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BackgroundReliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness.MethodsTight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (CCL) in real-time.Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed.ResultsThe four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells.ConclusionsUnder the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products.
In many real world applications, labeled data are in short supply. It often happens that obtaining labeled data in a new domain is expensive and time consuming, while there may be plenty of labeled data from a related but different domain. Traditional machine learning is not able to cope well with learning across different domains. In this paper, we address this problem for a text-mining task, where the labeled data are under one distribution in one domain known as in-domain data, while the unlabeled data are under a related but different domain known as out-of-domain data. Our general goal is to learn from the in-domain and apply the learned knowledge to out-of-domain. We propose a coclustering based classification (CoCC) algorithm to tackle this problem. Co-clustering is used as a bridge to propagate the class structure and knowledge from the in-domain to the out-of-domain. We present theoretical and empirical analysis to show that our algorithm is able to produce high quality classification results, even when the distributions between the two data are different. The experimental results show that our algorithm greatly improves the classification performance over the traditional learning algorithms.
The prosurvival activity of phosphoinositide 3 kinase (PI3K)/Akt (also known as protein kinase B, PKB) pathway has been investigated in great detail in human physiology and disease. Accumulating evidence is emerging that this signaling axis also actively engages with the migratory process in motile cells, including metastatic cancer cells. Interference with the role of PI3K/Akt-mediated cell motility impairs cellular development and attenuates malignant progression of cancer metastasis. Because metastasis is responsible for 90% of mortality in cancer patients, the acceleration of cancer cell spreading observed in association with hyperactivation of the PI3K pathway, triggered for example by chemotherapy/radiotherapy in the clinic, has heightened awareness of the conflict between "good drugs" and unfavorable effects. Here, we discuss recent studies on PI3K/Akt-regulated cell motility in both physiological and pathological settings, with the aim of a better understanding of how activities of the PI3K/Akt axis initiate and transmit "migratory signals" that stimulate cell movement. We focus in particular on its direct influence on cell migration and invasion, epithelial-mesenchymal transition, and cancer metastasis.
Protein kinase B (PKB/Akt) is a serine/threonine protein kinase that created serious interest when it was revealed as a mediator of the PI3K pathway. It comprises three isoforms that play both unique and redundant roles. Upon binding to phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) generated by PI3K, PKB is phosphorylated by PDK1 at T308. To achieve full kinase activity, PKB needs to be phosphorylated at a second key residue, S473, by members of the PI3K-related kinase family mTORC2 or DNA-PK, depending on the stimulus and the context. Besides, a number of phosphatases and interacting partners have been shown to further modulate its subcellular localization, phosphorylation, and kinase activity. This review aims at illustrating the remarkable complexity in the regulation of PKB signaling downstream of PI3K. Such regulation could be attributed to the specific roles of the PKB isoforms, their expression pattern, subcellular localization, targets, phosphorylation by upstream kinases in a stimulus- and context-dependent manner and by phosphatases, and interaction with binding partners. This allows this key kinase to fulfill physiological functions in numerous processes, including embryonic development, thymocyte development, adipocyte differentiation, glucose homeostasis, and to avoid pathological loss of control such as tumor formation.
MARCH 2012 CANCER DISCOVERY | 249The BATTLE Trial: Personalizing Therapy for Lung Cancer RESEARCH ARTICLE efficiently induces EMT. In a clinically relevant orthotopic breast cancer mouse model, Twist1 expression is essential for the formation of lung metastasis (5). In hepatocelluar carcinomas, overexpression of Twist1 correlates with EMTassociated changes and metastasis (6). Twist1 expression can also negatively regulate programmed cell death (7) and overcome oncogene-induced senescence (4).These studies indicate that Twist1 plays multiple roles in the regulation of antiapoptosis and metastasis during tumor progression. Active Akt directly phosphorylates Twist1 on serine 42 (S42) and protects cells from apoptosis induced by DNA damage (8). Twist1 promotes crucial prometastatic roles in tumor development and is frequently associated with aberrant hyperactivation of phosphoinositide 3-kinase (PI3K)/Akt. This prompted us to explore whether phosphorylation of Twist1 by Akt is part of a molecular mechanism contributing to Twist1 activity in cancer and particularly in metastasis.We analyzed Twist1 phosphorylation in human cancer cell lines (with different genetic backgrounds) and invasive human breast tumors and addressed its functional importance in a mouse model of spontaneous breast tumor metastasis. We show that Twist1 is ubiquitously phosphorylated in invasive human breast tumors and is required for breast cancer lung metastasis in vivo. Twist1 phosphorylation supports metastasis by transcriptionally upregulating TGF-β2, leading to enhanced activation of TGF-βR/Smad2 signaling, and these effects could be significantly suppressed by the knockdown of TGF-β2 expression. These data suggest that Twist1 phosphorylation not only mediates a feedback loop allowing cross-talk with TGF-β to maintain hyperactivation of PI3K/Akt but can also potentiate cancer metastasis, at least in part, through enhanced TGF-β signaling in cancer cells. Collectively, our data uncover a novel mechanism Metastatic breast tumor cells display an epithelial-mesenchymal transition (EMT) that increases cell motility, invasion, and dissemination. Although the transcription factor Twist1 has been shown to contribute to EMT and cancer metastasis, the signaling pathways regulating Twist1 activity are poorly understood. Here, we show that Twist1 is ubiquitously phosphorylated in 90% of 1,532 invasive human breast tumors. Akt/protein kinase B (PKB)-mediated Twist1 phosphorylation promotes EMT and breast cancer metastasis by modulating its transcriptional target TGF-β2, leading to enhanced TGF-β receptor signaling, which in turn maintains hyperactive phosphoinositide 3-kinase (PI3K)/Akt signaling. Preventing phosphorylation of Twist1, as well as depletion of TGF-β2, significantly impaired the metastatic potential of cancer cells in vivo, indicating a key role of phosphorylated Twist1 (phospho-Twist1) in mediating cross-talk between the PI3K/Akt and TGF-β/Smad signaling axes that supports metastatic tumor development. Our results describe a novel signalin...
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