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In JoVE (1)
- Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing
Other Publications (36)
- Accounts of Chemical Research
- Drug Discovery Today
- Acta Crystallographica. Section D, Biological Crystallography
- Leukemia Research
- Journal of Molecular Biology
- Leukemia Research
- The Journal of Investigative Dermatology
- Journal of Immunological Methods
- Methods in Molecular Medicine
- British Journal of Haematology
- Biochimica Et Biophysica Acta
- Journal of Molecular Biology
- Biochimica Et Biophysica Acta
- Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology
- Proteomics. Clinical Applications
- Methods in Molecular Biology (Clifton, N.J.)
- Leukemia Research
- FEBS Letters
- Current Opinion in Molecular Therapeutics
- Journal of Immunological Methods
- Journal of Immunological Methods
- International Journal of Molecular Sciences
- International Journal of Proteomics
- Leukemia & Lymphoma
- Journal of Proteome Research
- Journal of Proteome Research
- Leukemia & Lymphoma
- Journal of Proteomics
Articles by Richard I. Christopherson in JoVE
Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing
Jerry Zhou1, Larissa Belov1, Michael J. Solomon2, Charles Chan3, Stephen J. Clarke4, Richard I. Christopherson1
1School of Molecular Bioscience, University of Sydney, 2Department of Surgery, Royal Prince Alfred Hospital, 3Department of Anatomical Pathology, Department of Anatomical Pathology, 4Department of Medicine, Concord Repatriation General Hospital
We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.
Other articles by Richard I. Christopherson on PubMed
Accounts of Chemical Research. Nov, 2002 | Pubmed ID: 12437321
Potent inhibitors of enzymes catalyzing reactions in the de novo pathways for biosynthesis of purine and pyrimidine nucleotides are synthetic or natural-product analogues of pathway intermediates or, more recently, inhibitors rationally designed from a knowledge of the catalytic mechanism. Such inhibitors may be effective drugs against cancer, inflammatory disorders, or various infections. For human cancer, the purine pathway may be a better target for inhibition than the pyrimidine pathway, where toxic side effects are more apparent. Drugs such as methotrexate and 6-mercaptopurine have multiple sites of action, making it difficult to quantitatively predict their effects upon cells. Rational design of inhibitors based upon the X-ray structure of the target enzyme has the prospect of yielding drugs with only one site of action in human cells. Such a drug is VX-497, a potent inhibitor of the purine enzyme, IMP dehydrogenase.
Drug Discovery Today. Sep, 2002 | Pubmed ID: 12546881
Protein arrays are now an attractive proposition as they can measure a diverse range of protein interactions not possible with traditional DNA arrays. Antibody arrays are a specific subset of this technology. Originally conceived as multi-analyte detectors, antibody arrays are now used in a wide variety of applications. For instance, the potential of this technology to diagnose human diseases, such as leukemia, breast cancer and, potentially, heart failure, has stimulated much interest. Furthermore, identification of new protein targets in particular disease states will prove to be an invaluable tool in drug discovery and development. Patient prognosis and treatment are also potential applications of the technology. Antibody arrays have proved to be dynamic in response to these broad range of possibilities. This review examines variations in antibody array design and discusses current and potential applications of this novel and interesting technology.
Acta Crystallographica. Section D, Biological Crystallography. Feb, 2003 | Pubmed ID: 12554960
Dihydroorotase (DHOase) catalyses the formation of L-dihydroorotate (DHO) in the de novo pyrimidine biosynthetic pathway. The type I DHOase domain from hamster forms part of the trifunctional enzyme CAD. The hamster DHOase domain has been cloned and expressed in Escherichia coli. Solutions of the homodimeric protein convert to a homotetrameric species when incubated at ambient temperature. Formation of the tetrameric species is mediated via disulfide linkages between single free cysteine residues on the surface of each monomer. This process is also observed under conditions used for crystallization of the hamster DHOase domain; crystals composed exclusively of the tetrameric species grow from solutions containing as little as 10% tetramer. The crystallization of pure tetrameric DHOase results in two crystal forms: form I, with space group C222(1) and unit-cell parameters a = 127.1, b = 603.5, c = 144.7 A, and form II, with space group P2(1) and unit-cell parameters a = 260.5, b = 148.2, c = 308.0 A, beta = 102.2 degrees. Data have been recorded to 4.3 and 4.0 A resolution, respectively.
Proteomics. Nov, 2003 | Pubmed ID: 14595814
We have previously described a microarray of cluster of differentiation (CD) antibodies that enables concurrent determination of more than 60 CD antigens on leukocytes. This procedure does not require protein purification or labeling, or a secondary detection system. Whole cells are captured by a microarray of 10 nL antibody dots immobilized on a nitrocellulose film on a microscope slide. Distinct patterns of cell binding are observed for different leukemias or lymphomas. These haematological malignancies arise from precursor cells of T- or B-lymphocytic, or myeloid lineages of hematopoiesis. The dot patterns obtained from patients are distinct from those of peripheral blood leukocytes from normal subjects. This microarray technology has recently undergone a number of refinements. The microarray now contains more CD antibodies, and a scanner for imaging dot patterns and software for data analysis provide an extensive immunophenotype sufficient for diagnosis of common leukemias. The technology is being evaluated for diagnosis of leukemias with parallel use of conventional diagnostic criteria.
Metabolic Response Patterns of Nucleotides in B-cell Chronic Lymphocytic Leukaemias to Cladribine, Fludarabine and Deoxycoformycin
Leukemia Research. Jul, 2004 | Pubmed ID: 15158094
Nucleotides (NTPs and dNTPs) have been measured in patient-derived chronic lymphocytic leukaemia (CLL) cells stimulated with TPA plus ionomycin and then exposed to cladribine, fludarabine or deoxycoformycin (1 microM, 16 h). dNTPs were not measurable in 10(8) unstimulated CLL cells which are quiescent. Time-dependent changes in nucleotides in CLL cells showed that the mechanism of action changed as the drug triphosphate accumulated. dCf induced substantial accumulation of dATP in CLL cells in culture, and similar levels of dATP in the same patient during subsequent treatment with dCf. Determination of "metabolic response patterns" of nucleotides in CLL cells treated with drugs might distinguish patients with susceptible and refractory CLL prior to chemotherapy.
Journal of Molecular Biology. May, 2005 | Pubmed ID: 15826651
Escherichia coli dihydroorotase has been crystallized in the presence of the product, L-dihydroorotate (L-DHO), and the structure refined at 1.9A resolution. The structure confirms that previously reported (PDB entry 1J79), crystallized in the presence of the substrate N-carbamyl-D,L-aspartate (D, L-CA-asp), which had a dimer in the asymmetric unit, with one subunit having the substrate, L-CA-asp bound at the active site and the other having L-DHO. Importantly, no explanation for the unusual structure was given. Our results now show that a loop comprised of residues 105-115 has different conformations in the two subunits. In the case of the L-CA-asp-bound subunit, this loop reaches in toward the active site and makes hydrogen-bonding contact with the bound substrate molecule. For the L-DHO-bound subunit, the loop faces in the opposite direction and forms part of the surface of the protein. Analysis of the kinetics for conversion of L-DHO to L-CA-asp at low concentrations of L-DHO shows positive cooperativity with a Hill coefficient n=1.57(+/-0.13). Communication between subunits in the dimer may occur via cooperative conformational changes of the side-chains of a tripeptide from each subunit: Arg256-His257-Arg258, near the subunit interface.
Blood. Aug, 2005 | Pubmed ID: 15827132
Cluster of differentiation (CD) antigens are expressed on cells of myeloid and lymphoid lineages. As most disease processes involve immune system activation or suppression, these antigens offer unique opportunities for monitoring host responses. Immunophenotyping using limited numbers of CD antigens enables differentiation states of immune system cells to be determined. Extended phenotyping involving parallel measurement of multiple CD antigens may help identify expression pattern signatures associated with specific disease states. To explore this possibility we have made a CD monoclonal antibody array and scanner, enabling the parallel immunophenotyping of leukocyte cell suspensions in a single and rapid analysis. To demonstrate this approach, we used the specific example of patients infected with human immunodeficiency virus type-1 (HIV-1). An invariant HIV-induced CD antigen signature has been defined that is both robust and independent of clinical outcome, composed of a unique profile of CD antigen expression levels that are both increased and decreased relative to internal controls. The results indicate that HIV-induced changes in CD antigen expression are disease specific and independent of outcome. Their invariant nature indicates an irreversible component to retroviral infection and suggests the utility of CD antigen expression patterns in other disease settings.
Immunophenotypic Changes Induced on Human HL60 Leukaemia Cells by 1alpha,25-dihydroxyvitamin D3 and 12-O-tetradecanoyl Phorbol-13-acetate
Leukemia Research. Oct, 2005 | Pubmed ID: 16111532
1alpha,25-Dihydroxyvitamin D3 (1,25D3) induces HL60 cells to acquire a monocyte-like phenotype, while cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) resemble macrophages. Using a microarray of 82 CD antibodies, 24 cluster of differentiation (CD) antigens were detected on HL60 cells. 1,25D3 induced the following antigens in decreasing order of the change: CD14, CD11c, CD11b, CD54, CD86, CD38 and CD66c, with repression of CD117, CD71, CD95, CD45 and CD64. TPA induced the following antigens in decreasing order of the change: CD11c, CD9, CD11b, CD54, CD38, CD45RO and CD66c, with repression of CD4, CD117, CD95, CD71 and CD64. The results presented provide a basis for monitoring differentiation therapy of myeloid leukaemias in patients.
The Journal of Investigative Dermatology. Sep, 2005 | Pubmed ID: 16117789
Monocyte-derived dendritic cells (Mo-DC) express functional P2X7 receptors; however, the expression of these receptors on tissue-derived dendritic cells including epidermal Langerhans cells (LC) is unknown. Using immunolabeling and flow cytometry, we demonstrated that P2X7 was present on both human epidermal LC and monocyte-derived LC (Mo-LC), as well as on human keratinocytes. The ecto-ATPDase (CD39) was also present on LC, but not keratinocytes. The P2X7 agonists, 2'- and 3'-0(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) or ATP, but neither adenosine 5'-diphosphate (ADP) nor uridine 5'-triphosphate (UTP), induced ethidium+ uptake into these cells. Furthermore, ATP-induced ethidium+ uptake into epidermal LC, Mo-LC and keratinocytes was inhibited by the specific P2X7 antagonist, KN-62 (1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine). ATP-induced ethidium+ uptake into Mo-LC and Mo-DC was 2- and 3-fold greater, respectively, than that for fresh monocytes. P2X7 activation on LC induced downstream signaling events, as BzATP or ATP, but neither ADP nor UTP, induced shedding of the low-affinity receptor for IgE (CD23) from Mo-LC. This process was inhibited by KN-62. Finally, ATP-induced ethidium+ uptake and CD23 shedding were impaired in Mo-LC obtained from subjects homozygous for the loss-of-function Glu-496 to Ala polymorphism in the P2X7 receptor. These results demonstrate that human LC express functional P2X7 receptors, and suggest a role for this receptor in the skin immune system.
Screening Microarrays of Novel Monoclonal Antibodies for Binding to T-, B- and Myeloid Leukaemia Cells
Journal of Immunological Methods. Oct, 2005 | Pubmed ID: 16125720
We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.
Proteomics. Mar, 2006 | Pubmed ID: 16485257
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.
Methods in Molecular Medicine. 2006 | Pubmed ID: 16502589
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.
13C and 15N Isotope Effects for Conversion of L-dihydroorotate to N-carbamyl-L-aspartate Using Dihydroorotase from Hamster and Bacillus Caldolyticus
Biochemistry. Jun, 2006 | Pubmed ID: 16752903
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.
Thermodynamic Analysis of Catalysis by the Dihydroorotases from Hamster and Bacillus Caldolyticus, As Compared with the Uncatalyzed Reaction
Biochemistry. Jul, 2006 | Pubmed ID: 16819826
Dihydroorotase (DHOase, EC 188.8.131.52) from the extreme thermophile Bacillus caldolyticus has 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 Vmax and Ks 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 kcat/Ks 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(double dagger), making kcat/Ks 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(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -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 (delta deltaH(double dagger) = -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.
Pharmacogenomics. Jul, 2006 | Pubmed ID: 16886900
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.
Analysis of Human Leukaemias and Lymphomas Using Extensive Immunophenotypes from an Antibody Microarray
British Journal of Haematology. Oct, 2006 | Pubmed ID: 16939496
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.
Biochimica Et Biophysica Acta. Apr, 2007 | Pubmed ID: 17369005
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.
Structures of Ligand-free and Inhibitor Complexes of Dihydroorotase from Escherichia Coli: Implications for Loop Movement in Inhibitor Design
Journal of Molecular Biology. Jul, 2007 | Pubmed ID: 17550785
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.
Differentially Expressed Cytosolic Proteins in Human Leukemia and Lymphoma Cell Lines Correlate with Lineages and Functions
Biochimica Et Biophysica Acta. Sep, 2007 | Pubmed ID: 17698427
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.
Kinetic and Structural Analysis of Mutant Escherichia Coli Dihydroorotases: a Flexible Loop Stabilizes the Transition State
Biochemistry. Sep, 2007 | Pubmed ID: 17711307
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.
Cellular Physiology and Biochemistry : International Journal of Experimental Cellular Physiology, Biochemistry, and Pharmacology. 2007 | Pubmed ID: 17762184
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.
Differentially Expressed Nuclear Proteins in Human CCRF-CEM, HL-60, MEC-1 and Raji Cells Correlate with Cellular Properties
Proteomics. Clinical Applications. Oct, 2007 | Pubmed ID: 21136623
The human cell lines CCRF-CEM (T-cell acute lymphocytic leukemia), HL-60 (acute myeloid leukemia), MEC-1 (B-cell chronic lymphocytic leukemia) and Raji (Burkitt's B-cell lymphoma) have been analysed for differences in their nuclear proteomes. Using 2-D DIGE, 55 nuclear proteins have been identified that are differentially expressed (p<0.025) between the four cell lines, including proteins associated with transcription, proliferation, DNA repair and apoptosis. Of these 55 proteins, 22 were over-expressed in just one cell line, and four were down-regulated in one cell line. Proteins uniquely over-expressed between myeloid and lymphoid cell lines include those that may have use as markers for diagnosis, disease progression and B-cell maturation and differentiation. Expression of various proliferation-associated nuclear proteins correlated with relative growth rates of the cell lines, giving these proteins potential diagnostic applications for distinction of chronic versus acute subtypes of haematological malignancies. Identification of these differentially expressed nuclear proteins should facilitate elucidation of the molecular mechanisms underlying leukocyte differentiation and transformation to leukemias and lymphomas. The nuclear expression profiles should enable classification of subtypes of leukemia, and identify potential nuclear protein targets for development of diagnostic and therapeutic strategies.
Biochemistry. Mar, 2008 | Pubmed ID: 18303855
Orotidine 5'-monophosphate (OMP) decarboxylase from Plasmodium falciparum (PfODCase, EC 184.108.40.206) 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.
The Applicability of a Cluster of Differentiation Monoclonal Antibody Microarray to the Diagnosis of Human Disease
Methods in Molecular Biology (Clifton, N.J.). 2008 | Pubmed ID: 18370105
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.
All-trans Retinoic Acid Induces Different Immunophenotypic Changes on Human HL60 and NB4 Myeloid Leukaemias
Leukemia Research. Feb, 2008 | Pubmed ID: 17561254
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.
FEBS Letters. Jun, 2009 | Pubmed ID: 19298816
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.
Current Opinion in Molecular Therapeutics. Dec, 2009 | Pubmed ID: 20072937
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.
Surface Antigen Profiling of Colorectal Cancer Using Antibody Microarrays with Fluorescence Multiplexing
Journal of Immunological Methods. Apr, 2010 | Pubmed ID: 20156443
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.
Journal of Immunological Methods. Jun, 2010 | Pubmed ID: 20363224
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.
International Journal of Molecular Sciences. 2010 | Pubmed ID: 21339979
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.
Cladribine and Fludarabine Nucleoside Change the Levels of CD Antigens on B-Lymphoproliferative Disorders
International Journal of Proteomics. 2010 | Pubmed ID: 22084681
The purine analogs, fludarabine nucleoside (FdA), and cladribine (CdA) (1 μM, 24 hours), significantly changed the levels of some surface antigens on the human B-cell lines MEC2 and Raji. Changes in the surface proteins were identified using a Cluster of Differentiation (CD) antibody microarray that captures live cells and confirmed by flow cytometry. For Raji cells, CdA up-regulated CD10, CD54, CD80, and CD86, with repression of CD22, while FdA up-regulated CD20, CD54, CD80, CD86 and CD95. For MEC2 cells, CdA up-regulated CD11a, CD20, CD43, CD45, CD52, CD54, CD62L, CD80, CD86, and CD95, but FdA had no effect. Up-regulation of particular CD antigens induced on a B-cell lymphoproliferative disorder by a purine analog could provide targets for therapeutic antibodies with synergistic cell killing.
Leukemia & Lymphoma. Oct, 2011 | Pubmed ID: 22023531
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.
Journal of Proteome Research. Mar, 2011 | Pubmed ID: 21182289
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.
Journal of Proteome Research. Feb, 2012 | Pubmed ID: 22133146
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.
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
Leukemia & Lymphoma. Jan, 2012 | Pubmed ID: 22149137
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.
The Phosphoinositide 3-kinase Inhibitor LY294002, Decreases Aminoacyl-tRNA Synthetases, Chaperones and Glycolytic Enzymes in Human HT-29 Colorectal Cancer Cells
Journal of Proteomics. Feb, 2012 | Pubmed ID: 22172953
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 (10μM, 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-α, 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.