Richard I Christopherson

University of Sydney, Sydney, New South Wales, Australia

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Publications (170)524.27 Total impact

  • Kimberley Louise Kaufman · swetlana Mactier · Richard I Christopherson
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    ABSTRACT: An antibody microarray (DotScanTM) has been developed for profiling clinical melanoma specimens. Immobilized antibodies capture live cells expressing corresponding antigens to produce a dot pattern that represents the surface profile or immunophenotype. The unique signatures obtained may correlate with disease subtype, tumour progression and clinical outcome. Here we describe the rapid analysis of surgically resected metastatic melanoma. Leukocytes are separated from tumour cells using CD45 antibody-conjugated magnetic beads and separated cell populations are profiled on the microarray. This antibody microarray may be extended to include additional antibodies for cell surface biomarkers and therapeutic antibodies.
    No preview · Chapter · Dec 2015
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    ABSTRACT: Clinical trials of heat shock protein 90 (Hsp90) inhibitors have been limited by high toxicity. We previously showed that the Hsp90 inhibitor, SNX-7081, synergizes with and restores sensitivity to fludarabine nucleoside (2-FaraA) in human chronic lymphocytic leukemia (CLL) cells with lesions in the p53 pathway (Best OG, et al., Leukemia Lymphoma 53:1367-75, 2012). Here, we used label-free quantitative shotgun proteomics and comprehensive bioinformatic analysis to determine the mechanism of this synergy. We propose that 2-FaraA-induced DNA damage is compounded by SNX-7081-mediated inhibition of DNA repair, resulting in enhanced induction of apoptosis. DNA damage responses are impaired in part due to reductions in checkpoint regulators BRCA1 and cyclin D1, and cell death is triggered following reductions of MYC and nucleolin and an accumulation of apoptosis-inducing NFkB2 p100 subunit. Loss of nucleolin can activate Fas-mediated apoptosis, leading to the increase of pro-apoptotic proteins (BID, fas-associated factor-2) and subsequent apoptosis of p53-negative, 2-FaraA refractory CLL cells. A significant induction of DNA damage, indicated by increases in DNA damage marker γH2AX, was observed following the dual drug treatment of additional cell lines, indicating that a similar mechanism may operate in other p53-mutated human B-lymphoid cancers. These results provide valuable insight into the synergistic mechanism between SNX-7081 and 2-FaraA that may provide an alternative treatment for CLL patients with p53 mutations, for whom therapeutic options are currently limited. Moreover, this drug combination reduces the effective dose of the Hsp90 inhibitor and may therefore alleviate any toxicity encountered.
    Preview · Article · Nov 2015 · Oncotarget
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    ABSTRACT: Patients with a stable chronic lymphocytic leukemia (CLL) double their blood lymphocyte count in >5 years, but may develop progressive disease with lymphocytes doubling in <12 months. To identify a protein signature for progressive CLL, whole cell extracts of peripheral blood mononuclear cells from CLL patients (n = 27) were screened using iTRAQ (isobaric tags for relative and absolute quantification) analysis. Eighty-four differentially abundant proteins were identified from patients with stable and progressive CLL. Subsequently, 32 of these proteins were quantified by SRM (selected reaction monitoring) using extracts of purified CD19+ CLL cells from patients (n = 50). Hierarchical clustering of these protein profiles showed two clusters of patients that correlated with progressive and stable CLL, providing signatures that should be useful for triaging patients. Some of the proteins in the progressive cluster have not been linked with CLL, for example, glutamate dehydrogenase 1 and transcription intermediary factor 1-beta.
    No preview · Article · Sep 2015 · Leukemia & lymphoma
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    ABSTRACT: Glioblastoma (GBM) tumour invasion is facilitated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are actin-rich structures that protrude from the plasma membrane in direct contact with the ECM and are proposed to participate in epithelial-mesenchymal-transition. We have characterised the invasiveness of nine established GBM cell lines using an invadopodia assay and performed quantitative MS-based proteomic analyses on enriched membrane fractions. All GBM cells produced invadopodia, with a 65% difference between the most (U87MG) and least invasive (LN229) cells (p=0.0001). Overall, 1,141 proteins were identified in the GBM membrane proteome, the levels of 49 proteins correlated with cell invasiveness. Ingenuity pathway analysis predicted the activation ‘cell movement’ (z-score=2.608, p=3.94E-04) in more invasive cells and a network of invasion-associated proteins with direct links to key regulators of invadopodia formation was generated. Gene expression data relating to invasion-associated proteins, ITGA5, CD97 and ANXA1 show prognostic significance in independent GBM cohorts. Fluorescence microscopy demonstrated ITGA5, CD97 and ANXA1 localisations in invadopodia assays and siRNA-knockdown of ITGA5 reduced invadopodia formation in U87MG cells. Invasion-associated proteins, including ITGA5, may prove to be useful anti-invasive targets. The use of volociximab, a therapeutic antibody against integrin α5β1 should be assessed in a GBM setting.
    Full-text · Article · Apr 2015 · Journal of Neuropathology and Experimental Neurology
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    ABSTRACT: Hepatitis C virus (HCV) reinfection of the liver allograft after transplantation is universal, with some individuals suffering severe disease recurrence. Predictive markers of recurrent disease severity are urgently needed. In this study, we used a cluster of differentiation (CD) microarray to predict the severity of HCV recurrence after transplantation. The CD antibody microarray assays of live leukocytes were performed on peripheral blood taken in the first year after transplantation. The results were grouped into phases defined as; Pre-transplant (day 0), Early (day 3 to week 2), Mid (week 4 to week 10), and Late (week 12 to week 26). Hepatitis C virus severity was based on fibrosis stages in the first 2 years (F0-1 mild and F2-4 severe). Serial blood samples from 16 patients were taken before and after liver transplantation. A total of 98 assays were performed. Follow-up was 3 years or longer. Comparing recurrence severity, significantly greater numbers of CD antigens were differentially expressed on the pretransplant samples compared to any posttransplant timepoints. Five differentially expressed CD antigens before transplantation (CD27 PH, CD182, CD260, CD41, and CD34) were significantly expressed comparing severe to mild recurrence, whereas expression of only CD152 was significant in the late phase after transplantation. No relationship was observed between the donor or recipient interleukin-28B genotypes and HCV recurrence severity. This study shows that circulating leukocyte CD antigen expression has utility in assessing recurrent HCV disease severity after liver transplantation and serves as a proof of principle. Importantly, pretransplant CD antigen expression is most predictive of disease outcome.
    Full-text · Article · Feb 2015 · Transplantation
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    ABSTRACT: Extensive surface profiles of colorectal cancer (CRC) cells and tumour infiltrating lymphocytes (TIL) have been obtained from 45 surgical resection samples. Live cells were captured on an antibody microarray and stained with fluorescently-labeled antibodies. Minimal panels of 11 CRC antigens (CD13, CD24, CD26, CD49d, CD138, CD166, CA-125, CA19-9, EGFR, Galectin-4 and HLA-DR) and 11 T-cell antigens (CD10, CD11b, CD11c, CD25, CD31, CD95, CD151, CD181, Galectin-4, CA19-9, TSP-1) provide signatures for relapse and survival. Hierarchical clustering of profiles from CRC cells and TIL identified groups of patients for survival, systemic relapse and death. The groups from CRC and TIL profiles for systemic relapse showed 79.2% concordance, enabling prediction of relapse after surgery. The results demonstrate communication between CRC cells and TIL.
    Full-text · Article · Nov 2014 · Journal of Immunological Methods
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    Full-text · Conference Paper · Oct 2014
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    ABSTRACT: s P17.54. Glioblastoma multiforme (GBM) tumours are diffusely infiltrative making surgical resection virtually impossible. Invasion of brain parenchyma is facil-itated by cell migration and degradation of the extracellular matrix (ECM). Invadopodia are actin-rich organelles that protrude from the ventral side of the plasma membrane in direct contact with the ECM and play an important role in mesenchymal cell invasion. We have characterized the 'invasive poten-tial' of a panel of established GBM cell lines (n ¼ 9) using QCM gelatin inva-dopodia assay (Millipore) and performed comparative, quantitative membrane mass spectrometry-based proteomic analyses of highly invasive vs. less-invasive cell lines. All GBM cells produced invadopodia, and there was a significant difference between the most invasive (U87MG) and least in-vasive (LN229) cells (65%, percentage of total cell area; p ¼ 0.0001). Overall, 1667 quantifiable proteins were identified from duplicate analyses, of which 76% mapped to membrane structures using the David bioinformatics data-base (http://david.abcc.ncifcrf.gov). The differential abundance of 38 pro-teins significantly correlated with the degree of invasion (r 2 . 0.45 or r 2 , -0.45; n ≥ 5; p , 0.05) and are predominantly involved in cellular movement and cell-cell and interactions. Fluorescence microscopy demonstrates co-localisation of novel proteins to invadopodia structures and siRNA knock-down of a target protein confirmed its role in invadopodia-formation. Invadopodia-associated membrane proteins could be novel targets for anti-invasive GBM therapies. Neuro-Oncology 16:ii1 – ii112, 2014. doi:10.1093/neuonc/nou174
    Full-text · Conference Paper · Oct 2014
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    ABSTRACT: DIGE methods and protein function and network analysis.
    Preview · Dataset · Aug 2014
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    ABSTRACT: SRM methods and survival analysis.
    Preview · Dataset · Aug 2014
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    ABSTRACT: Representative protein map from DIGE analysis of proteins extracted from a lymph node melanoma metastasis. Figure S2. Density plot for the percentage of missing peptides for iTRAQ 2DLC-MS/MS data. Figure S3. Summary of identification of all 2031 proteins and 73 differentially abundant proteins identified bu iTRAQ 2DLC-MS/MS. Figure S4. Cellular location and biological processes of differentially abundant proteins between poor and good prognosis patients with melanoma lymph node metastases. Figure S5. SRM reproducibility between replicates 1 and 2. Figure S6. Scatter plots showing the distribution of protein levels in 33 lymph node melanoma metastases, verified by SRM. Figure S7. Western blot analysis of eukaryotic initiation factor 4A-I (eIF4A1) and mitochondrial superoxide dismutase [Mn] (SOD2).
    Preview · Dataset · Aug 2014
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    ABSTRACT: Clinico-pathologic characteristics of 33 AJCC stage III melanoma patients and their tumors. Table S2. Melanoma patient demographics, pathology, lymph node recurrence and survival parameters. Table S3. Differentially abundant proteins between poor and good prognosis AJCC stage IIIc melanoma patients identified using DIGE. Table S4. Summary of iTRAQ 2DLC-MS/MS analyses (number of peptides, proteins, percentage of spectra matched). Table S5. Reproducibility of iTRAQ 2DLC-MS/MS experiments. Table S7. Differentially abundant proteins between good and poor prognosis AJCC stage IIIc melanoma patients identified using iTRAQ and 2DLC-MS/MS. Table S8. Top ranking pathway maps and process networks associated with proteins differentially abundant between poor and good prognosis melanoma patients, based on GeneGo™ and MetaCore™ database analysis. Table S9. Parameters (Q1, Q3, DP and CE) for SRM assays.
    Preview · Dataset · Aug 2014
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    ABSTRACT: iTRAQ labeling and 2DLC-MS/MS methods.
    Preview · Dataset · Aug 2014
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    ABSTRACT: Outcomes for melanoma patients with stage III disease, differ widely even within the same sub-category. Molecular signatures that more accurately predict prognosis are needed to stratify patients according to risk. Proteomic analyses were used to identify differentially abundant proteins in extracts of surgically excised samples from patients with stage IIIc melanoma lymph node metastases. Analysis of samples from patients with poor (n = 14, < 1 year) and good (n = 19, > 4 years) survival outcomes identified 84 proteins that were differentially abundant between prognostic groups. Subsequent selected reaction monitoring analysis verified 21 proteins as potential biomarkers for survival. Poor prognosis patients are characterized by increased levels of proteins involved in protein metabolism, nucleic acid metabolism, angiogenesis, deregulation of cellular energetics and methylation processes, and decreased levels of proteins involved in apoptosis and immune response. These proteins are able to classify stage IIIc patients into prognostic sub-groups (p < 0.02). This is the first report of potential prognostic markers from stage III melanoma using proteomic analyses. Validation of these protein markers in larger patient cohorts should define protein signatures that enable better stratification of stage III melanoma patients.This article is protected by copyright. All rights reserved.
    Full-text · Article · Jul 2014 · Pigment Cell & Melanoma Research
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    ABSTRACT: Unlabelled: Altered glycosylation is commonly observed in colorectal cancer. In vitro models are frequently used to study this cancer but little is known about the differences that may exist between these model cell systems and tumour tissue. We have compared the membrane protein glycosylation of five colorectal cancer cell lines (SW1116, SW480, SW620, SW837, LS174T) with epithelial cells from colorectal tumours using liquid chromatography tandem mass spectrometry. Remarkably, there were five abundant O-glycans in the tumour cells that were undetected in the low-mucin producing cell lines, although two were found in the mucinous LS174T cells. The O-glycans included the well-known glycan cancer marker, sialyl-Tn, which has been associated with mucins. Using qRT-PCR, sialyl-Tn expression was found to be associated with an increase in α2,6-sialyltransferase gene (ST6GALNAC1) and a decrease in core 1 synthase gene (C1GALT1) in LS174T cells. The expression of a subset of mucins (MUC2, MUC6, MUC5B) was also correlated with sialyl-Tn expression in LS174T cells. Overall, the membrane protein glycosylation of the model cell lines was found to differ from each other and from the epithelial cells of tumour tissue. These findings should be noted in the design of biomarker discovery experiments particularly when cell surface targets are being investigated. Biological significance: The extent of protein glycosylation differences between in vitro cell lines and ex vivo tumours in colorectal cancer research is unknown. Our study expands current knowledge by characterising the membrane protein glycosylation profiles of five different colorectal cancer cell lines and of epithelial cells derived from resected colorectal cancer tumour tissue, using liquid chromatography tandem mass spectrometry. The detailed structural differences found in both N- and O-linked glycan structures on the membrane glycoproteins were determined and correlated with the mRNA expression of the relevant proteins in the cell lines. The glycosylation differences found between cultured cancer cell lines and epithelial cells from tumour tissue have important implications for glycan biomarker discovery.
    Full-text · Article · May 2014 · Journal of Proteomics
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    Full-text · Dataset · Apr 2014
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    ABSTRACT: 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.
    No preview · Article · Apr 2014 · Nucleosides Nucleotides & Nucleic Acids
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    ABSTRACT: There is an urgent need to identify more accurate prognostic biomarkers in melanoma patients, particularly in those with metastatic disease. This study aimed to identify melanoma and leukocyte surface antigens predictive of survival in a prospective series of AJCC stage IIIb/c melanoma patients (n = 29). Live cell suspensions were prepared from melanoma metastases within lymph nodes (LN). The suspensions were immuno-magnetically separated into CD45+ (leukocyte) and CD45- (non-hematopoietic, enriched melanoma cell) fractions. Surface antigens on CD45- and CD45+ cell populations were profiled using DotScan™ microarrays (Medsaic Pty. Ltd.) and showed differential abundance levels for 52 and 78 antigens respectively. Associations of the surface profiles with clinicopathologic and outcome data (median follow-up 35.4 months post LN resection) were sought using univariate (log-rank test) and multivariate (Wald's test; modelled with patient's age, gender and AJCC staging at LN recurrence) survival models. CD9 (p = 0.036), CD39 (p = 0.004) and CD55 (p = 0.005) on CD45+ leukocytes were independently associated with distant metastasis-free survival using multivariate analysis. Leukocytes with high CD39 levels were also significantly associated with increased overall survival (OS) in multivariate analysis (p = 0.016). LNs containing leukocytes expressing CD11b (p = 0.025), CD49d (p = 0.043) and CD79b (p = 0.044) were associated with reduced OS on univariate analysis. For enriched melanoma cells (CD45- cell populations), 11 surface antigens were significantly correlated with the disease-free interval (DFI) between diagnosis of culprit primary melanoma and LN metastasis resection. Nine antigens on CD45+ leukocytes also correlated with DFI. Following validation in independent datasets, surface markers identified here should enable more accurate determination of prognosis in stage III melanoma patients and provide better risk stratification of patients entering clinical trials.
    Full-text · Article · Jan 2014 · Clinical and Experimental Metastasis
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    ABSTRACT: Citation: Alomari M, Mactier S, Kaufman KL, Best OG, Mulligan SP, et al. (2014) Profiling the Lipid Raft Proteome from Human MEC1 Chronic Lymphocytic Leukemia Cells. J Proteomics Bioinform S7: 005. doi:10.4172/jpb.S7-005 Copyright: © 2014 Alomari M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
    Full-text · Article · Jan 2014 · Journal of Proteomics & Bioinformatics
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    ABSTRACT: There is an urgent need to identify more accurate prognostic biomarkers in melanoma patients, particularly in those with metastatic disease. This study aimed to identify melanoma and leukocyte surface antigens predictive of survival in a prospective series of AJCC stage IIIb/c melanoma patients (n=29). Live cell suspensions were prepared from melanoma metastases within lymph nodes (LN). The suspensions were immuno-magnetically separated into CD45+ (leukocyte) and CD45- (non-hematopoietic, enriched melanoma cell) fractions. Surface antigens on CD45- and CD45+ cell populations were profiled using DotScanTM microarrays (Medsaic Pty. Ltd.) and showed differential abundance levels for 52 and 78 antigens respectively. Associations of the surface profiles with clinicopathologic and outcome data (median follow-up 35.4 months post LN resection) were sought using univariate (log-rank test) and multivariate (Wald’s test; modelled with patient’s age, gender and AJCC staging at LN recurrence) survival models. CD9 (p=0.036), CD39 (p=0.004) and CD55 (p=0.005) on CD45+ leukocytes were independently associated with distant metastasis-free survival (DMFS) using multivariate analysis. Leukocytes with high CD39 levels were also significantly associated with increased overall survival (OS) in multivariate analysis (p=0.016). LNs containing leukocytes expressing CD11b (p=0.025), CD49d (p=0.043) and CD79b (p=0.044) were associated with reduced OS on univariate analysis. For enriched melanoma cells (CD45- cell populations), 11 surface antigens were significantly correlated with the disease-free interval (DFI) between diagnosis of culprit primary melanoma and LN metastasis resection. Nine antigens on CD45+ leukocytes also correlated with DFI. Following validation in independent datasets, surface markers identified here should enable more accurate determination of prognosis in stage III melanoma patients and provide better risk stratification of patients entering clinical trials.
    Full-text · Article · Jan 2014 · Clinical & experimental metastasis

Publication Stats

2k Citations
524.27 Total Impact Points

Institutions

  • 1989-2014
    • University of Sydney
      • School of Molecular Bioscience
      Sydney, New South Wales, Australia
    • University of New South Wales
      • School of Biotechnology and Biomolecular Sciences (BABS)
      Kensington, New South Wales, Australia
  • 1983-1985
    • Australian National University
      Canberra, Australian Capital Territory, Australia
  • 1977-1985
    • University of Melbourne
      Melbourne, Victoria, Australia
  • 1978-1981
    • University of North Carolina at Chapel Hill
      • Department of Medicine
      North Carolina, United States