Immunological systems biology: gene expression analysis of B-cell development in Ramos B-cells.

B-cell development into antibody producing cells is a complex process that relies on the tightly controlled production of hundreds of genes and proteins. A B-cell is activated through the B-cell receptor (BCR) and this activation is modified by different co-stimulatory or inhibitory co-receptors. The concerted action of signals from BCR and from co-receptors decides the fate of the B-cells. The majority of B-cells enter apoptosis, while some of them progress through the cell cycle and become, for example, antibody producing plasma cells. We studied BCR stimulated Ramos B-cells to explore the expression of BCR pathway, cell cycle and apoptosis related genes. We followed, using microarrays, the gene expression for several days after BCR engagement. Several bioinformatics methods were used to investigate the properties and common features of co-expressed genes. Certain gene ontologies have statistically significant enrichment into clusters of similarly expressed genes. The cell signaling pathways and gene expression data were combined to reveal detailed information about biological processes and B-cell systems biology. The results provide knowledge of the development of adaptive immunity and clues about how the pathways are affected by regulation of the expression of genes.

B cells.

B cells are an important component of adaptive immunity. They produce and secrete millions of different antibody molecules, each of which recognizes a different (foreign) antigen. The fact that humans express a very large repertoire of antibodies is due to the complex mechanism of V(D)J recombination of immunoglobulin (Ig) genes as well as other processes including somatic hypermutation, gene conversion and class switching. The B cell receptor (BCR) is an integral membrane protein complex that is composed of two Ig heavy chains, two Ig light chains and two heterodimers of Igalpha and Igbeta. To eliminate foreign antigens, B cells cooperate with other cells of the immune system including macrophages, dendritic cells and T cells. B cell development is a tightly controlled process in which over 75% of the developing cells become apoptotic because of inappropriate immunoglobulin gene rearrangements or recognition of self antigens by Igs. Hence, the majority of B cell-associated disorders are caused by the incorrect function of genes/proteins involved in B cell development.

Characterization of gene expression in major types of salivary gland carcinomas with epithelial differentiation.

Gene expression profiles were studied in 13 cases of salivary gland carcinoma including mucoepidermoid carcinoma (MEC), acinic cell carcinoma (ACC), and salivary duct carcinoma (SDC) using a cDNA array. A total of 162 genes were deregulated. Only 5 genes were overexpressed in all carcinomas including fibronectin 1 (FN1), tissue metalloproteinase inhibitor 1 (TIMP1), biglycan (BGN), tenascin-C (HXB), and insulin-like growth factor binding protein 5 (IGFBP5), whereas 16 genes were underexpressed. The small number of similarly deregulated genes in these carcinoma entities suggests an extensive genetic variation between them. This result agrees with the great histopathological diversity of different entities of salivary gland carcinoma. Furthermore, diversity in gene expression between the carcinoma types was identified also by hierarchical clustering. Each carcinoma entity was clustered together but MEC, SDC, and ACC were separated from each other. Significance analysis of microarrays identified 27 genes expressed differently between the groups. In MEC, overexpressed genes included those of cell proliferation (IL-6 and SFN) and cell adhesion (SEMA3F and COL6A3), whereas many underexpressed genes were related to DNA modification (NTHL1 and RBBP4). Apoptosis-related genes CASP10 and MMP11 were overexpressed in SDC, in accordance with the typical tumor necrosis seen in this entity. An intermediate filament protein of basal epithelial cells, cytokeratin 14 (KRT14) was clearly differently expressed between the 3 types of carcinoma, and can be used as an aid in their differential diagnosis. The array results were validated by RT-PCR and immunohistochemistry.

APECED-causing mutations in AIRE reveal the functional domains of the protein.

A defective form of the AIRE protein causes autoimmune destruction of target organs by disturbing the immunological tolerance of patients with a rare monogenic disease, autoimmune polyendocrinopathy (APE)-candidiasis (C)-ectodermal dystrophy (ED), APECED. Recently, experiments on knockout mice revealed that AIRE controls autoimmunity by regulating the transcription of peripheral tissue-restricted antigens in thymic medullary epithelial cells. Thus, AIRE provides a unique model for molecular studies of organ-specific autoimmunity. In order to analyze the molecular and cellular consequences of 16 disease-causing mutations in vitro, we studied the subcellular localization, transactivation capacity, homomultimerization, and complex formation of several mutant AIRE polypeptides. Most of the mutations altered the nucleus-cytoplasm distribution of AIRE and disturbed its association with nuclear dots and cytoplasmic filaments. While the PHD zinc fingers were necessary for the transactivation capacity of AIRE, other regions of AIRE also modulated this function. Consequently, most of the mutations decreased transactivation. The HSR domain was responsible for the homomultimerization activity of AIRE; all the missense mutations of the HSR and the SAND domains decreased this activity, but those in other domains did not. The AIRE protein was present in soluble high-molecular-weight complexes. Mutations in the HSR domain and deletion of PHD zinc fingers disturbed the formation of these complexes. In conclusion, we propose an in vitro model in which AIRE transactivates transcription through heteromeric molecular interactions that are regulated by homomultimerization and conditional localization of AIRE in the nucleus or in the cytoplasm.

Fast iterative gene clustering based on information theoretic criteria for selecting the cluster structure.

Grouping of genes into clusters according to their expression levels is important for deriving biological information, e.g., on gene functions based on microarray and other related analyses. The paper introduces the selection of the number of clusters based on the minimum description length (MDL) principle for the selection of the number of clusters in gene expression data. The main feature of the new method is the ability to evaluate in a fast way the number of clusters according to the sound MDL principle, without exhaustive evaluations over all possible partitions of the gene set. The estimation method can be used in conjunction with various clustering algorithms. A recent clustering algorithm using principal component analysis, the "gene shaving" (GS) procedure, can be modified to make use of the new MDL estimation method, replacing the Gap statistics originally used in GS algorithm. The resulting clustering algorithm is shown to perform better than GS-Gap and CEM (classification expectation maximization), in the simulations using artificial data. The proposed method is applied to B-cell differentiation data, and the resulting clusters are compared with those found by self-organizing maps (SOM).

Changes in apoptosis-related pathways in acute myelocytic leukemia.

Expression analysis of apoptotic genes was performed for 15 patients with acute myelocytic leukemia (AML) at the time of diagnosis to identify genes and signaling pathways involved in the regulation of cell survival and apoptosis during leukemogenesis. cDNA array analysis revealed 34 genes whose expression was significantly different compared to others. Tumor suppressor genes TP53 and CDKN2A were downregulated and protooncogenes JUN and GRB10 were upregulated. Furthermore, several cellular signaling pathways acting either in cell cycle regulation or in apoptosis were altered. Deregulation was found in pathways that contribute to genomic stability (by downregulation of either TP53 or CSE1L and by upregulation of GADD45A) and regulate cell cycle progression (by downregulation of CDKN2A and upregulation of RBBP4, CDC37, and NEDD5). Alterations at the transcriptional level were identified, namely, upregulation of JUN and E2F5. Abnormalities were observed in the regulation of the caspases through upregulation of CASP8 and by altered expression of BCL2-related pathway. Extrinsic apoptotic signals mediated by IGFs were deregulated and the glutathione detoxification pathway was downregulated. These findings provide insight into the regulation of balance between apoptosis and cell proliferation signals, and suggest that these genes and pathways may have an important role in the pathogenesis of AML.

Different gene expression in immunoglobulin-mutated and immunoglobulin-unmutated forms of chronic lymphocytic leukemia.

The mutation status of the immunoglobulin heavy chain variable regions (IgVH) has been found to be a good prognostic indicator for B-cell chronic lymphocytic leukemia (CLL) because unmutated VH genes are associated with rapid disease progression and shorter survival time. To study the differences in gene expression between the Ig-unmutated and Ig-mutated CLL subtypes, we performed gene expression profiling on 31 CLL cases and investigated the VH gene mutation status by sequencing. The array data showed that the greatest variances between the unmutated (20 cases) and the mutated (11 cases) group were in expressions of ZAP70, RAF1, PAX5, TCF1, CD44, SF1, S100A12, NUP214, DAF, GLVR1, MKK6, AF4, CX3CR1, NAFTC1, and HEX. ZAP70 was significantly more expressed in the Ig-unmutated CLL group, whereas the expression of all the other genes was higher in the Ig-mutated cases. These results corroborate a recent finding, according to which the expression of ZAP70 can predict the VH mutation status and suggest that RAF1, PAX5, and other differentially expressed genes may offer good markers for differentiating unmutated cases from mutated cases and thus serve as prognostic markers.

Microarray analysis of B-cell stimulation.

B-cell development to antibody-producing plasma cells requires the concerted function of a large number of genes and proteins. Genome-level expression profiling during human B-cell maturation was studied in anti-immunoglobulin M-stimulated Ramos cells. cDNA microarrays were used to follow changes in the transcriptome over several days. Close to 1500 genes had significantly altered expression at least at one time point. The genes were organized into clusters based on expression profiles and were further characterized based on the functions of the coded proteins. Several groups of genes important for B cells were analyzed. Here we concentrate on genes involved in signal transduction and cytokines and their receptors. The results provide knowledge on the development of humoral immunity. Several new genes were found to be essential for B-cell development. They can be used as targets for research and possibly for drug development.

Overexpression of translocation-associated fusion genes of FGFRI, MYC, NPMI, and DEK, but absence of the translocations in acute myeloid leukemia. A microarray analysis.

BACKGROUND AND OBJECTIVES: Translocation-associated gene fusions are well recognized in acute myeloid leukemia. Other molecular genetic changes are less well known. The novel cDNA technology has opened the avenue to large-scale gene expression analysis. Our aim was to perform cDNA microarray analysis of acute myeloid leukemia (AML). DESIGN AND METHODS: We performed cDNA microarray analysis using the Clontech hematology filter (containing 406 genes) on 15 patients to study gene expression profiling in AML. As reference, we used whole bone marrow from 5 healthy donors. RESULTS: Our results revealed 50 differentially expressed genes in at least 3 out of 15 patients. Twenty-two genes were upregulated (ratio > or =4), whereas 28 genes were downregulated (ratio < or =0.25). All but one of the 13 genes tested by real-time polymerase chain reaction (PCR) showed the same expression profiles. Among the overexpressed genes, several were those earlier associated with chromosomal translocations and gene fusions. These genes were FGFR1, MYC, NPM1, DEC, and BCL2. The expression of two upregulated genes, HOXA4 and CSF1R, was significantly higher in patients with a white blood cell count higher than 30 x 10(9)/L cells. In patients whose white blood cell count was higher than 100 x 10(9)/L cells, both CLC and GRN were significantly underexpressed, whereas HOXA4 and DAPK1 were overexpressed. FGFR1 and CAMLG were more frequently significantly overexpressed in patients with CD56 immunophenoytpe. INTERPRETATION AND CONCLUSIONS: Clinical and prognostic significance of differential gene expression should be studied with a larger series of patients by using other techniques, such as quantitative real-time PCR.