis plays an important role in cancer migration, invasion and metastasis. Research into the field of cancer cell chemotaxis is still in its infancy and traditional investigative tools have been developed with other cell types and purposes in mind. Direct visualisation chambers are considered the gold standard for investigating the behaviour of cells migrating in a chemotactic gradient. We therefore drew up a list of key attributes that a chemotaxis chamber should have for investigating cancer cell chemotaxis. These include (1) compatibility with thin cover slips for optimal optical properties and to allow use of high numerical aperture (NA) oil immersion objectives; (2) gradients that are relatively stable for at least 24 hours due to the slow migration of cancer cells; (3) gradients of different steepnesses in a single experiment, with defined, consistent directions to avoid the need for complicated analysis; and (4) simple handling and disposability for use with medical samples. Here we describe and characterise the Insall chamber, a novel direct visualisation chamber. We use it to show GFP-lifeact transfected MV3 melanoma cells chemotaxing using a 60x high NA oil immersion objective, which cannot usually be done with other chemotaxis chambers. Linear gradients gave very efficient chemotaxis, contradicting earlier results suggesting that only polynomial gradients were effective. In conclusion, the chamber satisfies our design criteria, most importantly allowing high NA oil immersion microscopy to track chemotaxing cancer cells in detail over 24 hours. © 2010 Muinonen-Martin et al.


Insall R.,CRUK Beatson Institute for Cancer Research
Current Opinion in Cell Biology | Year: 2013

Eukaryotic chemotaxis is extremely complex. Cells can sense a wide range of stimuli, and many intracellular pathways are simultaneously involved. Recent genetic analyses of the steps between receptors and cytoskeleton, and how the cell controls actin and pseudopod behaviour, have yielded exciting new data but still no coherent understanding of chemotaxis. However, concentrating on pseudopods themselves and the physical processes that regulate them, rather than the internal signalling pathways, can simplify the data and help resolve the underlying mechanism. Direct action of electric fields and physical forces on cell migration suggest that mechanical forces and force-generating proteins like actin and myosin are centrally important in steering cells during chemotaxis. © 2013 Elsevier Ltd.


PubMed | CRUK Beatson Institute for Cancer Research
Type: Journal Article | Journal: PloS one | Year: 2010

There has been a growing appreciation over the last decade that chemotaxis plays an important role in cancer migration, invasion and metastasis. Research into the field of cancer cell chemotaxis is still in its infancy and traditional investigative tools have been developed with other cell types and purposes in mind. Direct visualisation chambers are considered the gold standard for investigating the behaviour of cells migrating in a chemotactic gradient. We therefore drew up a list of key attributes that a chemotaxis chamber should have for investigating cancer cell chemotaxis. These include (1) compatibility with thin cover slips for optimal optical properties and to allow use of high numerical aperture (NA) oil immersion objectives; (2) gradients that are relatively stable for at least 24 hours due to the slow migration of cancer cells; (3) gradients of different steepnesses in a single experiment, with defined, consistent directions to avoid the need for complicated analysis; and (4) simple handling and disposability for use with medical samples. Here we describe and characterise the Insall chamber, a novel direct visualisation chamber. We use it to show GFP-lifeact transfected MV3 melanoma cells chemotaxing using a 60x high NA oil immersion objective, which cannot usually be done with other chemotaxis chambers. Linear gradients gave very efficient chemotaxis, contradicting earlier results suggesting that only polynomial gradients were effective. In conclusion, the chamber satisfies our design criteria, most importantly allowing high NA oil immersion microscopy to track chemotaxing cancer cells in detail over 24 hours.

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