90 research outputs found

    Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms

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    Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3′-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability

    Apoptosis control and proliferation marker in human normal and neoplastic adrenocortical tissues

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    We evaluated by immunohistochemistry the expression of the Bcl-2 and p53 proteins, as markers of apoptosis control, and of MIB-1, as a marker of cell proliferation, in a series of normal and neoplastic adrenocortical tissues. The specimens were 13 normal adrenals, 13 aldosterone-producing adenomas, 13 non-functioning adenomas and 16 carcinomas. Results were calculated as percentage of immunostained cells by using specific antibodies. No p53 protein was detected in any of the adrenocortical adenomas (functioning and non functioning) or normal adrenals, while p53 was overexpressed in 15 out of 16 carcinomas. In particular, 10 adrenal cancer specimens (62.5%) showed strong staining in a high percentage (range 10–50%) of the malignant cells. The percentage of Bcl-2 positive cells was higher (P<0.05 or less) in non-functioning adenomas (8.1±1.9%) and in carcinomas (14.9±5.6%) than in normals (2.9±0.9%) and aldosterone-producing adenomas (5.3±1.3%) since four specimens of the non-functioning adenomas-group (30.7%) and six of the carcinomas-group (37.5%) showed over 10% positivity (cut-off for normal values, set at 90th percentile of our controls). MIB-1 positivity was 0.50±0.36% in normals, 0.54±0.08% in non-functioning adenomas and 0.54±0.08% in aldosterone-producing adenomas. MIB-1 was expressed in all carcinomas with values (13.7±3.1%) significantly (P<0.0006) higher than in the other groups. In conclusion, the present data indicate that the apoptosis control and proliferation activity evaluated by the p53 and MIB-1 proteins are impaired in adrenal carcinomas but preserved in adenomas, independently of their functional status. Therefore, these immunohistochemical markers, overexpressed in carcinomas only, may be useful in the diagnosis of malignancy in adrenocortical tumours. Whether Bcl-2 positivity found in some carcinomas and non-functioning adenomas may constitute, in the latter, a negative prognostic marker is still unknown

    Initial Characterization of the FlgE Hook High Molecular Weight Complex of

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    The spirochete periplasmic flagellum has many unique attributes. One unusual characteristic is the flagellar hook. This structure serves as a universal joint coupling rotation of the membrane-bound motor to the flagellar filament. The hook is comprised of about 120 FlgE monomers, and in most bacteria these structures readily dissociate to monomers (∼ 50 kDa) when treated with heat and detergent. However, in spirochetes the FlgE monomers form a large mass of over 250 kDa [referred to as a high molecular weight complex (HMWC)] that is stable to these and other denaturing conditions. In this communication, we examined specific aspects with respect to the formation and structure of this complex. We found that the Lyme disease spirochete Borrelia burgdorferi synthesized the HMWC throughout the in vitro growth cycle, and also in vivo when implanted in dialysis membrane chambers in rats. The HMWC was stable to formic acid, which supports the concept that the stability of the HMWC is dependent on covalent cross-linking of individual FlgE subunits. Mass spectrometry analysis of the HMWC from both wild type periplasmic flagella and polyhooks from a newly constructed ΔfliK mutant indicated that other proteins besides FlgE were not covalently joined to the complex, and that FlgE was the sole component of the complex. In addition, mass spectrometry analysis also indicated that the HMWC was composed of a polymer of the FlgE protein with both the N- and C-terminal regions remaining intact. These initial studies set the stage for a detailed characterization of the HMWC. Covalent cross-linking of FlgE with the accompanying formation of the HMWC we propose strengthens the hook structure for optimal spirochete motility

    Electronic structure, linear, nonlinear optical susceptibilities and birefringence of CuInX2 (X = S, Se, Te) chalcopyrite-structure compounds

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    The electronic structure, linear and nonlinear optical properties have been calculated for CuInX2 (X=S, Se, Te) chalcopyrite-structure single crystals using the state-of-the-art full potential linear augmented plane wave (FP-LAPW) method. We present results for band structure, density of states, and imaginary part of the frequency-dependent linear and nonlinear optical susceptibilities. We find that these crystals are semiconductors with direct band gaps. We have calculated the birefringence of these crystals. The birefringence is negative for CuInS2 and CuInSe2 while it is positive for CuInTe2 in agreement with the experimental data. Calculations are reported for the frequency-dependent complex second-order non-linear optical susceptibilities . The intra-band and inter-band contributions to the second harmonic generation increase when we replace S by Se and decrease when we replace Se by Te. We find that smaller energy band gap compounds have larger values of in agreement with the experimental data and previous theoretical calculations.Comment: 17 pages, 6 figure

    Genomics and proteomics approaches to the study of cancer-stroma interactions

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    <p>Abstract</p> <p>Background</p> <p>The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.</p> <p>Methods</p> <p>The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.</p> <p>Results</p> <p>We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (<it>ARID4A</it>, <it>CALR</it>, <it>GNB2L1</it>, <it>RNF10</it>, <it>SQSTM1</it>, <it>USP9X</it>) were validated by real time PCR.</p> <p>Conclusions</p> <p>A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.</p
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