Metabolic perturbations resulting from excessive hepatic fat accumulation are poorly understood. Thus, in this study, leptin-deficient ob/ob mice, a mouse model of fatty liver disease, were used to ...investigate metabolic alterations in more detail. Metabolites were quantified in intact liver tissues of ob/ob (n = 8) and control (n = 8) mice using high-resolution magic angle spinning (HR-MAS)
H-NMR. In addition, after demonstrating that HR-MAS
H-NMR does not affect RNA integrity, transcriptional changes were measured by quantitative real-time PCR on RNA extracted from the same specimens after HR-MAS
H-NMR measurements. Importantly, the gene expression changes obtained agreed with those observed by Affymetrix microarray analysis performed on RNA isolated directly from fresh-frozen tissue. In total, 40 metabolites could be assigned in the spectra and subsequently quantified. Quantification of lactate was also possible after applying a lactate-editing pulse sequence that suppresses the lipid signal, which superimposes the lactate methyl resonance at 1.3 ppm. Significant differences were detected for creatinine, glutamate, glycine, glycolate, trimethylamine-N-oxide, dimethylglycine, ADP, AMP, betaine, phenylalanine, and uridine. Furthermore, alterations in one-carbon metabolism, supported by both metabolic and transcriptional changes, were observed. These included reduced demethylation of betaine to dimethylglycine and the reduced expression of genes coding for transsulfuration pathway enzymes, which appears to preserve methionine levels, but may limit glutathione synthesis. Overall, the combined approach is advantageous as it identifies changes not only at the single gene or metabolite level but also deregulated pathways, thus providing critical insight into changes accompanying fatty liver disease. Graphical abstract A Evaluation of RNA integrity before and after HR-MAS
H-NMR of intact mouse liver tissue. B Metabolite concentrations and gene expression levels assessed in ob/ob (steatotic) and ob/+ (control) mice using HR-MAS
H-NMR and qRT-PCR, respectively.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Transforming growth factor β (TGF-β) is a critical regulator of bone density owing to its multiple effects on cell growth and differentiation. Recently, we have shown that TGF-β
effectively blocks ...bone morphogenetic protein (BMP) induced maturation of osteoblasts by upregulating histone deacetylase (HDAC) activity. The current study aimed at investigating the effect of rhTGF-β
treatment on the expression of specific HDACs and their cellular effects, e.g., microtubule structures (primary cilia) and mechanosensation. Exposure to TGF-β
most significantly induced expression of HDAC6 both on gene and protein level. Being most abundant in the cytoplasm HDAC6 effectively deacetylates microtubule structures. Thus, TGF-β
-induced expression of HDAC6 led to deformation and shortening of primary cilia as well as to reduced numbers of ciliated cells. Primary cilia are described to sense mechanical stimuli. Thus, fluid flow was applied to the cells, which stimulated osteoblast function (AP activity and matrix mineralization). Compromised primary cilia in TGF-β
-treated cells were associated with reduced osteogenic function, despite exposure to fluid flow conditions. Chemical inhibition of HDAC6 with Tubacin restored primary cilium structure and length. These cells showed improved osteogenic function especially under fluid flow conditions. Summarizing our results, TGF-β
impairs human osteoblast maturation partially via HDAC6-mediated distortion and/or shortening of primary cilia. This knowledge opens up new treatment options for trauma patients with chronically elevated TGF-β
-levels (e.g., diabetics), which frequently suffer from delayed fracture healing despite adequate mechanical stimulation.
Exposure to TGF-β
induces expression of HDAC6 in human osteoblasts. TGF-β
exposed human osteoblasts show less and distorted primary cilia. TGF-β
exposed human osteoblasts are less sensitive towards mechanical stimulation. Mechanosensation can be recovered by HDAC6 inhibitor Tubacin in human osteoblasts.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic ...density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research.
Microarray pitch can be used to precisely modulate the proliferation rate, scale and metabolic state of tumour spheroids.
The DNA-repair protein O(6)-methylguanine-DNA methyltransferase (alkyltransferase; MGMT) is a major determinant of resistance of cells to various alkylating cytostatic drugs. Its expression in ...tissues is highly variable, indicating complex regulatory mechanisms involved. Transfection-mediated expression of wild-type p53 has been shown to negatively regulate basal promoter activity of MGMT in vitro. To elucidate whether p53 is involved in regulation of MGMT in tumor tissue, we examined MGMT expression and the p53 status of 140 primary ovarian carcinomas and analyzed the data as to the correlation between MGMT and p53, as well as the survival response of the patients after chemotherapy. We show that MGMT expression is highly variable in ovarian carcinomas, ranging from zero level up to 2500 fmol/mg protein. MGMT activity was significantly lower in tumors with wild-type p53 (p53wt) than in tumors with mutant p53 (p53mt) (p = 0.045). As expected, the percentage of tumors with p53mt increased with increasing histologic grade of the tumors. Thus, p53mt was observed in 4, 45 and 64% of grades 1, 2 and 3 tumors, respectively (p = 0.001). Increase in p53mt was accompanied by an increase in MGMT activity, which was, on average, 460 +/- 66, 624 +/- 63 and 662 +/- 60 fmol/mg protein in grades 1, 2 and 3 tumors, respectively (p = 0.047). In addition, MGMT activity as well as p53mt were associated with the FIGO stage of the tumors. Mean MGMT activity was 472 +/- 48 fmol/mg for patients with FIGO stages I and II, as compared with 675 +/- 50 fmol/mg for patients with FIGO stages III and IV, (p = 0.0179). The percentage of p53mt was 27% and 54% in ovarian tumors with FIGO stages I/II and FIGO stages III/IV, respectively (p = 0.004). Thus, progression of ovarian tumors was clearly associated with increase of both MGMT activity and the percentage of p53mt. In tumors expressing low MGMT (<100 fmol/mg), p53mt was very rarely found. No significant association was observed between MGMT level in ovarian carcinomas and the survival of patients treated with cyclophosphamide and carboplatin. On the other hand, a clear correlation was found between histological type, grading, residual tumor mass and p53wt expression and duration of the patient's survival. The finding that p53wt expression was associated with low MGMT level in primary ovarian cancer supports the view that down-regulation of basal MGMT promoter activity by p53wt is also relevant in tumor cells in vivo. Int. J. Cancer (Pred. Oncol.) 84:388-395, 1999.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Small cell lung cancer (SCLC) is usually classified into a two-stage system, limited (LD) and extensive disease (ED). However, the criteria for these two categories remain controversial. The widely ...used Veterans Administration Lung Study Group (VALG) definition of LD includes patients with primary tumor and nodal involvement limited to one hemithorax. In contrast, the International Association for the Study of Lung Cancer (IASLC) recommends that LD should additionally include all patients without distant metastasis. As a consequence, since treatment modalities for LD and ED could be different, individual clinical outcome of SCLC patients may be influenced by the staging system chosen. Among 109 consecutive SCLC patients treated in our clinic between 1989 and 1999 (mean age 68±9.1 years, 81% male) 23 patients (21%) could be either classified as LD or ED (LD-ED), depending on the staging system used. The prognosis of this overlapping group (LD-ED: median survival 291 days) was not statistically different from patients with limited disease defined by VALG criteria (LD-VALG: 385 days, log–rank test
P=0.42). On the other hand the survival difference between LD-ED patients and the ED-IASLC population was relevant (ED-IASLC: 208 days,
P=0.05), indicating that LD-ED patients should rather be included in the LD category. This is further supported by the results of a multivariate Cox regression analysis with all clinically relevant data. Only stage as defined by IASLC criteria was an independent prognostic factor in the likelihood-ratio-forward (hazard ratio=1.94, CI=1.26–2.99;
P=0.005) and backward model (hazard ratio=1.76, CI: 1.12–2.76;
P=0.012), confirming the higher discriminatory power of the IASLC definition. In conclusion, the IASLC staging criteria for SCLC patients have a higher prognostic impact and are therefore preferable in clinical practice and future therapeutic trials.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Signaling through the Wnt/beta-catenin pathway is a crucial determinant of hepatic zonal gene expression, liver development, regeneration, and tumorigenesis. Transgenic mice with hepatocyte-specific ...knockout of Ctnnb1 (encoding beta-catenin) have proven their usefulness in elucidating these processes. We now found that a small number of hepatocytes escape the Cre-mediated gene knockout in that mouse model. The remaining beta-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the beta-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated beta-catenin. Surprisingly, the expression pattern was observed independent of the cell's position within the liver lobule, suggesting a malfunction of physiological periportal repression of perivenously expressed genes in beta-catenin-deficient liver. Clusters of beta-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, beta-catenin-positive hepatocytes had no striking proliferative advantage, but started to grow out on treatment with phenobarbital, a tumor-promoting agent known to facilitate the formation of mouse liver adenoma with activating mutations of Ctnnb1. Progressive re-population of Ctnnb1 knockout livers with wild-type hepatocytes was seen in aged mice with a pre-cirrhotic phenotype. In these large clusters of beta-catenin-expressing hepatocytes, perivenous-specific gene expression was re-established. In summary, our data demonstrate that the zone-specificity of a hepatocyte's gene expression profile is dependent on the presence of beta-catenin, and that beta-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment. PUBLICATION ABSTRACT
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, VSZLJ, ZAGLJ
Stem cell-based in vitro test systems can recapitulate specific phases of human development. In the UKK test system, human pluripotent stem cells (hPSCs) randomly differentiate into cells of the ...three germ layers and their derivatives. In the UKN1 test system, hPSCs differentiate into early neural precursor cells. During the normal differentiation period (14 days) of the UKK system, 570 genes 849 probe sets (PSs) were regulated >fivefold; in the UKN1 system (6 days), 879 genes (1238 PSs) were regulated. We refer to these genes as 'developmental genes'. In the present study, we used genome-wide expression data of 12 test substances in the UKK and UKN1 test systems to understand the basic principles of how chemicals interfere with the spontaneous transcriptional development in both test systems. The set of test compounds included six histone deacetylase inhibitors (HDACis), six mercury-containing compounds ('mercurials') and thalidomide. All compounds were tested at the maximum non-cytotoxic concentration, while valproic acid and thalidomide were additionally tested over a wide range of concentrations. In total, 242 genes (252 PSs) in the UKK test system and 793 genes (1092 PSs) in the UKN1 test system were deregulated by the 12 test compounds. We identified sets of 'diagnostic genes' appropriate for the identification of the influence of HDACis or mercurials. Test compounds that interfered with the expression of developmental genes usually antagonized their spontaneous development, meaning that up-regulated developmental genes were suppressed and developmental genes whose expression normally decreases were induced. The fraction of compromised developmental genes varied widely between the test compounds, and it reached up to 60 %. To quantitatively describe disturbed development on a genome-wide basis, we recommend a concept of two indices, 'developmental potency' (D
) and 'developmental index' (D
), whereby D
is the fraction of all developmental genes that are up- or down-regulated by a test compound, and D
is the ratio of overrepresentation of developmental genes among all genes deregulated by a test compound. The use of D
makes hazard identification more sensitive because some compounds compromise the expression of only a relatively small number of genes but have a high propensity to deregulate developmental genes specifically, resulting in a low D
but a high D
. In conclusion, the concept based on the indices D
and D
offers the possibility to quantitatively express the propensity of test compounds to interfere with normal development.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The gene CTNNB1 encoding β-catenin is mutated in about 30% of hepatocellular carcinoma, generally often combined with other genetic alterations. In transgenic mice, it has been shown that activation ...of β-catenin in more than 70% of all hepatocytes causes immediate proliferation leading to hepatomegaly. In this study we established a novel mouse model where β-catenin is activated only in individual, dispersed hepatocytes. Hepatocyte-specific expression of activated point-mutated β-catenin (human β-catenin^sup S33Y^) was established using the Cre/loxP system. Expression of several downstream targets of β-catenin signaling such as glutamine synthetase and several cytochrome P450 isoforms was confirmed by immunostaining. Only a minor portion of hepatocytes expressed the β-catenin^sup S33Y^ transgene, which were mainly positioned as dispersed individual cells within the normal liver parenchyma. The hepatocytes with activated β-catenin did not show increased proliferation and the mice did not develop hepatomegaly. In conclusion, activated β-catenin in single hepatocytes induces a gene expression pattern in hepatocytes which is similar to that of Ctnnb1-mutated mouse liver tumors, but is apparently not sufficient to induce increased cell proliferation. Therefore, onset of proliferation seems to require concomitant activation of β-catenin in clusters of hepatocytes, suggesting a role of cell-cell communication in this process.PUBLICATION ABSTRACT
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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