Hardi Lührs

Dynamic interaction of HMGA1a proteins with chromatin

High-mobility-group proteins A1 (HMGA1; previously named HMGI/Y) function as architectural chromatin-binding proteins and are involved in the transcriptional regulation of several genes. We have used cells expressing proteins fused to green fluorescent protein (GFP) and fluorescence recovery after photobleaching (FRAP) to analyze the distribution and dynamics of HMGA1a in vivo. HMGA1-GFP proteins localize preferentially to heterochromatin and remain bound to chromosomes during mitosis. FRAP experiments showed that they are highly mobile components of euchromatin, heterochromatin and of mitotic chromosomes, although with different resident times. For a more-detailed investigation on the interaction of HMGA1a with chromatin, the contribution of the AT-hook DNA-binding motifs was analyzed using point-mutated HMGA1a-GFP proteins. Furthermore, by inhibiting kinase or histone deacetylase activities, and with the help of fusion proteins lacking specific phosphorylation sites, we analyzed the effect of reversible modifications of HMGA1a on chromatin binding. Collectively our data show that the kinetic properties of HMGA1a proteins are governed by the number of functional AT-hooks and are regulated by specific phosphorylation patterns. The higher residence time in heterochromatin and chromosomes, compared with euchromatic regions, correlates with an increased phosphorylation level of HMGA1a. The regulated dynamic properties of HMGA1a fusion proteins indicate that HMGA1 proteins are mechanistically involved in local and global changes in chromatin structure.

Butyrate Inhibits Leukocyte Adhesion to Endothelial Cells via Modulation of VCAM-1

BackgroundLeukocyte recruitment to areas of inflammation depends on Integrin-VCAM/ICAM interaction. Blocking the vascular cell adhesion molecule (VCAM-1) and the intracellular adhesion molecule (ICAM-1) may have therapeutic benefit for the inflammatory component of bowel disease. Notably, the induction of ICAM and VCAM is mediated by a nuclear factor kappaB (NF-&kgr;B)-dependent mechanism. We investigated whether the anti-inflammatory properties of butyrate are mediated via the modulation of VCAM and ICAM on human endothelial cells. MethodsVCAM-1 and ICAM-1 expression on human endothelial cells upon tumor necrosis factor-&agr; (TNF-&agr;) stimulation was assessd by FACS analysis. A monocyte adhesion assay was performed to evaluate the relevance of a modulated CAM-expression. Electrophoretic mobility shift assays were applied to investigate NF-&kgr;B activation. ResultsThe observed butyrate-associated inhibition of monocyte adhesion to endothelial cells is associated with an inhibition of NF-&kgr;B activation in human endothelial cells. In this context, the observed suppression of the TNF-&agr; induced VCAM-1 expression is likely to play an essential role. ConclusionsButyrate inhibits VCAM-1 mediated leukocyte adhesion to human endothelial cells. This inhibition may contribute to the anti-inflammatory effects of butyrate in patients with distal ulcerative colitis.

Butyrate inhibits interleukin-1-mediated nuclear factor-kappa B activation in human epithelial cells

Nuclear factor-kappa B (NF-κB) is a critical transcription factor for the inducible expression of multiple genes involved in inflammation. NF-κB is sequestered in the cytoplasm by inhibitory IκB proteins. Extracellular stimuli, notably interleukin-1β (IL-1β) and tumor necrosis factorα (TNF-α) activate NF-κB nuclear translocation via IκB phosphorylation and degradation. Since previous reports suggest that the short chain fatty acid butyrate has antiinflammatory properties, the effects of butyrate on NF-κB nuclear translocation in human epithelial cells (HeLa229) were tested. In cells pretreated with butyrate, a time- and dose-dependent inhibition of IL-1β-mediated NF-κB nuclear translocation was observed. However, IκBα phosphorylation and degradation occurred rapidly in both butyrate pretreated and nonpretreated cells, respectively. These data indicate that inhibition of IL-1β-induced NF-κB activation by butyrate does not require an intact IκBα protein.

Type I nitric oxide synthase in the human lung is predominantly expressed in capillary endothelial cells.

Nitric oxide (NO) has important functions in the regulation of pulmonary smooth muscle tone. In the human lung, published data on the expression and distribution of neuronal nitric oxide synthase (NOS-I) are contradictory. The aim of this study, therefore, was to determine the predominant cells expressing NOS-I in the human lung. Immunofluorescence double staining techniques were applied to normal human lung tissue using established monospecific antibodies directed against NOS-I. Suprisingly, capillary endothelial cells in the alveolar septa were identified as the major sites of NOS-I expression. Neither alveolar nor bronchiolar epithelium, nor the alveolar macrophages, expressed NOS-I. These results indicate that the predominant sites of NOS-I expression in the human lung are confined to non-neuronal, i.e. capillary endothelial cells and suggest a role for NO in the regulation of pulmonary endothelial cell permeability.

Spectral karyotyping of the human colon cancer cell lines SW480 and SW620

The cell lines SW480 and SW620, derived from different stages of colon carcinoma in the same patient, have been used for a number of biochemical, immunological, and genetic studies on colon cancer. A comparative analysis of their karyotypes may identify chromosomal aberrations that might represent markers for metastatic spread. In the present study spectral karyotyping (SKY) was applied to these two colon cancer cell lines. Compared to previously reported G-banded karyotypes, 9 (SW480) and 7 (SW620) markers were identical, 3 (SW480) and 3 (SW620) markers could be redefined, 5 (SW480) and 8 (SW620) markers were newly identified, and 4 (SW480) and 5 (SW620) of the previous described markers could not be confirmed. The redefined aberrations include very complex rearrangements, such as a der(16) t(3;16;1;16;8;16; 1;16;10) and a der(18)t(18;15;17)(q12; p11p13;??) in SW620 and a der(19)t(19;8;19;5) in SW480, that have not been identified by conventional banding techniques. The resulting chromosome gains (5q11→5q15, 7pter→q22, 11, 13q14→qter, 20pter→p12, X) and losses (8pter→p2, 18q12→qter, Y) found in both SW480 and SW620 were in good agreement with those frequently described in colorectal tumors as primary changes in the stem cell. Abnormalities found exclusively in SW620 cells only (gains of 5pter→5q11, 12q12→q23, 15p13→p11, and 16q21→q24 and losses of 2pter→2p24, 4q28→qter, and 6q25→qter) can be viewed as changes that occurred in a putative metastatic founder cell.

LOH and copy neutral LOH (cnLOH) act as alternative mechanism in sporadic colorectal cancers with chromosomal and microsatellite instability

BACKGROUND AND AIMS Tumor suppressor genes are often located in frequently deleted chromosomal regions of colorectal cancers (CRCs). In contrast to microsatellite stable (MSS) tumors, only few loss of heterozygosity (LOH) studies were performed in microsatellite instable (MSI) tumors, because MSI carcinomas are generally considered to be chromosomally stable and classical LOH studies are not feasible due to MSI. The single nucleotide polymorphism (SNP) array technique enables LOH studies also in MSI CRC. The aim of our study was to analyse tissue from MSI and MSS CRC for the existence of (frequently) deleted chromosomal regions and tumor suppressor genes located therein. METHODS AND RESULTS We analyzed tissues from 32 sporadic CRCs and their corresponding normal mucosa (16 MSS and 16 MSI tumors) by means of 50K SNP array analysis. MSS tumors displayed chromosomal instability that resulted in multiple deleted (LOH) and amplified regions and led to the identification of MTUS1 (8p22) as a candidate tumor suppressor gene in this region. Although the MSI tumors were chromosomally stable, we found several copy neutral LOHs (cnLOH) in the MSI tumors; these appear to be instrumental in the inactivation of the tumor suppressor gene hMLH1 and a gene located in chromosomal region 6pter-p22. DISCUSSION Our results suggest that in addition to classical LOH, cnLOH is an important mutational event in relation to the carcinogenesis of MSS and MSI tumors, causing the inactivation of a tumor suppressor gene without copy number alteration of the respective region; this is crucial for the development of MSI tumors and for some chromosomal regions in MSS tumors.

Effects of isomalt consumption on gastrointestinal and metabolic parameters in healthy volunteers

The polyol isomalt (Palatinit) is a well established sugar replacer. The impact of regular isomalt consumption on metabolism and parameters of gut function in nineteen healthy volunteers was examined in a randomised, double-blind, cross-over trial with two 4-week test periods. Volunteers received 30 g isomalt or 30 g sucrose daily as part of a controlled diet. In addition to clinical standard diagnostics, biomarkers and parameters currently discussed as risk factors for CHD, diabetes or obesity were analysed. Urine and stool Ca and phosphate excretions were measured. In addition, mean transit time, defecation frequency, stool consistency and weight were determined. Consumption of test products was affirmed by the urinary excretion of mannitol. Blood lipids were comparable in both phases, especially in volunteers with hyperlipidaemia, apart from lower apo A-1 (P=0.03) for all subjects. Remnant-like particles, oxidised LDL, NEFA, fructosamine and leptin were comparable and not influenced by isomalt. Ca and phosphate homeostasis was not affected. Stool frequency was moderately increased in the isomalt phase (P=0.006) without changes in stool consistency and stool water. This suggests that isomalt is well tolerated and that consumption of isomalt does not impair metabolic function or induce hypercalciuria. In addition, the study data indicate that isomalt could be useful in improving bowel function.

SNP-Array genotyping and spectral karyotyping reveal uniparental disomy as early mutational event in MSS- and MSI-colorectal cancer cell lines

In this study nine colorectal cancer cell lines were analysed by 10K SNP-arrays and spectral karyotyping (SKY). Complex chromosomal alterations and breakpoints of deleted or translocated fragments found by SKY could further be characterized by SNP-array analysis. Interestingly many monoallelic regions identified by SNP-array analysis display no copy number alterations, representing uniparental disomy (UPD). It was demonstrated that UPD seems to be involved in activation of early-acting tumor suppressor genes in MSS- (APC, CDKN2A) and MSI- (MLH1, MSH2, APC, CDKN2A) colorectal cancer cell lines. Genes involved later on in the adenoma-carcinoma sequence (i.e. TP53/SMAD4) were not found to be inactivated by UPD. Furthermore, identified amplified monoallelic regions may include oncogenes activated by allele-specific-amplification (i.e. Cyclin D1). However, at present, the majority of the monoallelic regions located in the present study have not yet been associated with known tumor suppressor genes and oncogenes. Further studies are warranted to identify relevant genes in the respective regions and to further verify the results presented here.

Modulation of HMG-N2 binding to chromatin by butyrate-induced acetylation in human colon adenocarcinoma cells.

Butyrate, a short chain fatty acid (SCFA), is generated by anaerobic fermentation of undigested carbohydrates within the colon. Butyrate enhances acetylation of core histones, a process directly linked to the formation of active chromatin and gene expression. However, additional chromatin components also contribute to the formation of transcriptionally active chromatin. The high mobility group protein N2 (HMG‐N2), a nonhistone protein, is involved in chromatin structure modulation. We examined the effects of butyrate on HMG‐N2 expression, hyperacetylation and chromatin binding. HT29 human adenocarcinoma cells were incubated with butyrate. Levels of HMG‐N2 mRNA and of total or acetylated HMG‐N2 protein were analyzed. Protein dynamics were investigated with transfected cells expressing HMG‐N2‐EGFP fusion proteins. Treatment of HT29 cells with butyrate led to significant hyperacetylation of HMG‐N2. Levels of HMG‐N2 protein remained unchanged. Northern blot analysis revealed a significant reduction in HMG‐N2 mRNA levels after treatment with butyrate. Analysis of HMG‐N2‐EGFP transfected HT29 cells demonstrated that butyrate treatment changes the binding properties of HMG‐N2‐EGFP to chromatin. In addition, butyrate treatment resulted in solubilization of endogenous acetylated HMG‐N2 into the supernatant of permeabilized cells. We demonstrate that butyrate treatment is associated with hyperacetylation of HMG‐N2 protein in HT29 cells. The modulation of this nonhistone chromatin protein resulted in altered binding properties to chromatin. This may represent an additional step in changing chromatin structure and composition with subsequent consequences for transcription and gene expression. Modulation of nonhistone chromatin proteins, like the ubiquitous HMG‐N2 proteins, may be partly responsible for the wide range of butyrate‐associated effects.

Establishment of a colonic adenoma cell line (GEKI-2): spectral karyotype analysis and functional characterization

Background and aimsOn the genetic level colonic carcinogenesis is best described by the adenoma-carcinoma sequence, but it may be modulated by exogenous factors, particularly by dietary factors and chemopreventive agents. The protective effects of exogenous factors are thought to be exerted rather in the early stages of the adenoma-carcinoma sequence. Thus, an in vitro model consisting of cells stemming from an colon adenoma would be desirable. However, establishing such a cell line has proven difficult.Materials and methodsWe report the establishment of a colon adenoma cell line. The cells were generated from a colon adenoma and propagated as a stable cell line for more than 40 passages. The cells are microsatellite stable and confirmed to be of epithelial origin by cytokeratin staining.ResultsIn contrast to commercially available colon cancer cell lines, cytogenetic analysis with spectral karyotype analysis revealed no chromosomal alterations in this adenoma cell line. Incubation with butyrate resulted in a time- and dose-dependent inhibition of proliferation and in an significant increase in cellular differentiation. The cdk inhibitor p21/waf which plays a pivotal role in growth inhibition and differentiation is expressed consecutively in GEKI-2 cells. The expression of cdk1 and cdk2, important regulatory elements in the cell cycle, is downregulated following treatment with butyrate.ConclusionThe presented colon adenoma cell line GEKI-2 may prove a versatile tool for further exploring the underlying mechanisms of protective and promoting factors in early colon cancerogenesis.