Biomedical subjects

Norbert Schweifer

Publications and source records attributed to Norbert Schweifer.

9 recordsLinked to original sources

PubMed2006

Negative and positive regulation of gene expression by mouse histone deacetylase 1.

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from core histones. Because of their capacity to induce local condensation of chromatin, HDACs are generally considered repressors of transcription. In this report, we analyzed the role of the class I histone deacetylase HDAC1 as a transcriptional regulator by comparing the expression profiles of wild-type and HDAC1-deficient embryonic stem cells. A specific subset of mouse genes (7%) was deregulated in the absence of HDAC1. We identified several putative tumor suppressors (JunB, Prss11, and Plagl1) and imprinted genes (Igf2, H19, and p57) as novel HDAC1 targets. The majority of HDAC1 target genes showed reduced expression accompanied by recruitment of HDAC1 and local reduction in histone acetylation at regulatory regions. At some target genes, the related deacetylase HDAC2 partially masks the loss of HDAC1. A second group of genes was found to be downregulated in HDAC1-deficient cells, predominantly by additional recruitment of HDAC2 in the absence of HDAC1. Finally, a small set of genes (Gja1, Irf1, and Gbp2) was found to require HDAC activity and recruitment of HDAC1 for their transcriptional activation. Our study reveals a regulatory cross talk between HDAC1 and HDAC2 and a novel function for HDAC1 as a transcriptional coactivator.

Animals↗

PubMed2006

Impact of constitutive IGF1/IGF2 stimulation on the transcriptional program of human breast cancer cells.

Insulin-like growth factor (IGF) signaling is a key regulator of breast development and breast cancer. We have analyzed the expression of the IGF signaling cascade in 17 human breast cancer and 4 mammary epithelial cell lines. Five cell lines expressed high levels of IGF1 receptor, insulin (INS)/IGF receptor substrate 1, IGF-binding proteins 2 and 4, as well as the estrogen receptor (ESR), indicating a co-activation of IGF and ESR signaling. Next, we stably overexpressed IGF1 and IGF2 in MCF7 breast cancer cells, which did not affect their epithelial characteristics and the expression and localization of the epithelial marker genes E-cadherin and beta-catenin. Conversely, IGF1 and IGF2 overexpression potently increased cellular proliferation rates and the efficiency of tumor formation in mouse xenograft experiments, whereas the resistance to chemotherapeutic drugs such as taxol was unaltered. Expression profiling of overexpressing cells with whole-genome oligonucleotide microarrays revealed that 21 genes were upregulated >2-fold by both IGF1 and IGF2, 9 by IGF1, and 9 by IGF2. Half of the genes found to be upregulated are involved in transport and biosynthesis of amino acids, including several amino acid transport proteins, argininosuccinate and asparagine synthetases, and methionyl-tRNA synthetase. Upregulation of these genes constitutes a novel mechanism apparently contributing to the stimulatory effects of IGF signaling on the global protein synthesis rate. We conclude that the induction of cell proliferation and tumor formation by long-term IGF stimulation may primarily be due to anabolic effects, in particular increased amino acid production and uptake.

Animals↗

PubMed2006

Laser capture microdissection of epithelial cancers guided by antibodies against fibroblast activation protein and endosialin.

Transcriptional profiling of cancer biopsies is used extensively to identify expression signatures for specific cancer types, diagnostic and prognostic subgroups, and novel molecular targets for therapy. To broaden these applications, several challenges remain. For example, the integrity of RNA extracted even from small tissue samples has to be insured and monitored. Moreover, total tumor RNA may hide the marked histologic heterogeneity of human cancers. A principle approach to this heterogeneity has been provided by laser capture microdissection performed on antibody-stained tissue sections (immuno-LCM; iLCM). In this study, we have established a procedure to assess the quality of RNA obtained from tissue sections, coupled with immunostaining using antibodies to different tumor stromal markers, and subsequent iLCM to selectively capture the cancer stroma compartments. The procedure was applied to 53 frozen specimens of human epithelial cancers. Sections were stained for histopathological evaluation, and RNA was isolated from adjacent serial sections. RNA quality was assessed by the Agilent-Bioanalyzer (Agilent, Palo Alto, CA) and by multiplex RT-PCR. Two thirds of the specimens were found to yield good to excellent RNA quality. For microdissection of the tumor stroma with reactive fibroblasts and tumor blood vessels, a rapid incubation protocol with antibodies against fibroblast activation protein (FAP) and against endosialin was developed to ensure RNA integrity for subsequent iLCM. Using these procedures, RNA from distinct tumor compartments can be isolated, analyzed, amplified, and used for transcription profiling.

Antibodies, Neoplasm↗

PubMed2006

Expression of stromal cell markers in distinct compartments of human skin cancers.

BACKGROUND: The importance of changes in the supporting tumor stroma for cancer initiation and progression is well established. The characteristics of an activated tumor stroma, however, are not completely understood. In an effort to better characterize the desmoplastic response to human skin tumors, we evaluated the expression pattern of three stromal cell markers, fibroblast-activation protein (FAP), endoglyx-1, and endosialin, in a series of melanocytic and epithelial skin tumors. METHODS: Immunohistochemistry using antibodies against FAP, endoglyx-1, and endosialin was carried out in skin samples obtained from 43 patients. Furthermore, microarray data from an independent set of human skin cancers were analyzed. RESULTS: FAP-positive fibroblasts were detected in all tumor tissues tested, including cases of melanocytic nevi, melanoma metastases, basal cell carcinomas, and squamous cell carcinomas. In cutaneous melanoma metastases, we identified different compartments within the stromal response on the basis of the regions of FAP expression. Endoglyx-1 expression was confined to normal and tumor blood vessel endothelium including 'hot spots' of neoangiogenesis within the cutaneous melanoma metastases. Endosialin was selectively induced in subsets of small- and medium-sized tumor blood vessels in melanoma metastases and squamous cell carcinomas. CONCLUSIONS: These data describe novel aspects of stromal marker expression in distinct compartments of human skin tumors and may point to potential targets for novel therapeutic strategies aimed at the tumor stroma.

Antigens, CD↗

PubMed2005

Characterization of cancer stroma markers: in silico analysis of an mRNA expression database for fibroblast activation protein and endosialin.

Standardized, high-throughput RNA detection with microarray chips allows for the construction of genome-wide databases for tissue specimens suitable for in silico electronic Northern blot (eNorthern) analysis of marker genes. We used the BioExpress database, which contains transcriptional profiles of normal and cancer samples, to examine two putative markers of cancer stroma: fibroblast activation protein-alpha (FAP-alpha) and endosialin. Analyses for FAP-alpha showed that normal tissues generally lack RNA signals, with the exception of endometrium. Typing of tumors revealed prominent FAP-alpha signals in cancer types marked by desmoplasia, and localization of FAP-alpha in reactive cancer stroma was confirmed by immunohistochemistry. A subset of sarcomas displayed prominent FAP-alpha signals localizing to the malignant cells. For endosialin, eNorthern analyses showed low to moderate RNA signals in many normal organs, whereas immunohistochemistry revealed endosialin in only some tissues, such as endometrium. Endosialin was detected at the RNA and protein level in sarcomas, notably malignant fibrous histiocytomas. Low to moderate endosialin RNA signals were found in epithelial cancer types for which immunostaining identifies expression in subsets of tumor capillaries or fibroblasts. These findings extend the FAP-alpha and endosialin profiling in silico to an unbiased tumor database and place both molecules in a novel context of endometrial biology and sarcoma subtyping. Our findings suggest that BioExpress can be searched directly for tumor stroma markers but may need prior enrichment for markers with narrow cellular representation, such as endosialin. Constructing databases from microdissected cancer tissues may be an essential step for tumor stroma-targeted therapies.

Adult↗

PubMed2005

Balance between NF-kappaB and JNK/AP-1 activity controls dendritic cell life and death.

The life cycle of dendritic cells (DCs) must be precisely regulated for proper functioning of adaptive immunity. However, signaling pathways actively mediating DC death remain enigmatic. Here we describe a novel mechanism of hierarchical transcriptional control of DC life and death. Ligation of tumor necrosis factor receptor superfamily (TNFR-SF) members on DCs and cognate contact with T cells resulted in quantitatively balanced nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK)-mediated activator protein-1 (AP-1) induction and strongly enhanced DC longevity. Specific blockade of NF-kappaB in DCs induced strongly augmented JNK/AP-1 activity because of elevated levels of reactive oxygen species. In this scenario, DC activation by TNFR-SF members or T cells induced DC apoptosis. Specific inhibition of JNK/AP-1 rescued DCs from this activation-induced cell death program and restored TNFR-SF member- and T-cell-mediated survival. We conclude that JNK/AP-1 activity is under negative feedback control of NF-kappaB and can execute apoptosis in DCs. Thus, feedback-controlled signaling amplitudes of 2 transcriptional pathways decide the fate of a DC.

CD40 Antigens↗

PubMed2004

Microarray gene expression profiling of B-cell chronic lymphocytic leukemia subgroups defined by genomic aberrations and VH mutation status.

PURPOSE: Genomic aberrations and mutational status of the immunoglobulin variable heavy chain (VH) gene have been shown to be among the most important predictors for outcome in patients with B-cell chronic lymphocytic leukemia (B-CLL). In this study, we report on differential gene expression patterns that are characteristic for genetically defined B-CLL subtypes. MATERIALS AND METHODS: One hundred genetically well-characterized B-CLL samples, together with 11 healthy control samples, were analyzed using oligonucleotide arrays, which test for the expression of some 12,000 human genes. RESULTS: Aiming at microarray-based subclassification, class predictors were constructed using sets of differentially expressed genes, which yielded in zero or low misclassification rates. Furthermore, a significant number of the differentially expressed genes clustered in chromosomal regions affected by the respective genomic losses/gains. Deletions affecting chromosome bands 11q22-q23 and 17p13 led to a reduced expression of the corresponding genes, such as ATM and p53, while trisomy 12 resulted in the upregulation of genes mapping to chromosome arm 12q. Using an unsupervised analysis algorithm, expression profiling allowed partitioning into predominantly VH-mutated versus unmutated patient groups; however, association of the expression profile with the VH mutational status could only be detected in male patients. CONCLUSION: The finding that the most significantly differentially expressed genes are located in the corresponding aberrant chromosomal regions indicates that a gene dosage effect may exert a pathogenic role in B-CLL. The significant difference in the partitioning of male and female B-CLL samples suggests that the genomic signature for the VH mutational status might be sex-related.

Adult↗

PubMed2004

CDCP1 identifies a broad spectrum of normal and malignant stem/progenitor cell subsets of hematopoietic and nonhematopoietic origin.

CUB-domain-containing protein 1 (CDCP1) is a novel transmembrane molecule that is expressed in metastatic colon and breast tumors as well as on the surface of hematopoietic stem cells. In this study, we used multiparameter flow cytometry and antibodies against CDCP1 to analyze the expression of CDCP1 on defined hematopoietic cell subsets of different sources. In addition, CDCP1 expression on leukemic blasts and on cells with nonhematopoietic stem/progenitor cell phenotypes was determined. Here we demonstrate that a subset of bone marrow (BM), cord blood (CB), and mobilized peripheral blood (PB) CD34+ cells expressed this marker and that CDCP1 was detected on CD34(+)CD38- BM stem/progenitor cells but not on mature PB cells. Analysis of leukemic blasts from patients with acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia in blast crisis revealed that CDCP1 is predominantly expressed on CD34(+)CD133+ myeloid leukemic blasts. However, CDCP1 was not strictly correlated with CD34 and/or CD133 expression, suggesting that CDCP1 is a novel marker for leukemia diagnosis. Stimulation of CD34+ BM cells with CDCP1-reactive monoclonal antibody CUB1 resulted in an increased (approximately twofold) formation of erythroid colony-forming units, indicating that CDCP1 plays an important role in early hematopoiesis. Finally, we show that CDCP1 is also expressed on cells phenotypically identical to mesenchymal stem/progenitor cells (MSCs) and neural progenitor cells (NPCs). In conclusion, CDCP1 is not only a novel marker for immature hematopoietic progenitor cell subsets but also unique in its property to recognize cells with phenotypes reminiscent of MSC and NPC.

AC133 Antigen↗

PubMed2003

CDCP1 is a novel marker for hematopoietic stem cells.

CDCP1 is a transmembrane protein that contains three CUB domains within the extracellular region and a hexalysine stretch within the cytoplasmic region. CDCP1 mRNA is highly expressed in lung and colon tumors and in the erythroleukemic cell line K562. To analyze CDCP1 protein expression, monoclonal antibodies against the extracellular domain of CDCP1 were raised. For this purpose, CDCP1 was overexpressed in NIH-3T3 cells. Balb/c mice were then immunized with the resultant cell line NIH-3T3/huCDCP1. After fusion of SP2/0 cells with immune spleen cells, hybridoma clones were selected that secreted antibodies reacting with NIH-3T3/huCDCP1 cells but not with parental cells. Four antibodies (CUB1-CUB4) were obtained that fulfilled these criteria. Screening of peripheral blood cells revealed that the antibodies did not recognize mature lymphocytes, monocytes, granulocytes, erythrocytes, or platelets. In contrast, multi-color analyses revealed that CDCP1 protein is almost exclusively expressed on a subset of CD34(+) stem/progenitor cells in bone marrow. Transplantation of purified CDCP1(+) cells into NOD/SCID mice resulted in engraftment of human cells with multi-lineage differentiation potential, suggesting that CDCP1 is a novel marker for hematopoietic stem cells.

3T3 Cells↗