%0 Journal Article
%~ PubMed
%A Kohnke, Philippa L
%A Mactier, Swetlana
%A Almazi, Juhura G
%A Crossett, Ben
%A Christopherson, Richard I
%T Fludarabine and Cladribine Induce Changes in Surface Proteins on Human B-Lymphoid Cell Lines Involved with Apoptosis, Cell Survival, and Antitumor Immunity.
%B Journal of Proteome Research
%D 2012
%C United States
%I American Chemical Society
%V 11
%N 9
%P 4436-4448
%@ 1535-3893
%X 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.
%Z FOR Codes: 111201 30406


%0 Journal Article
%~ PubMed
%A Almazi, Juhura G
%A Mactier, Swetlana
%A Best, O Giles
%A Crossett, Ben
%A Mulligan, Stephen P
%A Christopherson, Richard I
%T 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.
%B Proteomics - Clinical Applications
%D 2012
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 6
%N 5-6
%P 279-290
%@ 1862-8346
%X PURPOSE: Human Raji cells treated with fludarabine nucleoside (2-FaraA, 3 ??M) 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 4 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 ??M, 24 h, 48 h) and viability determined. Protein extracts of sub-cellular 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 3 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 sub-cellular 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.
%Z FOR Codes: 111204 30405 60109


%0 Journal Article
%~ PubMed
%A Huang, Pauline Y
%A Giles Best, O
%A Belov, L
%A Mulligan, Stephen P
%A Christopherson, Richard I
%T Surface profiles for sub-classification of chronic lymphocytic leukemia.
%B Leukemia and Lymphoma
%D 2012
%C Switzerland
%I Informa Healthcare
%V 53
%N 6
%P 1046-1056
%@ 1029-2403
%X Abstract   Chronic lymphocytic leukemia (CLL) has a variable clinical course, some patients have stable disease while others progress and require treatment. Levels of several cluster of differentiation (CD) antigens are known to correlate with prognosis and may be used to stratify patients according to risk. In this review, we summarize current information on surface CD antigens found on CLL, their pathological significance, and their detection using CD antibody microarrays. The use of extensive immunophenotypic patterns or surface profiles as disease signatures for CLL sub-classification, prognosis and patient management is discussed with a focus on triaging CLL patients with progressive disease.
%Z FOR Codes: 60110 1112


%0 Journal Article
%~ PubMed
%A Best, O Giles
%A Che, Yiping
%A Singh, Nisha
%A Forsyth, Cecily
%A Christopherson, Richard I
%A Mulligan, Stephen P
%T The Hsp90 inhibitor SNX-7081 synergizes with and restores sensitivity to fludarabine in chronic lymphocytic leukemia cells with lesions in the TP53 pathway: a potential treatment strategy for fludarabine refractory disease.
%B Leukemia & Lymphoma
%D 2012
%C Switzerland
%I Informa Healthcare
%V 53
%N 7
%P 1367-1375
%@ 1029-2403
%X Drug resistance in chronic lymphocytic leukemia (CLL) associated with lesions in the ATM/TP53 pathway represents a major challenge in clinical management. Evidence suggests that heat shock protein-90 (Hsp90) inhibitors may represent a therapeutic option in combination with more conventional therapies. We explored the effects of combining the Hsp90 inhibitor, SNX-7081, with fludarabine in vitro against CLL cells and hematological cell lines. In seven cell lines and 23 patient samples synergy between SNX-7081 and fludarabine (2-FaraA) was apparent in the three TP53 mutated cell lines and at significantly lower concentrations in TP53 or ATM dysfunctional patient cells. In 11/13 2-FaraA-resistant patient samples, SNX-7081 reduced the 50% inhibitory concentration to within a clinically achievable range. Synergy between SNX-7081 and 2-FaraA was evident in both the cell lines and patient samples as a significant decrease in cell viability. Our data suggest that combining SNX-7081 and fludarabine may be effective in the treatment of fludarabine-refractory CLL.
%Z FOR Codes: 60199


%0 Journal Article
%~ PubMed
%A Mallawaaratchy, Duthika M
%A Mactier, Swetlana
%A Kaufman, Kimberley L
%A Blomfield, Katherine
%A Christopherson, Richard I
%T The phosphoinositide 3-kinase inhibitor LY294002, decreases aminoacyl-tRNA synthetases, chaperones and glycolytic enzymes in human HT-29 colorectal cancer cells.
%B Journal of proteomics
%D 2012
%C Netherlands
%I Elsevier BV
%V 75
%N 5
%P 1590-9
%@ 1876-7737
%X The proposed anticancer drug LY294002, inhibits phosphoinositide-3 kinase (PI3K) that initiates a signalling pathway often activated in colorectal cancer (CRC). The effects of LY294002 (10??M, 48h) on the cytosolic, mitochondrial and nuclear proteomes of human HT-29 CRC cells have been determined using iTRAQ (isobaric tag for relative and absolute quantitation) and tandem mass spectrometry (MS/MS). Analysis of cells treated with LY294002 identified 26 differentially abundant proteins that indicate several mechanisms of action. The majority of protein changes were directly or indirectly associated with Myc and TNF-??, previously implicated in CRC progression. LY294002 decreased the levels of 6 aminoacyl-tRNA synthetases (average 0.39-fold) required for protein translation, 5 glycolytic enzymes (average 0.37-fold) required for ATP synthesis, and 3 chaperones required for protein folding. There was a 3.2-fold increase in lysozyme C involved in protein-glycoside hydrolysis. LY294002 increased cytosolic p53 with a concomitant decrease in nuclear p53, suggesting transfer of p53 to the cytosol where apoptosis might be initiated via the intrinsic mitochondrial pathway. Protein changes described here suggest that the anti-angiogenic effects of LY294002 may be related to p53; the mutational status of p53 in CRC may be an important determinant of the efficacy of PI3K inhibitors for treatment.
%Z FOR Codes: 111201


%0 Journal Article
%~ PubMed
%A Leong, Sharon
%A Nunez, Andrea C
%A Lin, Mike Z
%A Crossett, Ben
%A Christopherson, Richard I
%A Baxter, Robert C
%T iTRAQ-Based Proteomic Profiling of Breast Cancer Cell Response to Doxorubicin and TRAIL.
%B Journal of Proteome Research
%D 2012
%C United States
%I American Chemical Society
%V 11
%N 7
%P 3561-3572
%@ 1535-3893
%X Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC-MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer.
%Z FOR Codes: 111202 111201 100402


%0 Journal Article
%~ PubMed
%A Heath, E M L
%A Kaufman, K L
%A Christopherson, R I
%T B-RAF: A Contributor to the Melanoma Phenotype.
%B The international journal of biochemistry & cell biology
%D 2011
%C United Kingdom
%I Pergamon
%V 43
%N 1
%P 29-32
%@ 1357-2725
%X B-RAF, a serine-threonine protein kinase, is one of the three RAF paralogs in humans. B-RAF participates in the RAS-RAF-MEK-ERK pathway, a conserved protein kinase-signalling cascade that is involved in regulating a number of critical cellular functions. Mutated B-RAF is believed to play a crucial role in the development, maintenance and progression of melanoma, where it contributes to multiple aspects of the malignant phenotype, such as cell survival, proliferation and apoptosis resistance. Indeed, it is mutated in a high proportion of melanocytic skin lesions and B-RAF mutations are preserved through melanoma progression. Despite this, the direct inhibition of B-RAF has shown little success clinically in the treatment of melanoma, presumably due to the complexity of the RAS-RAF-MEK-ERK pathway. For this reason, alternative strategies must be developed to treat oncogenic B-RAF-induced melanomas.
%Z FOR Codes: 60105


%0 Journal Article
%~ PubMed
%A Leong, Sharon
%A McKay, Matthew J
%A Christopherson, Richard I
%A Baxter, Robert C
%T Biomarkers of breast cancer apoptosis induced by chemotherapy and TRAIL.
%B Journal of proteome research
%D 2011
%C United States
%I American Chemical Society
%V 11
%N 2
%P 1240-50
%@ 1535-3893
%X Treatment of breast cancer is complex and challenging due to the heterogeneity of the disease. To avoid significant toxicity and adverse side-effects of chemotherapy in patients who respond poorly, biomarkers predicting therapeutic response are essential. This study has utilized a proteomic approach integrating 2D-DIGE, LC-MS/MS, and bioinformatics to analyze the proteome of breast cancer (ZR-75-1 and MDA-MB-231) and breast epithelial (MCF-10A) cell lines induced to undergo apoptosis using a combination of doxorubicin and TRAIL administered in sequence (Dox-TRAIL). Apoptosis induction was confirmed using a caspase-3 activity assay. Comparative proteomic analysis between whole cell lysates of Dox-TRAIL and control samples revealed 56 differentially expressed spots (???2-fold change and p < 0.05) common to at least two cell lines. Of these, 19 proteins were identified yielding 11 unique protein identities: CFL1, EIF5A, HNRNPK, KRT8, KRT18, LMNA, MYH9, NACA, RPLP0, RPLP2, and RAD23B. A subset of the identified proteins was validated by selected reaction monitoring (SRM) and Western blotting. Pathway analysis revealed that the differentially abundant proteins were associated with cell death, cellular organization, integrin-linked kinase signaling, and actin cytoskeleton signaling pathways. The 2D-DIGE analysis has yielded candidate biomarkers of response to treatment in breast cancer cell models. Their clinical utility will depend on validation using patient breast biopsies pre- and post-treatment with anticancer drugs.
%Z FOR Codes: 110106 1112 60103


%0 Journal Article
%~ PubMed
%A Belov, Larissa
%A Zhou, Jerry
%A Christopherson, Richard I
%T Cell surface markers in colorectal cancer prognosis.
%B International journal of molecular sciences
%D 2011
%C Switzerland
%I Molecular Diversity Preservation International (M
%V 12
%N 1
%P 78-113
%@ 1422-0067
%X The classification of colorectal cancers (CRC) is currently based largely on histologically determined tumour characteristics, such as differentiation status and tumour stage, i.e., depth of tumour invasion, involvement of regional lymph nodes and the occurrence of metastatic spread to other organs. These are the conventional prognostic factors for patient survival and often determine the requirement for adjuvant therapy after surgical resection of the primary tumour. However, patients with the same CRC stage can have very different disease-related outcomes. For some, surgical removal of early-stage tumours leads to full recovery, while for others, disease recurrence and metastasis may occur regardless of adjuvant therapy. It is therefore important to understand the molecular processes that lead to disease progression and metastasis and to find more reliable prognostic markers and novel targets for therapy. This review focuses on cell surface proteins that correlate with tumour progression, metastasis and patient outcome, and discusses some of the challenges in finding prognostic protein markers in CRC.
%Z FOR Codes: 111203


%0 Journal Article
%~ PubMed
%A Zhou, Jerry
%A Belov, Larissa
%A Solomon, Michael J
%A Chan, Charles
%A Clarke, Stephen J
%A Christopherson, Richard I
%T Colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing.
%B Journal of Visualized Experiments
%D 2011
%C United States
%I Journal of Visualized Experiments
%V 0
%N 55
%P 3322
%@ 1940-087X
%X The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification(1). A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs). The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types(2,3). However, no single ''marker'' has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases.  A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple ''markers''. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods.  In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response(5), together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody(6).  For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells(7). The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.
%Z FOR Codes: 111202


%0 Journal Article
%~ PubMed
%A Mactier, Swetlana
%A Henrich, Silke
%A Che, Yiping
%A Kohnke, Philippa L
%A Christopherson, Richard I
%T Comprehensive proteomic analysis of the effects of purine analogs on human Raji B-cell lymphoma.
%B Journal of Proteome Research
%D 2011
%C United States
%I American Chemical Society
%V 10
%N 3
%P 1030-1042
%@ 1535-3893
%X Cladribine (CdA) and fludarabine (FdAMP) are purine analogs that induce apoptosis in chronic lymphocytic leukemia and non-Hodgkin''s lymphoma, but the mechanisms are undefined. The effects of CdA and fludarabine nucleoside (FdA) on the cytosolic, mitochondrial, and nuclear proteomes in human Raji lymphoma cells have been determined using two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry. Differentially abundant proteins have provided new insights into CdA- and FdA-induced apoptosis. Treatment with these purine analogs induced changes in proteins involved with intermediary metabolism, cell growth, signal transduction, protein metabolism, and regulation of nucleic acids. Differentially abundant mitochondrial 39S ribosomal protein L50, mTERF domain-containing protein 1, Chitinase-3 like 2 protein, and ubiquinone biosynthesis protein COQ9 have been identified in cells undergoing apoptosis. Up-regulation of several stress-associated proteins found in the endoplasmic reticulum (ER) including GRP78, ERp57, and ORP150 suggests that purine analog-induced apoptosis may result from ER stress and unfolded protein response. While mitochondria-dependent apoptosis has been associated with purine analog cytotoxicity, the likely involvement of the ER stress pathway in CdA- and FdA-induced apoptosis has been shown here for the first time.
%Z FOR Codes: 60109 111201


%0 Journal Article
%~ PubMed
%A Henrich, Silke
%A Mactier, Swetlana
%A Best, Giles
%A Mulligan, Stephen P
%A Crossett, Ben
%A Christopherson, Richard Ian
%T Fludarabine nucleoside modulates nuclear "survival and death" proteins in resistant chronic lymphocytic leukemia cells.
%B Nucleosides, Nucleotides and Nucleic Acids
%D 2011
%C United States
%I Taylor & Francis Inc.
%V 30
%N 12
%P 1181-1189
%@ 1532-2335
%X The nuclear mechanisms by which fludarabine nucleoside (F-ara-A) induces apoptosis have been investigated in human MEC1 cells derived from B-cell chronic lymphocytic leukemia. Upon treatment of cells with F-ara-A (100 ??M, 72 hours), 15 nuclear proteins changed in abundance by more than 2-fold. Nuclear proteins up-regulated included calmodulin (4.3-fold), prohibitin (3.9-fold), ??-actin variant (3.7-fold), and structure-specific recognition protein 1 (3.7-fold); those down-regulated included 60S ribosomal protein P2B (0.12-fold), fumarate hydratase (0.19-fold), splicing factor arginine/serine-rich 3 (0.35-fold), and replication protein A2 (0.42-fold). These changes in the levels of specific proteins promote survival or apoptosis; because the end result is apoptosis of MEC1 cells, apoptotic effects predominate.
%Z FOR Codes: 60103 111201


%0 Journal Article
%~ PubMed
%A Kaufman, Kimberley L
%A Belov, Larissa
%A Huang, Pauline
%A Mactier, Swetlana
%A Scolyer, Richard A
%A Mann, Graham J
%A Christopherson, Richard I
%T An extended antibody microarray for surface profiling metastatic melanoma.
%B Journal of immunological methods
%D 2010
%C Netherlands
%I Elsevier BV
%V 358
%N 1-2
%P 23-34
%@ 0022-1759
%X An antibody microarray was developed for profiling the surface proteome of melanoma cells, which may facilitate melanoma sub-classification and provide important prognostic information useful in predicting the clinical behavior of the melanoma (e.g., likely sites of metastatic spread), patient outcome and treatment response. Forty-eight antibodies were selected based on their correlation with melanoma development, progression and/or prognosis and printed on nitrocellulose slides. The immobilised antibodies capture live cells expressing corresponding antigens to produce a cell binding dot pattern representing the surface antigen profile (immunophenotype) of the melanoma. Surface antigen signatures were determined for a normal melanocyte and 6 melanoma cell lines and cell suspensions prepared from 10 surgically excised melanoma lymph node metastases. A procedure for obtaining separate surface antigen profiles for melanoma cells and leukocytes from clinical lymph node samples was also developed using anti-CD45 magnetic beads. The capture of live, bead-bound leukocytes on these antibody microarrays provides a significant enhancement of this microarray technology. The antibody microarray will be used to profile panels of surgically excised melanoma lymph node metastases (melanoma and leukocyte fractions) to determine whether the immunophenotypes correlate with clinicopathological characteristics, disease progression and clinical outcome.
%Z FOR Codes: 60109 111209 100402


%0 Journal Article
%A Cassano, Juan
%A Mactier, Swetlana
%A Mulligan, Stephen
%A Belov, Larissa
%A Huang, Pauline
%A Christopherson, Richard
%T Cladribine and Fludarabine Nucleoside Change the Levels of
CD Antigens on B-Lymphoproliferative Disorders
%B International Journal of Proteomics
%D 2010
%C United States
%I Hindawi Publishing Corporation
%V 2010
%N 
%P 964251
%@ 2090-2166
%X 
%Z FOR Codes: 60109


%0 Journal Article
%~ PubMed
%A Yang, F
%A Tay, K H
%A Dong, L
%A Thorne, R F
%A Jiang, C C
%A Yang, E
%A Tseng, H-Y
%A Liu, H
%A Christopherson, R
%A Hersey, P
%A Zhang, X D
%T Cystatin B inhibition of TRAIL-induced apoptosis is associated with the protection of FLIP(L) from degradation by the E3 ligase itch in human melanoma cells.
%B Cell Death and Differentiation
%D 2010
%C United Kingdom
%I Nature Publishing Group
%V 17
%N 8
%P 1354-1367
%@ 1476-5403
%X Past studies have identified a number of distinct mechanisms that contribute to the resistance of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). In this report we show that cystatin B is another endogenous inhibitor of TRAIL-induced apoptosis. Cystatin B-deficient melanoma cell lines established by shRNA knockdown displayed increased apoptosis that was associated with enhanced activation of caspase-8 induced by TRAIL. This was not related to the inhibitory effect of cystatin B on the lysosomal cysteine proteases, cathepsin B and L, as they did not have a role in TRAIL-induced apoptosis in most melanoma cell lines even when cystatin B was inhibited. Instead, sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of cystatin B appeared associated with decreased stability of FLIP(L) as the levels of FLIP(L) were reduced because of shortened half-life time in melanoma cells deficient in cystatin B. In contrast, over-expression of cystatin B increased the levels of FLIP(L), decreased the amount of the E3 ligase Itch associated with FLIP(L), and reduced FLIP(L) ubiquitination. Inhibition of Itch by siRNA restored the levels of FLIP(L) and blocked sensitization to TRAIL-induced apoptosis associated with deficiency in cystatin B. Taken together, these results indicate that cystatin B regulates Itch-mediated degradation of FLIP(L) and thereby TRAIL-induced apoptosis in melanoma cells.
%Z FOR Codes: 60103 111201


%0 Journal Article
%~ PubMed
%A Zhou, Jerry
%A Belov, Larissa
%A Huang, Pauline Y
%A Shin, Joo-Shik
%A Solomon, Michael J
%A Chapuis, Pierre H
%A Bokey, Leslie
%A Chan, Charles
%A Clarke, Candice
%A Clarke, Stephen J
%A Christopherson, Richard I
%T Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing.
%B Journal of Immunological Methods
%D 2010
%C Netherlands
%I Elsevier BV
%V 355
%N 1-2
%P 40-51
%@ 0022-1759
%X A procedure is described for the disaggregation of colorectal cancers (CRC) and normal intestinal mucosal tissues to produce suspensions of viable single cells, which are then captured on customized antibody microarrays recognising 122 different surface antigens (DotScan CRC microarray). Cell binding patterns recorded by optical scanning of microarrays provide a surface profile of antigens on the cells. Sub-populations of cells bound on the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with Quantum Dots or other fluorescent dyes. Surface profiles are presented for 6 CRC cell lines (T84, LIM1215, SW480, HT29, CaCo and SW620) and surgical samples from 40 CRC patients. Statistical analysis revealed significant differences between profiles for CRC samples and mucosal controls. Hierarchical clustering of CRC data identified several disease clusters that showed some correlation with clinico-pathological stage as determined by conventional histopathological analysis. Fluorescence multiplexing using Phycoerythrin- or Alexa Fluor 647-conjugated antibodies was more effective than multiplexing with antibodies labelled with Quantum Dots. This relatively simple method yields a large amount of information for each patient sample and, with further application, should provide disease signatures and enable the identification of patients with good or poor prognosis.
%Z FOR Codes: 111202


%0 Journal Article
%~ PubMed
%A Kohnke, Philippa L
%A Mulligan, Stephen P
%A Christopherson, Richard I
%T Membrane proteomics for leukemia classification and drug target identification.
%B Current Opinion in Molecular Therapeutics
%D 2009
%C United Kingdom
%I Current Opinion in Molecular Therapeutics
%V 11
%N 6
%P 603-610
%@ 2040-3445
%X Knowledge of protein expression in the plasma membrane of leukemia cells has contributed to improvements in the detection and treatment of hematological malignancies. Recently engineered antibodies against leukemia surface molecules have improved therapeutic efficacy compared with earlier agents, but there are still side effects. An increased understanding of the surface expression profiles and interactions of membrane proteins on leukemia cells will facilitate the expansion of the role of antibodies in therapy and enable the identification of novel biomarkers for the various stages of leukemogenesis and leukemia progression. Proteomic analysis enables the identification of thousands of proteins in a membrane extract and provides information on their relative abundance, interactions and post-translational modifications. Plasma membrane proteome analysis of leukemia cells can be used to define biomarkers for diagnosis, classification, prognosis and progression monitoring, as well as to predict therapeutic response or resistance. The effects of chemotherapy on the surface proteome and the functional consequences of perturbations to membrane protein networks can provide insights into leukemia cell signaling and survival mechanisms. Surface proteins that are differentially expressed on leukemia cells are prospective targets for the development of engineered antibodies or small-molecule therapeutics. This review focuses on recent discoveries in leukemia membrane proteomics and the potential for future research into leukemia classification and drug target identification.
%Z FOR Codes: 601


%0 Journal Article
%~ PubMed
%A Barber, Nicole
%A Gez, Swetlana
%A Belov, Larissa
%A Mulligan, Stephen P
%A Woolfson, Adrian
%A Christopherson, Richard I
%T Profiling CD antigens on leukaemias with an antibody microarray.
%B FEBS Letters
%D 2009
%C Netherlands, Denmark
%I Elsevier BV
%V 583
%N 11
%P 1785-1791
%@ 0014-5793
%X Cluster of differentiation (CD) antigens are defined when a surface molecule found on some members of a standard panel of human cells reacts with at least one novel antibody, and there is good accompanying molecular data. Monoclonal antibodies to surface CD antigens on leukocytes have been used for flow cytometry, and more recently to construct microarrays that capture live cells. These DotScan microarrays enable the rapid and highly parallel characterization of repertoires of CD antigens whose expression patterns may be correlated with discrete leukaemia subtypes, or used to define biomarker ''signatures'' for non-hematological diseases. DotScan with fluorescence multiplexing enables profiling of CD antigens for minor subsets of cells, such as colorectal cancer cells and tumour-infiltrating lymphocytes from a surgical sample.
%Z FOR Codes: 60109


%0 Journal Article
%~ PubMed
%A Langley, David B
%A Shojaei, Maryam
%A Chan, Camilla
%A Lok, Hiu Chuen
%A Mackay, Joel P
%A Traut, Thomas W
%A Guss, J Mitchell
%A Christopherson, Richard I
%T Structure and Inhibition of Orotidine 5'-Monophosphate Decarboxylase from Plasmodium falciparum.
%B Biochemistry
%D 2009
%C United States
%I American Chemical Society
%V 48
%N 11
%P 2570
%@ 0006-2960
%X 
%Z FOR Codes: 60199 60199


%0 Journal Article
%~ PubMed
%A Henrich, S
%A Gez, S
%A Crossett, B
%A Mulligan, S P
%A Christopherson, R I
%T Fludarabine Induces Differential Expression of Proteins in Human Leukemia and Lymphoma Cells.
%B Nucleosides, Nucleotides & Nucleic Acids
%D 2008
%C United States
%I Taylor & Francis Inc.
%V 27
%N 6
%P 634-640
%@ 1525-7770
%X The purine analog fludarabine (FdAMP) is widely used for chemotherapy of B-lymphoid malignancies, and multiple mechanisms of action leading to apoptosis have been proposed. We examined changes at the protein level induced in the Raji cell line (Burkitt''s lymphoma) by fludarabine nucleoside (FdA). Raji cells are sensitive to FdA. Raji cells treated with FdA (3 mu M, 24 hours), accumulate multiple phosphorylated forms of p53 in the nucleus that in turn degrade to phosphorylated forms of p40. Using CD antibody microarrays to determine surface expression profiles for Raji cells treated with FdA, we found up-regulation of the following CD antigens: CD20, CD54, CD80, CD86, and CD95. FdA thus induces changes in the genetic program of the cells that might be exploited to obtain synergy with therapeutic antibodies.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Ellmark, P
%A Högerkorp, C M
%A Ek, S
%A Belov, L
%A Berglund, M
%A Rosenquist, R
%A Christopherson, R I
%A Borrebaeck, C A K
%T Phenotypic protein profiling of different B cell sub-populations using antibody CD-microarrays.
%B Cancer Letters
%D 2008
%C CUSTOMER RELATIONS MANAGER, BAY 15, SHANNON INDUSTRIAL ESTATE CO, CLARE, IRELAN
%I Elsevier Sci Ireland Ltd
%V 265
%N 1
%P 98-106
%@ 0304-3835
%X Antibody microarrays enable extensive protein expression profiling, and provide a valuable complement to DNA microarray-based gene expression profiling. In this study, we used DotScan antibody microarrays that contain antibodies against 82 different cell surface antigens, to determine phenotypic protein expression profiles for human B cell sub-populations. We then demonstrated that the B cell protein profile can be used to delineate the relationship between normal B cells and malignant counterparts. Principle component analysis showed that the lymphomas did not cluster with the normal memory B cells or germinal centre B cells, but they did cluster with germinal centre founder cells and naïve B cells.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Langley, David B
%A Shojaei, Maryam
%A Chan, Camilla
%A Lok, Hiu Chuen
%A Mackay, Joel P
%A Traut, Thomas W
%A Guss, J Mitchell
%A Christopherson, Richard I
%T Structure and inhibition of orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum.
%B Biochemistry
%D 2008
%C United States
%I American Chemical Society
%V 47
%N 12
%P 3842-3854
%@ 0006-2960
%X Orotidine 5''-monophosphate (OMP) decarboxylase from Plasmodium falciparum (PfODCase, EC 4.1.1.23) has been overexpressed, purified, subjected to kinetic and biochemical analysis, and crystallized. The native enzyme is a homodimer with a subunit molecular mass of 38 kDa. The saturation curve for OMP as a substrate conformed to Michaelis-Menten kinetics with K m = 350 +/- 60 nM and V max = 2.70 +/- 0.10 micromol/min/mg protein. Inhibition patterns for nucleoside 5''-monophosphate analogues were linear competitive with respect to OMP with a decreasing potency of inhibition of PfODCase in the order: pyrazofurin 5''-monophosphate ( K i = 3.6 +/- 0.7 nM) > xanthosine 5''-monophosphate (XMP, K i = 4.4 +/- 0.7 nM) > 6-azauridine 5''-monophosphate (AzaUMP, K i = 12 +/- 3 nM) > allopurinol-3-riboside 5''-monophosphate ( K i = 240 +/- 20 nM). XMP is an approximately 150-fold more potent inhibitor of PfODCase compared with the human enzyme. The structure of PfODCase was solved in the absence of ligand and displays a classic TIM-barrel fold characteristic of the enzyme. Both the phosphate-binding loop and the betaalpha5-loop have conformational flexibility, which may be associated with substrate capture and product release along the reaction pathway.
%Z FOR Codes: 60199


%0 Journal Article
%~ PubMed
%A Ellmark, Peter
%A Woolfson, Adrian
%A Belov, Larissa
%A Christopherson, Richard I
%T The applicability of a cluster of differentiation monoclonal antibody microarray to the diagnosis of human disease.
%B Methods in Molecular Biology
%D 2008
%C United States
%I Humana Press, Inc.
%V 439
%N 
%P 199-209
%@ 1064-3745
%X Recent advances in antibody microarray technology have facilitated the development of multiplexed diagnostic platforms. Highly parallel antigen expression data obtained from these arrays allow disease states to be characterized using protein patterns rather than individual protein markers. The development of an antibody microarray platform of general applicability requires careful consideration of the array content. The human cluster of differentiation (CD) antigens constitute a promising candidate set, being united by their common expression at the leukocyte cell surface and the fact that the majority perform critical functions in the human immune response. The diagnostic potential of a microarray, containing 82 cluster of differentiation monoclonal antibodies (DotScan microarrays) has been demonstrated for a variety of infectious and neoplastic disease states, including HIV, many acute and chronic leukemias, and colorectal cancer. It is likely that these microarrays will have more general utility that extends to other pathological categories, including autoimmune, metabolic, and degenerative diseases.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Barber, Nicole
%A Belov, Larissa
%A Christopherson, Richard I
%T All-trans retinoic acid induces different immunophenotypic changes on human HL60 and NB4 myeloid leukaemias.
%B Leukemia research
%D 2007
%C England
%I Pergamon Press
%V 32
%N 0
%P 315-22
%@ 0145-2126
%X All-trans retinoic acid (ATRA) is used to treat patients with acute promyelocytic leukaemia (APL), inducing APL cells to differentiate into abnormal neutrophils. To investigate the possible relationship between the chromosome translocation t(15;17) found in APL and ATRA treatment, the human myeloid leukaemia cell lines HL60 and NB4, that are PML-RARalpha negative and positive, respectively, were treated with ATRA and immunophenotyped using a CD antibody microarray. For HL60 cells, ATRA induced major increases in descending order of CD38, CD11b, CD45RO, CD11c, CD54 and CD36 with repression of CD117 and CD44. For NB4 cells, ATRA induced major increases in descending order of CD11c, CD54, CD11a, CD11b, CD53, CD65, CD138, CD66c and T-cell receptor alpha/beta (TCRalpha/beta), with repression of CD38 and CD9. The induction of a number of these CD antigens is consistent with the known differentiation of these leukaemias to abnormal neutrophils. Approximately half of the antigens up-regulated by ATRA on NB4 cells were adhesion molecules, including CD11a, CD11b, CD11c, CD54, CD66c and CD138, consistent with the increased adhesiveness of leukaemia cells observed for APL patients treated with ATRA. On HL60 cells, ATRA induced expression of CD38, CD43 and CD45RO and repressed CD117, while the converse was true on NB4 cells that contain chimeric PML-RARalpha. For NB4 cells, ATRA induced some remarkable increases in CD antigens not seen for HL60: CD14 (16.6-fold), CD32 (27.8), CD53 (20.5), CD65 (139), CD66c (79.7), CD126 (15.1), and CD138 (57.6). The expression of these antigens may be regulated by PML-RARalpha in the presence of ATRA. Such CD antigens could be targets for synergistic treatment of APL with therapeutic antibodies following ATRA treatment.
%Z FOR Codes: 110106 110106


%0 Journal Article
%~ PubMed
%A Gez, Swetlana
%A Crossett, Ben
%A Christopherson, Richard I
%T Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions.
%B Biochimica et biophysica acta
%D 2007
%C Netherlands
%I Elsevier BV
%V 1774
%N 9
%P 1173-83
%@ 0006-3002
%X Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt''s lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.
%Z FOR Codes: 110106


%0 Journal Article
%~ Isi
%A Henrich, S
%A Crossett, B
%A Christopherson, RI
%T Differentially expressed nuclear proteins in human CCRF-CEM, HL-60, MECA and Raji cells correlate with cellular properties
%B PROTEOMICS CLINICAL APPLICATIONS
%D 2007
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 1
%N 10
%P 1252-1265
%@ 
%X 
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Lee, Mihwa
%A Maher, Megan
%A Christopherson, Richard
%A Guss, J
%T Kinetic and Structural Analysis of Mutant Escherichia coli Dihydroorotases: A Flexible Loop Stabilizes the Transition State(,).
%B Biochemistry
%D 2007
%C United States
%I American Chemical Society
%V 46
%N 37
%P 10538-50
%@ 0006-2960
%X Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-l-aspartate (CA-asp) to l-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. Two different conformations of the surface loop (residues 105-115) were found in the dimeric Escherichia coli DHOase crystallized in the presence of DHO (PDB code 1XGE). The loop asymmetry reflected that of the active site contents of the two subunits: the product, DHO, was bound in the active site of one subunit and the substrate, CA-asp, in the active site of the other. In the substrate- (CA-asp-) bound subunit, the surface loop reaches in toward the active site and makes hydrogen bonds with the bound CA-asp via two threonine residues (Thr109 and Thr110), whereas the loop forms part of the surface of the protein in the product- (DHO-) bound subunit. To investigate the relationship between the structural states of this loop and the catalytic mechanism of the enzyme, a series of mutant DHOases including deletion of the flexible loop were generated and characterized kinetically and structurally. Disruption of the hydrogen bonds between the surface loop and the substrate results in significant loss of catalytic activity. Furthermore, structures of these mutants with low catalytic activity have no interpretable electron density for parts of the flexible loop. The structure of the mutant (Delta107-116), in which the flexible loop is deleted, shows only small differences in positions of other substrate binding residues and in the binuclear zinc center compared with the native structure, yet the enzyme has negligible activity. The kinetic and structural analyses suggest that Thr109 and Thr110 in the flexible loop provide productive binding of substrate and stabilize the transition-state intermediate, thereby increasing catalytic activity.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Henrich, S
%A Christopherson, R I
%T Multiple forms of nuclear p53 formed in human Raji and MEC1 cells treated with fludarabine.
%B Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
%D 2007
%C England
%I Nature Publishing Group, Specialist Journals
%V 22
%N 0
%P 657-60
%@ 0887-6924
%X 
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Leong, Sharon
%A Christopherson, Richard
%A Baxter, Robert
%T Profiling of Apoptotic Changes in Human Breast Cancer Cells Using SELDI-TOF Mass Spectrometry.
%B Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
%D 2007
%C Switzerland
%I S. Karger
%V 20
%N 5
%P 579-90
%@ 1015-8987
%X Apoptosis is a key process in the response of tumours to chemotherapeutic agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many tumor cells, while sparing most normal cells. Several chemotherapeutic drugs synergize with TRAIL in reducing tumor growth and inducing apoptosis. Because some tumour cells respond poorly to these treatments, biomarkers that predict clinical responsiveness are needed. This study used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify novel apoptotic markers in TRAIL and etoposide (T+E)-treated MDA-MB-231 and ZR-75-1 breast cancer cells and MCF-10A non-transformed breast cells. T+E induced apoptosis, increasing caspase-3 activity at 4-8h, in all cell lines. Protein profiles revealed two prominent peaks, m/z 10090 and 8560, which decreased significantly during apoptosis. Mass spectrometry sequencing of tryptic peptides identified these proteins as S100A6 (confirmed immunologically) and ubiquitin (confirmed against a purified standard), respectively. Caspase inhibition prevented the decrease in both proteins during T+E-induced apoptosis whereas proteasome inhibition combined with T+E further decreased ubiquitin, possibly by preventing its recycling. Using SELDI-TOF MS we have identified S100A6 and ubiquitin as potential protein markers of apoptosis. Further validation using patient samples is required to confirm their potential utility in monitoring the effectiveness of anti-cancer drugs in inducing tumour cell apoptosis. Copyright (c) 2007 S. Karger AG, Basel.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Lee, Mihwa
%A Chan, Camilla W
%A Graham, Stephen C
%A Christopherson, Richard I
%A Guss, J Mitchell
%A Maher, Megan J
%T Structures of Ligand-free and Inhibitor Complexes of Dihydroorotase from Escherichia coli: Implications for Loop Movement in Inhibitor Design.
%B Journal of molecular biology
%D 2007
%C United Kingdom
%I Academic Press Ltd Elsevier Science Ltd
%V 370
%N 5
%P 812-825
%@ 1089-8638
%X Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-l-aspartate (CA-asp) to l-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. DHOase is a potential anti-malarial drug target as malarial parasites can only synthesize pyrimidines via the de novo pathway and do not possess a salvage pathway. Here we report the structures of Escherichia coli DHOase crystallized without ligand (1.7 A resolution) and in the presence of the inhibitors 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate (HDDP; 2.0 A) and 5-fluoroorotate (FOA, 2.2 A). These are the first crystal structures of DHOase-inhibitor complexes, providing structural information on the mode of inhibitor binding. HDDP possesses features of both the substrate and product, and ligates the Zn atoms in the active site. In addition, HDDP forms hydrogen bonds to the flexible loop (residues 105-115) stabilizing the "loop-in" conformation of the flexible loop normally associated with the presence of CA-asp in the active site. By contrast, FOA, a product-like inhibitor, binds to the active site in a similar fashion to DHO but does not ligate the Zn atoms directly nor stabilize the loop-in conformation. These structures define the necessary features for the future design of improved inhibitors of DHOase.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Henrich, Silke
%A Cordwell, Stuart J
%A Crossett, Ben
%A Baker, Mark S
%A Christopherson, Richard I
%T The nuclear proteome and DNA-binding fraction of human Raji lymphoma cells.
%B Biochimica et biophysica acta
%D 2007
%C Netherlands
%I Elsevier BV
%V 1774
%N 4
%P 413-432
%@ 0006-3002
%X Purification of organelles and analysis of their proteins is an important initial step for biological proteomics, simplifying the proteome prior to analysis by established techniques such as two-dimensional liquid chromatography (2-DLC) or two-dimensional gel electrophoresis (2-DE). Nuclear proteins play a central role in regulating gene expression, but are often under-represented in proteomic studies due to their lower abundance in comparison to cellular ''housekeeping'' metabolic enzymes and structural proteins. A reliable procedure for separation and proteomic analysis of nuclear proteins would be useful for investigations of cell proliferation and differentiation during disease processes (e.g., human cancer). In this study, we have purified nuclei from the human Burkitt''s lymphoma B-cell line, Raji, using sucrose density gradient centrifugation. The integrity and purity of the nuclei were assessed by light microscopy and proteins from the nuclear fractions were separated by 2-DE and identified using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). A total of 124 unique proteins were identified, of which 91% (n=110) were predicted to be nuclear using PSORT. Proteins from the nuclear fraction were subjected to affinity chromatography on DNA-agarose to isolate DNA-binding proteins. From this purified fraction, 131 unique proteins were identified, of which 69% (n=90) were known or predicted DNA-binding proteins. Purification of nuclei and subsequent enrichment of DNA-binding proteins allowed identification of a total of 209 unique proteins, many involved in transcription and/or correlated with lymphoma, leukemia or cancer in general. The data obtained should be valuable for identification of biomarkers and targets for cancer therapy, and for furthering our understanding of the molecular mechanisms underlying lymphoma development and progression.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Anderson, Mark A
%A Cleland, W Wallace
%A Huang, Danny T
%A Chan, Camilla
%A Shojaei, Maryam
%A Christopherson, Richard I
%T 13C and 15N isotope effects for conversion of L-dihydroorotate to N-carbamyl-L-aspartate using dihydroorotase from hamster and Bacillus caldolyticus.
%B Biochemistry
%D 2006
%C 1155 16Th St, Nw, Wa
%I Amer Chemical Soc
%V 45
%N 23
%P 7132-9
%@ 0006-2960
%X In the pyrimidine biosynthetic pathway, N-carbamyl-L-aspartate (CA-asp) is converted to L-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 degrees C for hamster DHOase; 37, 50, and 60 degrees C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the beta-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO --> CA-asp have also been measured (secondary 13K(eq) = 1.0028 +/- 0.0002, primary 13K(eq) = 1.0053 +/- 0.0003, primary 15K(eq) = 1.0027 +/- 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the physiological reaction (CA-asp --> DHO) have been calculated. These values indicate that the carbon of the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the physiological reaction. It appears that the ring opening and closing is the slow step of the reaction.
%Z FOR Codes:


%0 Journal Article
%~ PubMed
%A Belov, Larissa
%A Mulligan, Stephen P
%A Barber, Nicole
%A Woolfson, Adrian
%A Scott, Mike
%A Stoner, Kerryn
%A Chrisp, Jeremy S
%A Sewell, William A
%A Bradstock, Kenneth F
%A Bendall, Linda
%A Pascovici, Dana S
%A Thomas, Mervyn
%A Erber, Wendy
%A Huang, Pauline
%A Sartor, Mary
%A Young, Graham A R
%A Wiley, James S
%A Juneja, Surender
%A Wierda, William G
%A Green, Anthony R
%A Keating, Michael J
%A Christopherson, Richard I
%T Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray.
%B British journal of haematology
%D 2006
%C United Kingdom
%I Blackwell Publishing Ltd.
%V 135
%N 2
%P 184-97
%@ 0007-1048
%X A novel antibody microarray has been developed that provides an extensive immunophenotype of leukaemia cells. The assay is a solid phase cell-capture technique in which 82 antigens are studied simultaneously. This paper presents the analysis of 733 patients with a variety of leukaemias and lymphomas from peripheral blood and bone marrow. Discriminant Function Analysis of the expression profiles from these 733 patients and 63 normal subjects were clustered and showed high levels of consistency with diagnoses obtained using conventional clinical and laboratory criteria. The overall levels of consensus for classification using the microarray compared with established criteria were 93.9% (495/527 patients) for peripheral blood and 97.6% (201/206 patients) for bone marrow aspirates, showing that the extensive phenotype alone was frequently able to classify the disease when the leukaemic clone was the dominant cell population present. Immunophenotypes for neoplastic cells were distinguishable from normal cells when the leukaemic cell count was at least 5 x 10(9) cells/l in peripheral blood, or 20% of cells obtained from bone marrow aspirates. This technique may be a useful adjunct to flow cytometry and other methods when an extensive phenotype of the leukaemia cell is desired for clinical trials, research and prognostic factor analysis.
%Z FOR Codes:


%0 Journal Article
%~ PubMed
%A Christopherson, Richard I
%A Stoner, Kerryn
%A Barber, Nicole
%A Belov, Larissa
%A Woolfson, Adrian
%A Scott, Mike
%A Bendall, Linda
%A Mulligan, Stephen P
%T Classification of AML using a monoclonal antibody microarray.
%B Methods in molecular medicine
%D 2006
%C USA
%I Humana Press, Inc.
%V 125
%N 
%P 241-51
%@ 1543-1894
%X A cluster of differentiation (CD) antibody microarray called the DotScan microarray has been developed that enables an extensive immunophenotype to be obtained for a suspension of leukocytes in a single analysis. For a leukemia with a leukemia count of greater than 10 x 10(9)/L, the immunophenotype obtained is essentially that of the leukemic clone. The antibody microarray is printed as microscopic (10 nL) dots on a nitrocellulose film on a microscope slide. Cells are captured by the immobilized antibodies and a dot pattern is recorded with an optical array reader giving the immunophenotype of the leukemia. Procedures are being developed that should enable diagnosis of myeloid leukemias by comparison of the dot pattern obtained from an unknown blood sample with a library of consensus patterns for the common leukemias.
%Z FOR Codes:


%0 Journal Article
%~ PubMed
%A Ellmark, Peter
%A Belov, Larissa
%A Huang, Pauline
%A Lee, C Soon
%A Solomon, Michael J
%A Morgan, Daniel K
%A Christopherson, Richard I
%T Multiplex detection of surface molecules on colorectal cancers.
%B Proteomics
%D 2006
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 6
%N 6
%P 1791-802
%@ 1615-9853
%X A technique of fluorescence multiplexing is described for analysis of the plasma membrane proteome of colorectal cancer cells from surgically resected specimens, enabling detection and immunophenotyping when the cancer cells are in the minority. A single-cell suspension was prepared from a colorectal tumour, and the mixed population of cells was captured on a CD antibody microarray. The cancer cells were detected using a fluorescently tagged antibody for carcinoembryonic antigen (CEA-Alexa647) or epithelial cell adhesion marker (EpCAM-Alexa488). Using this multiplexing procedure, dot patterns from colorectal cancers were distinct from those of adjacent normal tissue. Subtraction of the expression levels for each antigen from normal tissue from those for the cancer shows differential expression in the cancer of CD66c, CD15s, CD55, CD45, CD71, CD45RO, CD11b and CEA, in descending order. Cells captured on the same microarray were also labelled with fluorescent CD3-phycoerythrin antibody revealing the presence of tumour-infiltrating lymphocytes. The immunophenotypes of T lymphocytes from the tumour samples showed differential expression of HLA-DR, TCR alpha/beta, CD49d, CD52, CD49e, CD5, CD95, CD28, CD38 and CD71, in descending order. Fluorescence multiplexing of mixed cell populations captured on a single antibody microarray enables expression profiling of multiple sub-populations of cells within a tumour sample.
%Z FOR Codes: 111201


%0 Journal Article
%~ PubMed
%A Woolfson, Adrian
%A Ellmark, Peter
%A Chrisp, Jeremy S
%A A Scott, Mike
%A Christopherson, Richard I
%T The application of CD antigen proteomics to pharmacogenomics.
%B Pharmacogenomics
%D 2006
%C LONDON, ENGLAND
%I Future Medicine Ltd
%V 7
%N 5
%P 759-771
%@ 1462-2416
%X The advent of multiplexing technologies has raised the possibility that disease states can be defined using discrete genomic and proteomic patterns or signatures. However, this emerging area has been limited by the ''content problem'', arising from the uncertainty of which molecules to focus on. The human cluster of differentiation (CD) antigens are expressed on cells of the human immune system (leukocytes) and on other cell types. These heterogeneous molecules perform a host of roles essential to immune function and to the physiology of other lineages. The 339 defined CD antigens and their, as yet, undefined counterparts constitute key components of the expressed human cell surface proteome. We propose that CD antigen expression patterns will form the basis of a rational, discrete and generalized diagnostic and prognostic system. Furthermore, disease-specific CD antigen proteomic signatures are likely to be more robust than corresponding genomic signatures and will also help to identify molecular targets for therapeutic intervention.
%Z FOR Codes:


%0 Journal Article
%~ PubMed
%A Huang, Danny T
%A Kaplan, Jacob
%A Menz, R Ian
%A Katis, Vittorio L
%A Wake, R Gerry
%A Zhao, Feng
%A Wolfenden, Richard
%A Christopherson, Richard I
%T Thermodynamic Analysis of Catalysis by the Dihydroorotases from Hamster and Bacillus caldolyticus, As Compared with the Uncatalyzed Reaction.
%B Biochemistry
%D 2006
%C 1155 16Th St, Nw, Wa
%I Amer Chemical Soc
%V 45
%N 27
%P 8275-83
%@ 0006-2960
%X Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticushas been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher V(max) and K(s) values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of l-dihydroorotate were determined at 25 degrees C for hamster DHOase (DeltaG = -6.9 kcal/mol, DeltaH = -11.5 kcal/mol, TDeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (DeltaG = -5.6 kcal/mol, DeltaH = -4.2 kcal/mol, TDeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the k(cat)/K(s) value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(), making k(cat)/K(s) almost temperature independent. The pseudo-first-order rate constant for water attack on l-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-)(11) s(-)(1) at 25 degrees C, with DeltaH() = 24.7 kcal/mol and TDeltaS() = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (DeltaDeltaH() = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.
%Z FOR Codes: