Functional gene expression profile underlying methotrexate-induced senescence in human colon cancer cells

Cellular functions accompanying establishment of premature senescence in methotrexate-treated human colon cancer C85 cells are deciphered in the present study from validated competitive expression microarray data, analyzed with the use of Ingenuity Pathways Analysis (IPA) software. The nitrosative/oxidative stress, inferred from upregulated expression of inducible nitric oxide synthase (iNOS) and mitochondrial dysfunction-associated genes, including monoamine oxidases MAOA and MAOB, β-amyloid precursor protein (APP) and presenilin 1 (PSEN1), is identified as the main determinant of signaling pathways operating during senescence establishment. Activation of p53-signaling pathway is found associated with both apoptotic and autophagic components contributing to this process. Activation of nuclear factor κB (NF-κB), resulting from interferon γ (IFNγ), integrin, interleukin 1β (IL-1β), IL-4, IL-13, IL-22, Toll-like receptors (TLRs) 1, 2 and 3, growth factors and tumor necrosis factor (TNF) superfamily members signaling, is found to underpin inflammatory properties of senescent C85 cells. Upregulation of p21-activated kinases (PAK2 and PAK6), several Rho molecules and myosin regulatory light chains MYL12A and MYL12B, indicates acquisition of motility by those cells. Mitogen-activated protein kinase p38 MAPK β, extracellular signal-regulated kinases ERK2 and ERK5, protein kinase B AKT1, as well as calcium, are identified as factors coordinating signaling pathways in senescent C85 cells

The place of values in the aims of school science education

Debates about the aims of school science education are perennial (e.g., Reiss & White, 2014; see also Kidman & Fensham, Chapter “ Intended, Achieved and Unachieved Values of Science Education” this volume), particularly in Western cultures. In this chapter we review some of these arguments about the aims of school science education, and look at what has changed in the last decade since one of us (Michael) considered a similar debate (see Reiss, 2007). We have situated this review of arguments in current global circumstances including rapid technological advances, a continuing demand for workers with STEM (Science, Technology, Engineering and Mathematics) qualifications and the increasing acknowledgement of the deeply worrying effects that humans have on the Earth’s ecology, and indeed its future. Part of our argument is that decisions about the aims of school science education are inevitably decisions about values in education in general and values in school science education more specifically. This means that for a country, a group of schools, an individual school or a classroom teacher to come to a view about the aims of science education in the classroom is to have made a judgement, implicitly or explicitly, about values

The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12).In this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-alpha, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-alpha, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with >or=50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-alpha are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs.The HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-alpha release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen

The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation

The ataxia telangiectasia mutated (ATM) and the ATM- related (ATR) kinases play a central role in facilitating the resistance of cancer cells to genotoxic treatment regimens. The components of the ATM and ATR regulated signaling pathways thus provide attractive pharmacological targets, since their inhibition enhances cellular sensitivity to chemo- and radiotherapy. Caffeine as well as more specific inhibitors of ATM (KU55933) or ATM and ATR (CGK733) have recently been shown to induce cell death in drug-induced senescent tumor cells. Addition of these agents to cancer cells previously rendered senescent by exposure to genotoxins suppressed the ATM mediated p21 expression required for the survival of these cells. The precise molecular pharmacology of these agents however, is not well characterized. Herein, we report that caffeine, CGK733, and to a lesser extent KU55933, inhibit the proliferation of otherwise untreated human cancer and non-transformed mouse fibroblast cell lines. Exposure of human cancer cell lines to caffeine and CGK733 was associated with a rapid decline in cyclin D1 protein levels and a reduction in the levels of both phosphorylated and total retinoblastoma protein (RB). Our studies suggest that observations based on the effects of these compounds on cell proliferation and survival must be interpreted with caution. The differential effects of caffeine/CGK733 and KU55933 on cyclin D1 protein levels suggest that these agents will exhibit dissimilar molecular pharmacological profiles

Endocytosis of plasma-derived factor V by megakaryocytes occurs via a clathrin-dependent, specific membrane binding event

Megakaryocytes were analyzed for their ability to endocytose factor V to define the cellular mechanisms regulating this process. In contrast to fibrinogen, factor V was endocytosed by megakaryocytes derived from CD34 + cells or megakaryocyte-like cell lines, but not by platelets. CD41 + ex vivo -derived megakaryocytes endocytosed factor V, as did subpopulations of the megakaryocyte-like cells MEG-01, and CMK. Similar observations were made for fibrinogen. Phorbol diester-induced megakaryocytic differentiation of the cell lines resulted in a substantial increase in endocytosis of both proteins as compared to untreated cells that did not merely reflect their disparate plasma concentrations. Factor IX, which does not associate with platelets or megakaryocytes, was not endocytosed by any of the cells examined. Endocytosis of factor V by megakaryocytes proceeds through a specific and independent mechanism as CHRF-288 cells endocytosed fibrinogen but not factor V, and the presence of other plasma proteins had no effect on the endocytosis of factor V by MEG-01 cells. Furthermore, as the endocytosis of factor V was also demonstrated to occur through a clathrin-dependent mechanism, these combined data demonstrate that endocytosis of factor V by megakaryocytes occurs via a specific, independent, and most probably receptor-mediated, event.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75473/1/j.1538-7836.2005.01190.x.pd

Importance of Intracellular pH in Determining the Uptake and Efficacy of the Weakly Basic Chemotherapeutic Drug, Doxorubicin

Low extracellular pH (pHe), that is characteristic of many tumours, tends to reduce the uptake of weakly basic drugs, such as doxorubicin, thereby conferring a degree of physiological resistance to chemotherapy. It has been assumed, from pH-partition theory, that the effect of intracellular pH (pHi) is symmetrically opposite, although this has not been tested experimentally. Doxorubicin uptake into colon HCT116 cells was measured using the drug's intrinsic fluorescence under conditions that alter pHi and pHe or pHi alone. Acutely, doxorubicin influx across the cell-membrane correlates with the trans-membrane pH-gradient (facilitated at alkaline pHe and acidic pHi). However, the protonated molecule is not completely membrane-impermeant and, therefore, overall drug uptake is less pHe-sensitive than expected from pH-partitioning. Once inside cells, doxorubicin associates with slowly-releasing nuclear binding sites. The occupancy of these sites increases with pHi, such that steady-state drug uptake can be greater with alkaline cytoplasm, in contradiction to pH-partition theory. Measurements of cell proliferation demonstrate that doxorubicin efficacy is enhanced at alkaline pHi and that pH-partition theory is inadequate to account for this. The limitations in the predictive power of pH-partition theory arise because it only accounts for the pHi/pHe-sensitivity of drug entry into cells but not the drug's subsequent interactions that, independently, show pHi-dependence. In summary, doxorubicin uptake into cells is favoured by high pHe and high pHi. This modified formalism should be taken into account when designing manoeuvres aimed at increasing doxorubicin efficacy

Caspase-dependent and -independent suppression of apoptosis by monoHER in Doxorubicin treated cells

Doxorubicin (DOX) is an antitumour agent for different types of cancer, but the dose-related cardiotoxicity limits its clinical use. To prevent this side effect we have developed the flavonoid monohydroxyethylrutoside (monoHER), a promising protective agent, which did not interfere with the antitumour activity of DOX. To obtain more insight in the mechanism underlying the selective protective effects of monoHER, we investigated whether monoHER (1 mM) affects DOX-induced apoptosis in neonatal rat cardiac myocytes (NeRCaMs), human endothelial cells (HUVECs) and the ovarian cancer cell lines A2780 and OVCAR-3. DOX-induced cell death was effectively reduced by monoHER in heart, endothelial and A2780 cells. OVCAR-3 cells were highly resistant to DOX-induced apoptosis. Experiments with the caspase-inhibitor zVAD-fmk showed that DOX-induced apoptosis was caspase-dependent in HUVECs and A2780 cells, whereas caspase-independent mechanisms seem to be important in NeRCaMs. MonoHER suppressed DOX-dependent activation of the mitochondrial apoptotic pathway in normal and A2780 cells as illustrated by p53 accumulation and activation of caspase-9 and -3 cleavage. Thus, monoHER acts by suppressing the activation of molecular mechanisms that mediate either caspase-dependent or -independent cell death. In light of the current work and our previous studies, the use of clinically achievable concentrations of monoHER has no influence on the antitumour activity of DOX whereas higher concentrations as used in the present study could influence the antitumour activity of DOX

DNA damage induced by cis- and carboplatin as indicator for in vitro sensitivity of ovarian carcinoma cells

<p>Abstract</p> <p>Background</p> <p>The DNA damage by platinum cytostatics is thought to be the main cause of their cytotoxicity. Therefore the measurement of the DNA damage induced by cis- and carboplatin should reflect the sensitivity of cancer cells toward the platinum chemotherapeutics.</p> <p>Methods</p> <p>DNA damage induced by cis- and carboplatin in primary cells of ovarian carcinomas was determined by the alkaline comet assay. In parallel, the reduction of cell viability was measured by the fluorescein diacetate (FDA) hydrolysis assay.</p> <p>Results</p> <p>While in the comet assay the isolated cells showed a high degree of DNA damage after a 24 h treatment, cell viability revealed no cytotoxicity after that incubation time. The individual sensitivities to DNA damage of 12 tumour biopsies differed up to a factor of about 3. DNA damage after a one day treatment with cis- or carboplatin correlated well with the cytotoxic effects after a 7 day treatment (r = 0,942 for cisplatin r = 0.971 for carboplatin). In contrast to the platinum compounds the correlation of DNA damage and cytotoxicity induced by adriamycin was low (r = 0,692), or did not exist for gemcitabine.</p> <p>Conclusion</p> <p>The measurement of DNA damage induced by cis- and carboplatin is an accurate method to determine the in vitro chemosensitivity of ovarian cancer cells towards these cytostatics, because of its quickness, sensitivity, and low cell number needed.</p

Pneumocystis cell wall β-glucan stimulates calcium-dependent signaling of IL-8 secretion by human airway epithelial cells

<p>Abstract</p> <p>Background</p> <p>Respiratory failure secondary to alveolar inflammation during <it>Pneumocystis </it>pneumonia is a major cause of death in immunocompromised patients. Neutrophil infiltration in the lung of patients with <it>Pneumocystis </it>infection predicts severity of the infection and death. Several previous studies indicate that airway epithelial cells release the neutrophil chemoattractant proteins, MIP-2 (rodents) and IL-8 (humans), in response to <it>Pneumocystis </it>and purified <it>Pneumocystis </it>cell wall β-glucans (PCBG) through the NF-κB-dependent pathway. However, little is known about the molecular mechanisms that are involved in the activation of airway epithelium cells by PCBG resulting in the secretion of IL-8.</p> <p>Method</p> <p>To address this, we have studied the activation of different calcium-dependent mitogen-activated protein kinases (MAPKs) in 1HAEo^-cells, a human airway epithelial cell line.</p> <p>Results</p> <p>Our data provide evidence that PCBG induces phosphorylation of the MAPKs, ERK, and p38, the activation of NF-κB and the subsequently secretion of IL-8 in a calcium-dependent manner. Further, we evaluated the role of glycosphingolipids as possible receptors for β-glucans in human airway epithelial cells. Preincubation of the cells with D-<it>threo</it>-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) a potent inhibitor of the glycosphingolipids synthesis, prior to PCBG stimulation, significantly decreased IL-8 production.</p> <p>Conclusion</p> <p>These data indicate that PCBG activates calcium dependent MAPK signaling resulting in the release of IL-8 in a process that requires glycosphingolipid for optimal signaling.</p