High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients.To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p < 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation.In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study.Acute leukemia (AL)¹ is the most common childhood cancer, accounting for a quarter of all pediatric malignancies (1). Accumulated chromosomal translocations and mutations in proto-oncogenes alter proliferation, differentiation, apoptosis and death in developing hematogones, ultimately leading to the development of leukemia (2, 3). The most recent understanding of these cancer-related changes is based on molecular genetic studies that focused primarily on DNA and mRNA alterations. High-throughput molecular genetic technologies, such as mRNA expression profiling and next generation sequencing, are widely used in clinical research. These techniques can provide new classification schemes, define new prognostic subgroups and outline the background of some pathological mechanisms (2, 4, 5, 6, 7) but they cannot easily elucidate the functional consequences at the cellular level. Proteins are the principal carriers of cellular functions. Thus, the analysis of proteins and protein modifications can elucidate the pathological mechanisms of leukemia or clarify the response mechanisms to current and emerging therapies. Currently, flow cytometry is used in clinical laboratories to analyze dozens of proteins that are expressed by leukemic cells (8, 9). These proteins, which are mostly surface CD markers, can reflect lineage commitment, developmental status and even the underlying genetic lesion (10, 11) but they do not carry information about the intracellular processes that control malignant transformation. Moreover, many cancer alterations are manifested only at the functional level, including changes in subcellular localization, post-translational modification (e.g. phosphorylation), protein cleavage, or protein–protein interactions (12). Proteomic techniques that can capture disease-associated changes are needed. Mass spectrometry (MS) is presently the technique of choice for large-scale proteomic analysis. MS can uncover thousands of molecules without an a priori probe selection, e.g. new disease-associated features in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) (13, 14). Despite its tremendous analytical power, MS is complex and not widely accessible. Unlike MS, affinity proteomics is a simple technology suitable for large-scale protein analysis in primary cancer samples in the clinical laboratories. Recently, a technique linking size exclusion chromatography (SEC) to microsphere-based antibody arrays (microsphere-based affinity proteomics (MAP)) has been developed (15, 16). SEC-MAP enables the detection of hundreds of proteins in a single sample and provides essential information about protein size. Because only five to ten million cells are necessary, SEC-MAP can serve as a sensitive, sample-sparing and high-content tool for protein profiling in leukemia samples probing the relative amounts of different proteins, as well as protein size and cleavage (17). Our in-house-assembled MAP array is a set of 1152 populations of fluorescent-labeled microbeads, each carrying an antibody against a single human antigen. Native cellular proteins (and their complexes) are isolated from cellular compartments using detergents, labeled with biotin (biotin-PEO4-NHS) and subjected to SEC to obtain 24 size fractions. The SEC fractions are incubated with MAP microbeads, and antibody-protein binding is detected using phycoerythrin (PE)-labeled streptavidin with flow cytometry. The flow cytometer resolves the color code of each microbead population and reads the amount of bound protein. The data from 24 SEC fractions are combined, and a protein''s binding relative to its size is detected as a “protein entity.” Data are analyzed with in-house R-based software. This approach permits automatic batch processing of raw flow cytometry standard (FCS) files in addition to advanced analyses including quality control steps (the minimal number of microspheres required in a population and the unimodality of the signal in the PE channel is checked) (17). We wanted to find out whether SEC-MAP can be used in the clinical laboratory to bring a biologically important information, e.g. to classify acute leukemias or to find the marker with a prognostic relevance. We assembled MAP arrays to carry antibodies against proteins that are known to be important for leukemia diagnostics (18, 9) and against components of intracellular signaling networks (16). Through extensive testing on leukemia samples, we have identified antibodies that are suitable for immunoprecipitation-based techniques. Furthermore, we have improved the software tools to allow for large-scale data normalization, fast automatic protein entity detection with manual correction, and the discovery of differentially expressed entities in multiple samples. Using innovative software tools, we have identified entities that were differentially expressed between particular AL subgroups. To ensure the specificity we have validated the data collected by SEC-MAP with classical flow cytometry-based immunophenotyping (FACS), Western blot (WB) and quantitative real-time PCR (qRT-PCR). Moreover, we have addressed practical sample processing issues related to patient material handling and logistics. Based on the protein size profile, we were able to discriminate proteolytically degraded samples from those with an uncleaved proteome. Importantly, proteolysis would be missed by conventional protein load controls in Western blots. Thus, the SEC-MAP array was demonstrated to be a useful, reproducible and accurate high-content proteomic tool for the assessment of primary leukemia samples.     

Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

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CD36 Protein Influences Myocardial Ca2+ Homeostasis and Phospholipid Metabolism: CONDUCTION ANOMALIES IN CD36-DEFICIENT MICE DURING FASTING*

Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca²⁺ flux and activation of Ca²⁺-dependent phospholipases A₂ that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca²⁺ handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18–22 h) CD36^−/− and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36^−/− mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36^−/− mice documented prolongation of Ca²⁺ transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca²⁺. Hearts of CD36^−/− mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36^−/− hearts. Abnormal Ca²⁺ homeostasis in the CD36^−/− myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A₂ activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca²⁺ homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.     

Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure

CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N6-isopentenyladenine, trans-zeatin, kinetin, p-topolin and N-methyl-isopentenyladenine) were studied. By using stopped-flow analysis of the reductive half-reaction, spectral intermediates were observed indicative of the transient formation of a binary enzyme-product complex between the cytokinin imine and the reduced enzyme. The reduction rate was high for isoprenoid cytokinins that showed formation of a charge-transfer complex of reduced enzyme with bound cytokinin imine. For the other cytokinins, flavin reduction was slow and no charge-transfer intermediates were observed. The binary complex of reduced enzyme and imine product intermediate decays relatively slowly to form an unbound product, cytokinin imine, which accumulates in the reaction mixture. The imine product only very slowly hydrolyses to adenine and an aldehyde derived from the cytokinin N6 side-chain. Mixing of the substrate-reduced enzyme with Cu2+/imidazole as an electron acceptor to monitor the oxidative half-reaction revealed a high rate of electron transfer for this type of electron acceptor when using N6-isopentenyladenine. The stability of the cytokinin imine products allowed their fragmentation analysis and structure assessment by Q-TOF (quadrupole-time-of-flight) MS/MS. Correlations of the kinetic data with the known crystal structure are discussed for reactions with different cytokinins.     

Identification of molecular targets for selective elimination of TRAIL‐resistant leukemia cells. From spots to in vitro assays using TOP15 charts

The resistance of malignant cells to chemotherapy calls for the development of novel anti‐cancer drugs. TNF‐related apoptosis‐inducing ligand (TRAIL) is a pro‐apoptotic cytokine, which selectively induces apoptosis in malignant cells. We derived two TRAIL‐resistant HL‐60 subclones, HL‐60/P1 and HL‐60/P2, from a TRAIL‐sensitive HL‐60 acute promyelocytic leukemia cell line. To identify therapeutically exploitable “weaknesses” of the TRAIL‐resistant leukemia cells that could be used as molecular targets for their elimination, we performed proteomic (2‐DE) analysis and compared both TRAIL‐resistant subclones with the original TRAIL‐sensitive HL‐60 cells. We identified over 40 differentially expressed proteins. To significantly narrow the lists of candidate proteins, we excluded proteins that are known to be often differentially expressed, regardless of experiment type and tissue (the so‐called “TOP15” proteins). Decreased expression of DNA replication and maintenance proteins MCM7 and RPA32 in HL‐60/P1 cells, and the marked down‐regulation of enzyme adenosine deaminase in HL‐60/P2 cells, suggests increased sensitivity of these cells to DNA‐interfering drugs, and adenosine and its homologues, respectively. In a series of in vitro assays, we confirmed the increased toxicity of etoposide and cisplatin to TRAIL resistant HL‐60/P1 cells, and adenosine and vidarabine to HL‐60/P2, compared with TRAIL‐sensitive HL‐60 cells.     

Human MRCKα is regulated by cellular iron levels and interferes with transferrin iron uptake

Myotonic dystrophy kinase-related Cdc42-binding kinase α (MRCKα, formally known as CDC42BPA) is a serine/threonine kinase that can regulate actin/myosin assembly and activity. Recently, it has been shown that it possesses a functional iron responsive element (IRE) in the 3′-untranslated region (UTR) of its mRNA, suggesting that it may be involved in iron metabolism. Here we report that MRCKα protein expression is also regulated by iron levels; MRCKα colocalizes with transferrin (Tf)-loaded transferrin receptors (TfR), and attenuation of MRCKα expression by a short hairpin RNA silencing construct leads to a significant decrease in Tf-mediated iron uptake. Our results thus indicate that MRCKα takes part in Tf-iron uptake, probably via regulation of Tf-TfR endocytosis/endosome trafficking that is dependent on the cellular cytoskeleton. Regulation of the MRCKα activity by intracellular iron levels could thus represent another molecular feedback mechanism cells could use to finely tune iron uptake to actual needs.     

A Subset of Cytokinin Two-component Signaling System Plays a Role in Cold Temperature Stress Response in Arabidopsis

A multistep two-component signaling system is established as a key element of cytokinin signaling in Arabidopsis. Here, we provide evidence for a function of the two-component signaling system in cold stress response in Arabidopsis. Cold significantly induced the expression of a subset of A-type ARR genes and of GUS in Pro_ARR7:GUS transgenic Arabidopsis. AHK2 and AHK3 were found to be primarily involved in mediating cold to express A-type ARRs despite cytokinin deficiency. Cold neither significantly induced AHK2 and AHK3 expression nor altered the cytokinin contents of wild type within the 4 h during which the A-type ARR genes exhibited peak expression in response to cold, indicating that cold might induce ARR expression via the AHK2 and AHK3 proteins without alterations in cytokinin levels. The ahk2 ahk3 and ahk3 ahk4 mutants exhibited enhanced freezing tolerance compared with wild type. These ahk double mutants acclimated as efficiently to cold as did wild type. The overexpression of the cold-inducible ARR7 in Arabidopsis resulted in a hypersensitivity response to freezing temperatures under cold-acclimated conditions. The expression of C-repeat/dehydration-responsive element target genes was not affected by ARR7 overexpression as well as in ahk double mutants. By contrast, the arr7 mutants showed increased freezing tolerance. The ahk2 ahk3 and arr7 mutants showed hypersensitive response to abscisic acid (ABA) for germination, whereas ARR7 overexpression lines exhibited insensitive response to ABA. These results suggest that AHK2 and AHK3 and the cold-inducible A-type ARRs play a negative regulatory role in cold stress signaling via inhibition of ABA response, occurring independently of the cold acclimation pathway.