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Biomedical subjects

Michael Stock

Publications and source records attributed to Michael Stock.

12 recordsLinked to original sources
PubMed2006

Inhibitors of DNA methylation and histone deacetylation independently relieve AML1/ETO-mediated lysozyme repression.

The human lysozyme (LZM) gene is highly methylated in LZM-nonexpressor immature myeloid and in nonmyeloid cells and unmethylated only in LZM-expressing cells. Extended methylation analyses of the CpG-poor 5' flanking region and of the exon 4 CpG island (both containing Alu elements) of the LZM gene were now performed. Marked demethylation was noted after treatment of AML1/ETO-positive Kasumi-1 cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-azaCdR), not associated with cellular differentiation. LZM mRNA in Kasumi-1, but not in several AML1/ETO-negative myeloid cell lines, was specifically and independently up-regulated upon treatment with 5-azaCdR and, to a lesser extent, with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Increased chromatin accessibility within the 5' LZM gene was observed concomitantly with 5-azaCdR-induced demethylation. In contrast, TSA treatment had no effect on chromatin accessibility, but, as shown by chromatin immunoprecipitation, resulted in increased acetylation of histones H3 and H4. Repression of LZM transcription is mediated by conditional AML1/ETO expression in an inducible cell line model (U-937), and is reversed by siRNA "knock-down" of AML1/ETO in Kasumi-1 cells (Dunne et al., Oncogene 25: 2006). Antagonization of LZM repression following conditional expression of AML1/ETO was achieved by TSA. In conclusion, we demonstrate complex interactions between DNA methylation and histone modifications in mediating LZM repression, which implicate AML1/ETO as one component involved in local chromatin remodeling. Interestingly, inhibitors of DNMTs and HDACs independently relieve repression of this CpG-poor gene in AML1/ETO-positive cells.

Acetylation↗
PubMed2006

Twisted gastrulation modulates bone morphogenetic protein-induced collagen II and X expression in chondrocytes in vitro and in vivo.

Twisted gastrulation (TSG) is an extracellular modulator of bone morphogenetic protein (BMP) activity and regulates dorsoventral axis formation in early Drosophila and Xenopus development. Studies on tsg-deficient mice also indicated a role of this protein in skeletal growth, but the mechanism of TSG activity in this process has not yet been investigated. Here we show for the first time by in situ hybridization and immunohistochemistry that TSG is strongly expressed in bovine and mouse growth plate cartilage as well as in fetal ribs, vertebral cartilage, and cartilage anlagen of the skull. Furthermore we provide evidence that TSG is directly involved in BMP-regulated chondrocyte differentiation and maturation. In vitro, TSG impaired the dose-dependent BMP-2 stimulation of collagen II and X expression in cultures of MC615 chondrocytes and primary mouse chondrocytes. In the presence of chordin, a BMP antagonist, the inhibitory effect of TSG was further enhanced. TSG also inhibited BMP-2-stimulated phosphorylation of Smad factors in chondrocytes, confirming the role of TSG as a modulator of BMP signaling. For analysis of TSG functions in cartilage development in vivo, the gene was overexpressed in transgenic mice under the control of the cartilage-specific Col2a1 promoter. As a result, Col10a1 expression was significantly reduced in the growth plates of transgenic embryos and newborns in comparison with wild type littermates as shown by in situ hybridization and by real time PCR analysis. The data suggest that TSG is an important modulator of BMP-regulated cartilage development and chondrocyte differentiation.

Animals↗
PubMed2006

Reduced levels of antiinflammatory cytokines in patients with chronic widespread pain.

OBJECTIVE: The term chronic widespread pain refers to a group of painful diseases of poorly understood pathophysiology. One major subgroup is fibromyalgia (FM), as defined by the criteria of the American College of Rheumatology. Among other hypotheses, a potential pathophysiologic role of cytokines in chronic widespread pain has been proposed. We undertook this study to investigate whether cytokine profiles differ in patients with chronic widespread pain and controls. METHODS: We analyzed cytokine expression patterns in 40 patients with chronic widespread pain (26 of whom had FM), 40 age- and sex-matched healthy controls, and an additional 15 patients with chronic widespread pain who were recruited from a different center. Expression of messenger RNA (mRNA) for interleukin-2 (IL-2), IL-4, IL-8, IL-10, tumor necrosis factor alpha (TNFalpha), and transforming growth factor beta1 (TGFbeta1) in peripheral blood was analyzed using quantitative real-time polymerase chain reaction (PCR). Serum protein levels were measured by enzyme-linked immunosorbent assay. RESULTS: We found significantly lower relative gene expression (P < 0.0001 for IL-4; P = 0.03 for IL-10) and lower levels of serum protein concentrations (P < 0.0001 for IL-4; P = 0.04 for IL-10) of the Th2 cytokines IL-4 and IL-10 in patients with chronic widespread pain than in the control group. This finding was corroborated in an additional group of 15 patients with chronic widespread pain. There were no significant differences between the groups in levels of mRNA for IL-2, IL-8, TNFalpha, or TGFbeta1. Protein data paralleled the real-time PCR results. CONCLUSION: Chronic widespread pain is associated with a lack of antiinflammatory and analgesic Th2 cytokine activity, which may contribute to its pathogenesis.

Adult↗
PubMed2005

Gene deregulation in gastric cancer.

Despite its decreasing frequency in the Western world during recent decades, gastric cancer is still one of the leading causes of cancer-related deaths worldwide. Due to the oligosymptomatic course of early gastric cancer, most cases are diagnosed in the advanced stages of the disease. The curative potential of current standard treatment continues to be unsatisfactory, despite multimodal approaches involving surgery, chemotherapy and radiotherapy. Novel therapeutics including small molecules and monoclonal antibodies are being developed and have been partially introduced into clinical use in connection with neoplastic diseases such as chronic myeloid leukemia, non-Hodgkin's lymphoma and colorectal cancer. Thorough understanding of the changes in gene expression occurring during gastric carcinogenesis may help to develop targeted therapies and improve the treatment of this disease. Novel molecular biology techniques have generated a wealth of data on up- and down-regulation, activation and inhibition of specific pathways in gastric cancer. Here, we provide an overview of the different aspects of aberrant gene expression patterns in gastric cancer.

Animals↗
PubMed2005

Control of RUNX2 isoform expression: the role of promoters and enhancers.

The three mammalian RUNX genes constitute the family of runt domain transcription factors that are involved in the regulation of a number of developmental processes such as haematopoiesis, osteogenesis and neuronal differentiation. All three genes show a complex temporo-spatial pattern of expression. Since the three proteins are probably mutually interchangeable with regard to function, most of the specificity of each family member seems to be based on a tightly controlled regulation of expression. While RUNX gene expression is driven by two promoters for each gene, the promoter sequence alone does not seem to suffice for a proper expressional control. This review focuses on the available evidence for the existence of such control mechanisms and studies aiming at discovering cis-acting regulatory sequences of the RUNX2 gene.

Animals↗
PubMed2004

Williams-Beuren syndrome critical region-5/non-T-cell activation linker: a novel target gene of AML1/ETO.

The chromosomal translocation t(8;21) fuses the AML1 (RUNX1) gene on chromosome 21 and the ETO gene on chromosome 8 in human acute myeloid leukemias (AMLs), resulting in expression of the chimeric transcription factor AML1/ETO. AML1/ETO-mediated dysregulation of target genes critical for hematopoietic differentiation and proliferation is thought to contribute to the leukemic phenotype. Several mechanisms, including recruitment of histone deacetylases (HDACs) to AML1 target genes, may be responsible for altered gene expression. We used an ecdysone-inducible expression system in the human monoblastic U-937 cell line to isolate genes that were differentially expressed upon induction of AML1/ETO expression. By representational difference analysis (cDNA-RDA), we identified 26 genes whose expression levels were significantly modulated following AML1/ETO induction for 48 h. None of these genes has previously been described as a target of AML1, ETO or AML1/ETO. One gene downregulated by AML1/ETO in vitro, Williams Beuren syndrome critical region 5 (WBSCR5), was expressed in primary t(8;21)-negative AML blasts but not in primary t(8;21)-positive AML blasts, strongly implying a role of this gene in the phenotype of t(8;21)-positive AML. Four upregulated and four downregulated genes were further studied with all-trans-retinoic acid (ATRA), an inducer of differentiation of U-937 cells, and Trichostatin A (TSA), an HDAC inhibitor. Three out of eight genes including WBSCR5 were regulated during ATRA-induced monocytic differentiation of U-937 cells, however, none of them antagonistically, upon both ATRA treatment and AML1/ETO induction. AML1/ETO-associated dysregulation of gene expression was not mediated by a TSA-sensitive mechanism. The identified genes provide a useful model to study the mechanism by which the AML1/ETO fusion protein exerts its function in transcriptional dysregulation in AML. The possible role of WBSCR5 in normal and malignant hematopoiesis warrants further study.

Cell Differentiation↗
PubMed2004

Identification of novel genes of the bone-specific transcription factor Runx2.

INTRODUCTION: The transcription factor Runx2 is a key regulator of osteoblast development and plays a role in chondrocyte maturation. The identification of transcriptional target genes of Runx2 may yield insight into how osteoblastic differentiation is achieved on a molecular level. MATERIALS AND METHODS: Using a differential hybridization technique (selective amplification through biotin and restriction-mediated enrichment [SABRE]) and cDNA microarray analysis, 15 differentially expressed genes were identified using mRNA from C3H 10Tl/2 cells with constitutive and inducible overexpression of Runx2. RESULTS AND CONCLUSIONS: Among the 15 genes identified, 4 encode the extracellular matrix proteins Ecml, Mgp, Fbn5, and Osf-2, three represent the transcription factors Esxl, Osrl, and Sox9, whereas others were Ptn, Npdc-1, Higl, and Tem l. The gene for Pttg1ip was upregulated in Runx2-expressing cells. Pttg1ip is widely expressed during development, but at highest levels in limbs and gonads. The Pttg1ip promoter binds Runx2 in a sequence specific manner, and Runx2 is able to transactivate the Pttg lip promoter in MC3T3-El cells. Therefore, Pttg1ip is likely tobe a novel direct transcriptional target gene of Runx2. In conclusion, the genes identified in this study are important candidates for mediating Runx2 induced cellular differentiation.

Animals↗
PubMed2003

Upstream and downstream targets of RUNX proteins.

In recent years, the in vivo role of the three members of the RUNX family of transcription factors has in part been elucidated. While Runx1 is essential for mature haematopoiesis and Runx2 for osteochondrogenesis, Runx3 has a function in the nervous system. Translocations and mutations affecting the RUNX1 gene are clearly implicated in leukemogenesis whereas recent data suggest that changed expression levels of RUNX3 may be involved in gastric carcinogenesis. Germ line mutations in RUNX2 have been identified in patients with an autosomal dominant skeletal disorder, cleidocranial dysplasia. While a number of pathways have been delineated that regulate RUNX activity, transcription factors binding to RUNX promoters are only beginning to be identified. A growing number of genes have been characterised that are being regulated in their transcriptional activity by different RUNX proteins. Whether a particular RUNX protein specifically targets a defined subset of downstream genes or whether there is some redundancy as to which RUNX protein activates which target promoter remains to be elucidated.

Animals↗
PubMed2003

Expression of galectin-3 in skeletal tissues is controlled by Runx2.

The beta-galatoside-specific lectin galectin-3 is expressed in vivo in osteoblasts as well as in epiphyseal cartilage. Here we show that in vitro, galectin-3 expression is up-regulated in the preosteoblastic cell line MC3T3-E1 during the matrix maturation stage of the osteoblast developmental sequence. Expression persists into late differentiation stages when the mature osteoblastic phenotype is established. The skeletal expression pattern of galectin-3 overlaps at many sites with that of the transcription factor Runx2. Runx2 is a key regulator of osteoblast development and necessary for chondrocyte differentiation in the growth plate. Both human and mouse galectin-3 promoters contain putative Runx-binding sites. The constitutive or inducible forced expression of Runx2 is sufficient for the onset of galectin-3 transcription in the mesenchymal precursor cell line C3H10T1/2. Moreover, Runx2 is able to bind to at least two sites in the galectin-3 promoter region. The crucial role of Runx2 was confirmed in Runx2-deficient mice, which are devoid of galectin-3 expression in skeletal cells. The overlapping expression pattern of galectin-3 with the other two members of the Runt family of transcription factors (Runx1 and Runx3) points to a potential regulation of the galectin-3 gene (LGALS3) by these factors in hematopoietic, skin, and dorsal root ganglial cells.

Animals↗
PubMed2002

Genomic characterization of the RUNX2 gene of Fugu rubripes.

A 105 kb Fugu rubripes genomic region containing the RUNX2 ortholog (frunx2) was sequenced and analysed. Spanning 32 kb, frunx2 is seven times smaller than its human orthologue (223 kb). By comparison of Fugu and human genomic environment a stretch of conserved synteny, comprising the neighbouring genes on both sides, was identified. Except one exon that is alternatively spliced in human RUNX2, all other seven exons could be identified in frunx2. The predicted protein sequence of frunx2 shows a high degree of sequence conservation compared with RUNX2 (83% identity). Like all human paralogues, frunx2 possesses two promoter regions separated by a large intron. Both promoter regions are conserved between the two species and contain several RUNX binding sites pointing to a self-regulatory function. Three further conserved non-coding regions were identified possibly functioning as enhancer elements for tissue-specific expression of RUNX2.

Amino Acid Sequence↗