Can supervise: YES
Baratchi, S, Almazi, JG, Darby, W, Tovar-Lopez, FJ, Mitchell, A & McIntyre, P 2016, 'Shear stress mediates exocytosis of functional TRPV4 channels in endothelial cells', Cellular and Molecular Life Sciences, vol. 73, no. 3, pp. 649-666.View/Download from: Publisher's site
© 2015 Springer Basel. Mechanosensitive ion channels are implicated in the biology of touch, pain, hearing and vascular reactivity; however, the identity of these ion channels and the molecular basis of their activation is poorly understood. We previously found that transient receptor potential vanilloid 4 (TRPV4) is a receptor operated ion channel that is sensitised and activated by mechanical stress. Here, we investigated the effects of mechanical stimulation on TRPV4 localisation and activation in native and recombinant TRPV4-expressing cells. We used a combination of total internal reflection fluorescence microscopy, cell surface biotinylation assay and Ca2+ imaging with laser scanning confocal microscope to show that TRPV4 is expressed in primary vascular endothelial cells and that shear stress sensitises the response of TRPV4 to its agonist, GSK1016790A. The sensitisation was attributed to the recruitment of intracellular pools of TRPV4 to the plasma membrane, through the clathrin and dynamin-mediated exocytosis. The translocation was dependent on ILK/Akt signalling pathway, release of Ca2+ from intracellular stores and we demonstrated that shear stress stimulated phosphorylation of TRPV4 at tyrosine Y110. Our findings implicate calcium-sensitive TRPV4 translocation in the regulation of endothelial responses to mechanical stimulation.
Baratchi, S, Tovar-Lopez, FJ, Khoshmanesh, K, Grace, MS, Darby, W, Almazi, J, Mitchell, A & McIntyre, P 2014, 'Examination of the role of transient receptor potential vanilloid type 4 in endothelial responses to shear forces', BIOMICROFLUIDICS, vol. 8, no. 4.View/Download from: Publisher's site
Christopherson, RI, Mactier, S, Almazi, JG, Kohnke, PL, Best, OG & Mulligan, SP 2014, 'Mechanisms of action of fludarabine nucleoside against human raji lymphoma cells', Nucleosides, Nucleotides and Nucleic Acids, vol. 33, no. 4-6, pp. 375-383.View/Download from: Publisher's site
Fludarabine (2-FaraAMP) is a purine analog that is effective against chronic lymphocytic leukemia (CLL) and non-Hodgkins lymphoma (NHL). For some cases of CLL, 2-FaraAMP as a single agent can clear the blood of leukemia cells, but leukemia stem cells usually remain protected in sanctuary sites. It is clear that 2-FaraAMP has multiple mechanisms of action that may collectively result in strand breaks in DNA, accumulation of phosphorylated p53 and apoptosis. We have demonstrated using the human Burkitt's lymphoma B-cell line, Raji, that p53, p63 and p73 all accumulate in the nucleus, following treatment of cells with fludarabine nucleoside (2-FaraA). In addition, phosphorylated p53 accumulates in the cytosol and at mitochondria. Using sophisticated methods of proteomic analysis with mass spectrometry, proteins that become differentially abundant after treatment of cells with 2-FaraA have been identified, providing considerable additional information about the cellular responses of B-lymphoid cancers to this purine analog. The levels of proteins involved in the unfolded protein response increase, indicating that endoplasmic reticulum stress is likely to be one mechanism for induction of apoptosis. The levels of a number of proteins found on the outer plasma membrane change on cells treated with 2-FaraA, suggesting that signaling from the B-cell antigen receptor (BCR) is stimulated, resulting in induction of apoptosis through the intrinsic pathway. Increased levels of the cell surface proteins, CD50, CD100 and ECE-1, would promote survival of these cells; the balance between these survival and death responses would determine the fate of the cell. © 2014 Taylor and Francis Group, LLC.
Huang, PY, Best, OG, Almazi, JG, Belov, L, Davis, ZA, Majid, A, Dyer, MJ, Pascovici, D, Mulligan, SP & Christopherson, RI 2014, 'Cell surface phenotype profiles distinguish stable and progressive chronic lymphocytic leukemia', Leukemia and Lymphoma, vol. 55, no. 9, pp. 2085-2092.View/Download from: Publisher's site
Chronic lymphocytic leukemia (CLL) is clinically heterogeneous. While some patients have indolent disease for many years, 20-30% will progress and ultimately die of their disease. CLL may be classified by the Rai or Binet staging system, mutational status of the immunoglobulin variable heavy-chain gene (IGVH), ZAP-70 overexpression, cytogenetic abnormalities (13q-, + 12, 11q-, 17p-) and expression of several cell surface antigens (CD38, CD49d) that correlate with risk of disease progression. However, none of these markers identify all cases of CLL at risk. In a recent review, we summarized those CD antigens known to correlate with the prognosis of CLL. The present study has identified surface profiles of CD antigens that distinguish clinically progressive CLL from slow-progressive and stable CLL. Using an extended DotScan™ CLL antibody microarray (Version 3; 182 CD antibodies), and with refined analysis of purified CD19 + B-cells, the following 27 CD antigens were differentially abundant for progressive CLL: CD11a, CD11b, CD11c, CD18, CD19, CD20 (two epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD180, CD196 and CD270. The extensive surface profiles obtained provide disease signatures with an accuracy of 79.2%, a sensitivity of 83.9% and a specificity of 72.5% that could provide the basis for a rapid test to triage patients with CLL according to probability of clinical progression and potential earlier requirement for treatment. © 2014 Informa UK, Ltd.
Almazi, JG, Mactier, S, Best, OG, Crossett, B, Mulligan, SP & Christopherson, RI 2012, 'Fludarabine nucleoside induces accumulations of p53, p63 and p73 in the nuclei of human B-lymphoid cell lines, with cytosolic and mitochondrial increases in p53', Proteomics - Clinical Applications, vol. 6, no. 5-6, pp. 279-290.View/Download from: Publisher's site
Purpose: Human Raji cells treated with fludarabine nucleoside (2-FaraA, 3 μM) undergo apoptosis with accumulation of p53 in the nuclei as multiple phosphorylated isoforms and C-terminal truncated derivatives. Changes induced by 2-FaraA in the levels of p53, p63 and p73 in the nuclear, cytosolic and mitochondrial fractions have been determined in four human B-lymphoid cell lines that are TP53-functional (Raji and IM9) and TP53-mutated (MEC1 and U266). Experimental design: The B-lymphoid cell lines were treated with 2-FaraA (3 μM, 24 h, 48 h) and viability determined. Protein extracts of subcellular fractions from 2-FaraA-treated cells were analysed by 1D and 2D electrophoresis; multiple phosphorylated isoforms and truncated derivatives were identified by Western blots for p53, p63 and p73. Results: p53 and p63 were present in all three fractions, while p73 was only detected in nuclei. After treatment with 2-FaraA, nuclear p53, p63 and p73 accumulated as multiple phosphorylated isoforms and truncated derivatives. The association of p63 with mitochondria in human cells is novel. Conclusions and clinical relevance: Comprehensive information on the subcellular distributions and responses of p53, p63 and p73 to 2-FaraA provides additional insight into mechanisms for induction of apoptosis in the treatment of B-lymphoproliferative disorders with fludarabine. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Kohnke, PL, MacTier, S, Almazi, JG, Crossett, B & Christopherson, RI 2012, 'Fludarabine and cladribine induce changes in surface proteins on human B-lymphoid cell lines involved with apoptosis, cell survival, and antitumor immunity', Journal of Proteome Research, vol. 11, no. 9, pp. 4436-4448.View/Download from: Publisher's site
Fludarabine and cladribine are purine analogues used to treat hematological malignancies. Alone or in combination with therapeutic antibodies, they are effective in treating patients with chronic lymphocytic leukemia and non-Hodgkin's lymphoma. However, the mechanisms of action of these drugs are not well understood. Plasma membrane proteins perform a variety of essential functions that can be affected by malignancy and perturbed by chemotherapy. Analysis of surface proteins may contribute to an understanding of the mechanisms of action of purine analogues and identify biomarkers for targeted therapy. The surface of human cells is rich in N-linked glycoproteins, enabling use of a hydrazide-coupling technique to enrich for glycoproteins, with iTRAQ labeling for quantitative comparison. A number of plasma membrane proteins on human leukemia and lymphoma cells were affected by treatment with a purine analogue, including decreases in CD22 (an adhesion and signaling molecule) and increases in CD205 (a "damaged cell marker") and CD80 and CD50 (T-cell interaction molecules). Purine analogues may affect B-cell receptor (BCR) signaling and costimulatory molecules, leading to multiple signals for apoptosis and cell clearance. Fludarabine and cladribine induce differential effects, with some cell survival proteins (ECE-1 and CD100) more abundant after fludarabine treatment. Cell surface proteins induced by fludarabine and cladribine may be targets for therapeutic antibodies. © 2012 American Chemical Society.