939 resultados para alterations
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Purpose: The cytomegalovirus (CMV) promoter is one of the most commonly used promoters for expression of transgenes in mammalian cells. The aim of our study was to evaluate the role of methylation and upregulation of the CMV promoter by irradiation and the chemotherapeutic agent cisplatin in vivo using non-invasive fluorescence in vivo imaging. Procedures: Murine fibrosarcoma LPB and mammary carcinoma TS/A cells were stably transfected with plasmids encoding CMV and p21 promoter-driven green fluorescent protein (GFP) gene. Solid TS/A tumors were induced by subcutaneous injection of fluorescent tumor cells, while leg muscles were transiently transfected with plasmid encoding GFP under the control of the CMV promoter. Cells, tumors, and legs were treated either by DNA methylation inhibitor 5-azacytidine, irradiation, or cisplatin. GFP expression was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging in vivo. Results: Treatment of cells, tumors, and legs with 5-azacytidine (re)activated the CMV promoter. Furthermore, treatment with irradiation or cisplatin resulted in significant upregulation of GFP expression both in vitro and in vivo. Conclusions: Observed alterations in the activity of the CMV promoter limit the usefulness of this widely used promoter as a constitutive promoter. On the other hand, inducibility of CMV promoters can be beneficially used in gene therapy when combined with standard cancer treatment, such as radiotherapy and chemotherapy. © 2010 The Author(s).
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Nowadays, the emergence of resistance to the current available chemotherapeutic drugs by cancer cells makes the development of new agents imperative. The skin secretion of amphibians is a natural rich source of antimicrobial peptides (AMP), and researchers have shown that some of these wide spectrum molecules are also toxic to cancer cells. The aim of this study was to verify a putative anticancer activity of the AMP pentadactylin isolated for the first time from the skin secretion of the frog Leptodactylus labyrinthicus and also to study its cytotoxic mechanism to the murine melanoma cell line B16F10. The results have shown that pentadactylin reduces the cell viability of B16F10 cells in a dose-dependent manner. It was also cytotoxic to normal human fibroblast cells; nevertheless, pentadactylin was more potent in the first case. The studies of action mechanism revealed that pentadactylin causes cell morphology alterations (e.g., round shape and shrinkage morphology), membrane disruption, DNA fragmentation, cell cycle arrest at the S phase, and alteration of mitochondrial membrane potential, suggesting that B16F10 cells die by apoptosis. The exact mechanism that causes reduction of cell viability and cytotoxicity after treatment with pentadactylin is still unknown. In conclusion, as cancer cells become resilient to death, it is worthwhile the discovery of new drugs such as pentadactylin that induces apoptosis.
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Until recently, the low-abundance (LA) range of the serum proteome was an unexplored reservoir of diagnostic information. Today it is increasingly appreciated that a diagnostic goldmine of LA biomarkers resides in the blood stream in complexed association with more abundant higher molecular weight carrier proteins such as albumin and immunoglobulins. As we now look to the possibility of harvesting these LA biomarkers more efficiently through engineered nano-scale particles, mathematical approaches are needed in order to reveal the mechanisms by which blood carrier proteins act as molecular 'mops' for LA diagnostic cargo, and the functional relationships between bound LA biomarker concentrations and other variables of interest such as biomarker intravasation and clearance rates and protein half-lives in the bloodstream. Here we show, by simple mathematical modeling, how the relative abundance of large carrier proteins and their longer half-lives in the bloodstream work together to amplify the total blood concentration of these tiny biomarkers. The analysis further suggests that alterations in the production of biomarkers lead to gradual rather than immediate changes in biomarker levels in the blood circulation. The model analysis also points to the characteristics of artificial nano-particles that would render them more efficient harvesters of tumor biomarkers in the circulation, opening up possibilities for the early detection of curable disease, rather than simply better detection of advanced disease.
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Web servers are accessible by anyone who can access the Internet. Although this universal accessibility is attractive for all kinds of Web-based applications, Web servers are exposed to attackers who may want to alter their contents. Alterations range from humorous additions or changes, which are typically easy to spot, to more sinister tampering, such as providing false or damaging information.
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The thesis investigates “where were the auditors in asset securitizations”, a criticism of the audit profession before and after the onset of the global financial crisis (GFC). Asset securitizations increase audit complexity and audit risks, which are expected to increase audit fees. Using US bank holding company data from 2003 to 2009, this study examines the association between asset securitization risks and audit fees, and its changes during the global financial crisis. The main test is based on an ordinary least squares (OLS) model, which is adapted from the Fields et al. (2004) bank audit fee model. I employ a principal components analysis to address high correlations among asset securitization risks. Individual securitization risks are also separately tested. A suite of sensitivity tests indicate the results are robust. These include model alterations, sample variations, further controls in the tests, and correcting for the securitizer self-selection problem. A partial least squares (PLS) path modelling methodology is introduced as a separate test, which allows for high intercorrelations, self-selection correction, and sequential order hypotheses in one simultaneous model. The PLS results are consistent with the main results. The study finds significant and positive associations between securitization risks and audit fees. After the commencement of the global financial crisis in 2007, there was an increased focus on the role of audits on asset securitization risks resulting from bank failures; therefore I expect that auditors would become more sensitive to bank asset securitization risks after the commencement of the crisis. I find that auditors appear to focus on different aspects of asset securitization risks during the crisis and that auditors appear to charge a GFC premium for banks. Overall, the results support the view that auditors consider asset securitization risks and market changes, and adjust their audit effort and risk considerations accordingly.
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Molecular interactions that underlie pathophysiological states are being elucidated using techniques that profile proteomicend points in cellular systems. Within the field of cancer research, protein interaction networks play pivotal roles in the establishment and maintenance of the hallmarks of malignancy, including cell division, invasion, and migration. Multiple complementary tools enable a multifaceted view of how signal protein pathway alterations contribute to pathophysiological states.One pivotal technique is signal pathway profiling of patient tissue specimens. This microanalysis technology provides a proteomic snapshot at one point in time of cells directly procured from the native context of a tumor micro environment. To study the adaptive patterns of signal pathway events over time, before and after experimental therapy, it is necessary to obtain biopsies from patients before, during, and after therapy. A complementary approach is the profiling of cultured cell lines with and without treatment. Cultured cell models provide the opportunity to study short-term signal changes occurring over minutes to hours. Through this type of system, the effects of particular pharmacological agents may be used to test the effects of signal pathway inhibition or activation on multiple endpoints within a pathway. The complexity of the data generated has necessitated the development of mathematical models for optimal interpretation of interrelated signaling pathways. In combination,clinical proteomic biopsy profiling, tissue culture proteomic profiling, and mathematical modeling synergistically enable a deeper understanding of how protein associations lead to disease states and present new insights into the design of therapeutic regimens.
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miR-126 has been implicated in the processes of inflammation and angiogenesis. Through these processes, miR-126 is implicated in cancer biology, but its role there has not been well reviewed. The aim of this review is to examine the molecular mechanisms and clinicopathological significance of miR-126 in human cancers. miR-126 was shown to have roles in cancers of the gastrointestinal tract, genital tracts, breast, thyroid, lung and some other cancers. Its expression was suppressed in most of the cancers studied. The molecular mechanisms that are known to cause aberrant expression of miR-126 include alterations in gene sequence, epigenetic modification and alteration of dicer abundance. miR-126 can inhibit progression of some cancers via negative control of proliferation, migration, invasion, and cell survival. In some instances, however, miR-126 supports cancer progression via promotion of blood vessel formation. Downregulation of miR-126 induces cancer cell proliferation, migration, and invasion via targeting specific oncogenes. Also, reduced levels of miR-126 are a significant predictor of poor survival of patients in many cancers. In addition, miR-126 can alter a multitude of cellular mechanisms in cancer pathogenesis via suppressing gene translation of numerous validated targets such as PI3K, KRAS, EGFL7, CRK, ADAM9, HOXA9, IRS-1, SOX-2, SLC7A5 and VEGF. To conclude, miR-126 is commonly down-regulated in cancer, most likely due to its ability to inhibit cancer cell growth, adhesion, migration, and invasion through suppressing a range of important gene targets. Understanding these mechanisms by which miR-126 is involved with cancer pathogenesis will be useful in the development of therapeutic targets for the management of patients with cancer.
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PURPOSE Colorectal signet-ring cell carcinoma (SRCC) is rare, and very little detailed information on the molecular biology of the disease is available. METHODS The literature on the clinical, pathological and, in particular, the molecular biology of this rare entity was critically reviewed. The reviewed articles take into account a total of 1,817 cases of SRCC, but only 143 cases have molecular data available. The characteristics of two patients with colorectal SRCC were also discussed. RESULTS Colorectal SRCC mostly occurs in younger patients, is larger and has different site predilection compared with conventional colorectal adenocarcinoma. It can occur as one of the synchronous cancers in the colorectum. The cancer is usually diagnosed at advanced stages because of the late manifestation of symptoms, and aggressive treatment strategy is required. Limited reports in the literature have shown that the variant of colorectal cancer demonstrated a different pattern of genetic alterations of common growth kinase-related oncogenes (K-ras, BRAF), tumour suppressor genes (p53, p16), gene methylation and cell adhesion-related genes related to the Wingless signalling pathway (E-cadherin and beta-catenin) from conventional colorectal adenocarcinoma. Colorectal SRCC also showed high expression of mucin-related genes and genes related to the gastrointestinal system. There was also a higher prevalence of microsatellite instability-high tumours and low Cox-2 expression in colorectal SRCC as opposed to conventional adenocarcinoma. CONCLUSIONS Colorectal SRCC has unique molecular pathological features. The unique molecular profiles in SRCC may provide molecular-based improvements to patient management in colorectal SRCC.
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Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial dysfunction and vascular remodeling. Recently, bone marrow progenitor cells have been localized to PAH lungs, raising the question of their role in disease progression. Independently, serotonin (5-HT) and its receptors have been identified as contributors to the PAH pathogenesis. We hypothesized that 1 of these receptors, 5-HT(2B), is involved in bone marrow stem cell mobilization that participates in the development of PAH and pulmonary vascular remodeling. A first study revealed expression of 5-HT(2B) receptors by circulating c-kit(+) precursor cells, whereas mice lacking 5-HT(2B) receptors showed alterations in platelets and monocyte-macrophage numbers, and in myeloid lineages of bone marrow. Strikingly, mice with restricted expression of 5-HT(2B) receptors in bone marrow cells developed hypoxia or monocrotaline-induced increase in pulmonary pressure and vascular remodeling, whereas restricted elimination of 5-HT(2B) receptors on bone marrow cells confers a complete resistance. Moreover, ex vivo culture of human CD34(+) or mice c-kit(+) progenitor cells in the presence of a 5-HT(2B) receptor antagonist resulted in altered myeloid differentiation potential. Thus, we demonstrate that activation of 5-HT(2B) receptors on bone marrow lineage progenitors is critical for the development of PAH.
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Purpose We examined the age-dependent alterations and longitudinal course of subbasal nerve plexus (SNP) morphology in healthy individuals. Methods Laser-scanning corneal confocal microscopy, ocular screening, and health and metabolic assessment were performed on 64 healthy participants at baseline and at 12-month intervals for 3 years. At each annual visit, eight central corneal images of the SNP were selected and analyzed using a fully-automated analysis system to quantify corneal nerve fiber length (CNFL). Two linear mixed model approaches were fitted to examine the relationship between age and CNFL, and the longitudinal changes of CNFL over three years. Results At baseline, mean age was 51.9 ± 14.7 years. The cohort was sex balanced (χ2 = 0.56, P = 0.45). Age (t = 1.6, P = 0.12) and CNFL (t = -0.50, P = 0.62) did not differ between sexes. A total of 52 participants completed the 36-month visit and 49 participants completed all visits. Age had a significant effect on CNFL (F1,33 = 5.67, P = 0.02) with a linear decrease of 0.05 mm/mm2 in CNFL per one year increase in age. No significant change in CNFL was observed over the 36-month period (F1,55 = 0.69, P = 0.41). Conclusions The CNFL showed a stable course over a 36-month period in healthy individuals, although there was a slight linear reduction in CNFL with age. The findings of this study have implications for understanding the time-course of the effect of pathology and surgical or therapeutic interventions on the morphology of the SNP, and serves to confirm the suitability of CNFL as a screening/monitoring marker for peripheral neuropathies.
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Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.
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Lymphatic vessels guide interstitial fluid, modulate immune responses by regulating leukocyte and antigen trafficking to lymph nodes, and in a cancer setting enable tumor cells to track to regional lymph nodes. The aim of the study was to determine whether primary murine lymphatic endothelial cells (mLECs) show conserved vascular endothelial growth factor (VEGF) signaling pathways with human LECs (hLECs). LECs were successfully isolated from murine dermis and prostate. Similar to hLECs, vascular endothelial growth factor (VEGF) family ligands activated MAPK and pAkt intracellular signaling pathways in mLECs. We describe a robust protocol for isolation of mLECs which, by harnessing the power of transgenic and knockout mouse models, will be a useful tool to study how LEC phenotype contributes to alterations in lymphatic vessel formation and function.
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Chronic kidney disease (CKD) in ageing is a burden on health systems worldwide. Rat models of age-related CKD linked with obesity and hypertension were used to investigate alterations in oxidant handling and energy metabolism to identify gene targets or markers for age-related CKD. Young adult (3 months) and old (21–24 months) spontaneously-hypertensive (SHR), normotensive Wistar-Kyoto (WKY) and Wistar rats (normotensive, obese in ageing) were compared for renal functional and physiological parameters, renal fibrosis and inflammation, oxidative stress (hemeoxygenase-1/HO-1), apoptosis and cell injury (including Bax:Bcl-2), phosphorylated and non-phosphorylated forms of oxidant and energy sensing proteins (p66Shc, AMPK), signal transduction proteins (ERK1/2, PKB), and transcription factors (NF-κB, FoxO1). All old rats were normoglycemic. Renal fibrosis, tubular epithelial apoptosis, interstitial macrophages and myofibroblasts (all p < 0.05), p66Shc/phospho-p66 (p < 0.05), Bax/Bcl-2 ratio (p < 0.05) and NF-κB expression (p < 0.01) were highest in old obese Wistars. Expression of phospho-FoxO/FoxO was elevated in old Wistars (p < 0.001) and WKYs (p < 0.01). SHRs had high levels in young and old rats. Expression of PKB, phospho-PKB, ERK1/2 and phospho-ERK1/2 were significantly elevated in all aged animals. These results suggest that obesity and hypertension have differing oxidant handling and signalling pathways that act in the pathogenesis of age-related CKD
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The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.
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Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.