[Identification of VHL-associated changes in clear cell renal carcinoma: the application of combined genome and expression analyses]. / Identifizierung VHL-assoziierter Veränderungen im klarzelligen Nierenzellkarzinom: Anwendung von kombinierten Genom- und Expressionsanalysen.

Sporadic renal cell carcinoma (RCC) represents a heterogeneous tumor, which is traditionally classified into subtypes based on morphological criteria. In recent years high-throughput molecular analyses have been able to identify genomic and proteomic alterations in tumor cells. These markers are the basis for a molecular classification of RCC and bear prognostic value. However, an isolated consideration of genomic and proteomic alterations prevents deeper insights into the complex processes of carcinogenesis. Here we summarize recent studies focussing on this aspect of RCC and present a systems biology concept for the identification of novel tumor markers. These could be applied to improve future diagnosis and therapy of RCC.

Immunophenotyping without antibodies. New perspectives for lymphoma characterization.

AIMS: Accurate classification of haematological malignancies is a prerequisite for their correct diagnosis, prognosis and therapy. Clear classification of lymphomas is often hindered by the limited number of available cell surface protein markers that are suitable for immunophenotyping. A systematic and quantitative analysis of cell surface proteins is thus required to identify new protein markers on lymphoma subtypes in an unbiased and discovery-driven approach. METHODS: Nine Hodgkin and non-Hodgkin B cell lines of diffuse large cell type and mediastinal type were investigated by cell surface capture (CSC) technology, a mass spectrometry-based method to identify cell surface glycoproteins. Selected proteins are verified by antibody-based methods, including flow cytometry and immunohistochemistry on cell line arrays. RESULTS: A total of 747 predicted transmembrane proteins were identified from all cell lines, including 142 CD (cluster of differentiation) annotated proteins. A group of differentially expressed cell surface glycoproteins between Hodgkin and non-Hodgkin B cell lines was revealed via quantitative CSC technology. In addition to classical and expected CD molecules such as CD20 and CD30, less frequently expressed molecules such as CD2 on Hodgkin lymphoma (HL) cell lines were identified by CSC and verified by immunohistochemistry in cell lines and primary lymphoma tissue. A panel of CSC-identified differentiation glycoprotein candidates is currently under investigation on tissue microarrays (TMAs) from patient samples.

Association of SLP-65/BLNK with the B cell antigen receptor through a non-ITAM tyrosine of Ig-alpha.

The cytoplasmic adaptor protein SLP-65 (BLNK or BASH) is a critical downstream effector of the B cell antigen receptor (BCR). Tyrosine-phosphorylated SLP-65 assembles intracellular signaling complexes such as the Ca(2 +) initiation complex encompassing phospholipase C-gamma2 and Bruton's tyrosine kinase. It is, however, unclear how the SLP-65 signaling module can be recruited to the plasma membrane. Here we show that following B cell stimulation, SLP-65 associates directly with the BCR signaling subunit, the Ig-alpha / Ig-beta heterodimer. The interaction is mediated by the Src homology 2 domain of SLP-65 and the phosphorylated Ig-alpha tyrosine 204, which is located outside of the immunoreceptor tyrosine-based activation motif. Our data identify an unexpected BCR phosphorylation pattern and indicate that Ig-alpha has the capability to serve as transmembrane adaptor in BCR signaling.

Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65.

During signal transduction through the B cell antigen receptor (BCR), several signaling elements are brought together by the adaptor protein SLP-65. We have investigated the role of SLP-65 in B cell maturation and function in mice deficient for SLP-65. While the mice are viable, B cell development is affected at several stages. SLP-65-deficient mice show increased proportions of pre-B cells in the bone marrow and immature B cells in peripheral lymphoid organs. B1 B cells are lacking. The mice show lower IgM and IgG3 serum titers and poor IgM but normal IgG immune responses. Mutant B cells show reduced Ca2+ mobilization and reduced proliferative responses to B cell mitogens. We conclude that while playing an important role, SLP-65 is not always required for signaling from the BCR.

Characterization of the B cell-specific adaptor SLP-65 and other protein tyrosine kinase substrates by two-dimensional gel electrophoresis.

The identification of substrates for protein tyrosine kinases in B cells is a critical step to a better understanding of the molecular mechanism(s) of lymphocyte activation through the antigen receptor. The substrate proteins were immunopurified from stimulated B cells and separated by two-dimensional gel electrophoresis techniques using either the isoelectric focussing (IEF)/SDS-PAGE or the non-equilibrium PH gradient electrophoresis (NEPHGE)/SDS-PAGE method. The biochemical characteristics of the proteins (isoelectric point and relative molecular mass) obtained and the subsequent use of antibodies that are specific for different cellular proteins confirmed the participation of HS1, Vav, Ig-alpha, Lyn and Btk in antigen receptor-mediated signal transduction. The heat shock cognate protein HSC70 was identified as a novel substrate protein in activated B cells. An important signaling function has previously been suggested for a 65-kDa protein (p65), whose phosphorylation can be detected before that of other substrate proteins. The analysis identified p65 as a so far unknown protein. Based on p65 peptide sequences, the full length cDNA was isolated and found to encode a B cell-specific adaptor protein, called SLP-65.

SH3P7 is a cytoskeleton adapter protein and is coupled to signal transduction from lymphocyte antigen receptors.

Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.

SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-alpha/Ig-beta heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H2O2. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H2O2-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.