Preclinical Testing of an Oncolytic Parvovirus: Standard Protoparvovirus H-1PV Efficiently Induces Osteosarcoma Cell Lysis In Vitro.

PubMed

Geiss, Carsten; Kis, Zoltán; Leuchs, Barbara; Frank-Stöhr, Monika; Schlehofer, Jörg R; Rommelaere, Jean; Dinsart, Christiane; Lacroix, Jeannine

2017-10-17

Osteosarcoma is the most frequent malignant disease of the bone. On the basis of early clinical experience in the 1960s with H-1 protoparvovirus (H-1PV) in osteosarcoma patients, this effective oncolytic virus was selected for systematic preclinical testing on various osteosarcoma cell cultures. A panel of five human osteosarcoma cell lines (CAL 72, H-OS, MG-63, SaOS-2, U-2OS) was tested. Virus oncoselectivity was confirmed by infecting non-malignant human neonatal fibroblasts and osteoblasts used as culture models of non-transformed mesenchymal cells. H-1PV was found to enter osteosarcoma cells and to induce viral DNA replication, transcription of viral genes, and translation to viral proteins. After H-1PV infection, release of infectious viral particles from osteosarcoma cells into the supernatant indicated successful viral assembly and egress. Crystal violet staining revealed progressive cytomorphological changes in all osteosarcoma cell lines. Infection of osteosarcoma cell lines with the standard H-1PV caused an arrest of the cell cycle in the G2 phase, and these lines had a limited capacity for standard H-1PV virus replication. The cytotoxicity of wild-type H-1PV virus towards osteosarcoma cells was compared in vitro with that of two variants, Del H-1PV and DM H-1PV, previously described as fitness variants displaying higher infectivity and spreading in human transformed cell lines of different origins. Surprisingly, wild-type H-1PV displayed the strongest cytostatic and cytotoxic effects in this analysis and thus seems the most promising for the next preclinical validation steps in vivo.

The anticancer properties of iron core–gold shell nanoparticles in colorectal cancer cells

PubMed Central

Wu, Ya-Na; Wu, Ping-Ching; Yang, Li-Xing; Ratinac, Kyle R; Thordarson, Pall; Jahn, Kristina A; Chen, Dong-Hwang; Shieh, Dar-Bin; Braet, Filip

2013-01-01

Previously, iron core–gold shell nanoparticles (Fe@Au) have been shown to possess cancer-preferential cytotoxicity in oral and colorectal cancer (CRC) cells. However, CRC cell lines are less sensitive to Fe@Au treatment when compared with oral cancer cell lines. In this research, Fe@Au are found to decrease the cell viability of CRC cell lines, including Caco-2, HT-29, and SW480, through growth inhibition rather than the induction of cell death. The cytotoxicity induced by Fe@Au in CRC cells uses different subcellular pathways to the mitochondria-mediated autophagy found in Fe@Au-treated oral cancer cells, OECM1. Interestingly, the Caco-2 cell line shows a similar response to OECM1 cells and is thus more sensitive to Fe@Au treatment than the other CRC cell lines studied. We have investigated the underlying cell resistance mechanisms of Fe@Au-treated CRC cells. The resistance of CRC cells to Fe@Au does not result from the total amount of Fe@Au internalized. Instead, the different amounts of Fe and Au internalized appear to determine the different response to treatment with Fe-only nanoparticles in Fe@Au-resistant CRC cells compared with the Fe@Au-sensitive OECM1 cells. The only moderately cytotoxic effect of Fe@Au nanoparticles on CRC cells, when compared to the highly sensitive OECM1 cells, appears to arise from the CRC cells’ relative insensitivity to Fe, as is demonstrated by our Fe-only treatments. This is a surprising outcome, given that Fe has thus far been considered to be the “active” component of Fe@Au nanoparticles. Instead, we have found that the Au coatings, previously considered only as a passivating coating to protect the Fe cores from oxidation, significantly enhance the cytotoxicity of Fe@Au in certain CRC cells. Therefore, we conclude that both the Fe and Au in these core–shell nanoparticles are essential for the anticancer properties observed in CRC cells. PMID:24039416

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells.

PubMed

Li, Bing; Su, Trent; Ferrari, Roberto; Li, Jing-Yu; Kurdistani, Siavash K

2014-02-01

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.

Knockdown of Oncogenic KRAS in Non-Small Cell Lung Cancers Suppresses Tumor Growth and Sensitizes Tumor Cells to Targeted Therapy

PubMed Central

Sunaga, Noriaki; Shames, David S.; Girard, Luc; Peyton, Michael; Larsen, Jill E.; Imai, Hisao; Soh, Junichi; Sato, Mitsuo; Yanagitani, Noriko; Kaira, Kyoichi; Xie, Yang; Gazdar, Adi F.; Mori, Masatomo; Minna, John D.

2011-01-01

Oncogenic KRAS is found in >25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we performed microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines (HBECs) with and without oncogenic KRAS. We found that while the MAPK pathway is significantly down-regulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-EGFR, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment but may offer possibilities of combining anti-KRAS strategies with other targeted drugs. PMID:21306997

Interactions of Nitroxide-Conjugated and Non-Conjugated Glycodendrimers with Normal and Cancer Cells and Biocompatibility Studies.

PubMed

Andreozzi, Elisa; Antonelli, Antonella; Cangiotti, Michela; Canonico, Barbara; Sfara, Carla; Pianetti, Anna; Bruscolini, Francesca; Sahre, Karin; Appelhans, Dietmar; Papa, Stefano; Ottaviani, Maria Francesca

2017-02-15

Poly(propyleneimine) glycodendrimers fully modified with maltose units were administered to different cancer cell lines and their effect on cell viability was evaluated by using MTS assay and flow cytometry. The mechanism of dendrimer-cell interactions was investigated by the electron paramagnetic resonance (EPR) technique by using a new nitroxide-conjugated glycodendrimer. The nitroxide groups did not modify both the biological properties (cell viability and apoptosis degree) of the dendrimers in the presence of the cells and the dendrimer-cell interactions. Since this class of dendrimers is already known to be biocompatible for human healthy cells, noncancer cells such as human peripheral blood mononuclear cells (PBMCs) and macrophages were also treated with the glycodendrimer, and EPR spectra of the nitroxide-conjugated glycodendrimer were compared for cancer and noncancer cells. It was found that this dendrimer selectively affects the cell viability of tumor cells, while, surprisingly, PBMC proliferation is induced. Moreover, H-bond-active glycodendrimer-cell interactions were different for the different cancer cell lines and noncancer cells. The nitroxide-conjugated glycodendrimer was able to interact with the cell membrane and eventually cross it, getting in contact with cytosol antioxidants. This study helps to clarify the potential anticancer effect of this class of dendrimers opening to future applications of these macromolecules as new antitumor agents.

Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism

PubMed Central

Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G.; Ronimus, Ron S.; McGee, Sean L.; Wilson, William R.

2013-01-01

Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines. PMID:23799003

A Novel Isoform of the B Cell Tyrosine Kinase BTK Protects Breast Cancer Cells from Apoptosis

PubMed Central

Eifert, Cheryl; Wang, Xianhui; Kokabee, Leila; Kourtidis, Antonis; Jain, Ritu; Gerdes, Michael J.; Conklin, Douglas S.

2016-01-01

Tyrosine kinases orchestrate key cellular signaling pathways and their dysregulation is often associated with cellular transformation. Several recent cases in which inhibitors of tyrosine kinases have been successfully used as anticancer agents have underscored the importance of this class of proteins in the development of targeted cancer therapies. We have carried out a large-scale loss-of-function analysis of the human tyrosine kinases using RNA interference to identify novel survival factors for breast cancer cells. In addition to kinases with known roles in breast and other cancers, we identified several kinases that were previously unknown to be required for breast cancer cell survival. The most surprising of these was the cytosolic, nonreceptor tyrosine kinase, Bruton’s tyrosine kinase (BTK), which has been extensively studied in B cell development. Down regulation of this protein with RNAi or inhibition with pharmacological inhibitors causes apoptosis; overexpression inhibits apoptosis induced by Doxorubicin in breast cancer cells. Our results surprisingly show that BTK is expressed in several breast cancer cell lines and tumors. The predominant form of BTK found in tumor cells is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. This alternate form of BTK is expressed at significantly higher levels in tumorigenic breast cells than in normal breast cells. Since this protein is a survival factor for these cells, it represents both a potential marker and novel therapeutic target for breast cancer. PMID:23913792

Mitochondrial fluctuations as a measure of active biomechanical properties of mammalian cells

NASA Astrophysics Data System (ADS)

Xu, Wenlong; Alizadeh, Elaheh; Castle, Jordan; Prasad, Ashok

A single-cell assay of mechanical properties would give significant insights into cellular processes. Force spectrum microscopy is one such technique, which involves both active and passive particle tracking microrheology on the same cells. Since active microrheology requires expensive instruments, it is of great interest to develop simpler alternatives. Here we study an alternative using endogenous mitochondrial fluctuations, rather than fluorescent beads, in particle tracking microrheology. Mitochondria of the C3H-10T1/2 cell line are labeled and tracked using confocal microscopy, their mean square displacement (MSD) measured, and mechanical parameters calculated. Active fluctuations are distinguished from passive fluctuations by treatment with ATP synthesis inhibitors. We find that the MSD of mitochondria resembles that of particles in viscoelastic media. However, comparisons of MSD between controls and cells disrupted in the actin or microtubule network showed surprisingly small effects, while ATP-depleted cells showed significantly decreased MSD, and characteristics of thermally driven fluctuations. Both active and ATP-depleted parameters showed heterogeneity among cells and between cell lines. This method is potentially very useful due to its simplicity. We gratefully acknowledge support from NSF CAREER Grant PHY-1151454 awarded to Ashok Prasad.

Evolution of radiation resistance in a complex microenvironment

NASA Astrophysics Data System (ADS)

Kim, So Hyun; Austin, Robert; Mehta, Monal; Kahn, Atif

2013-03-01

Radiation treatment responses in brain cancers are typically associated with short progression-free intervals in highly lethal malignancies such as glioblastomas. Even as patients routinely progress through second and third line salvage therapies, which are usually empirically selected, surprisingly little information exists on how cancer cells evolve resistance. We will present experimental results showing how in the presence of complex radiation gradients evolution of resistance to radiation occurs. Sponsored by the NCI/NIH Physical Sciences Oncology Centers

In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining.

PubMed

Zilla, P; Fasol, R; Dudeck, U; Siedler, S; Preiss, P; Fischlein, T; Müller-Glauser, W; Baitella, G; Sanan, D; Odell, J

1990-08-01

A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).

NK cells interactions with dendritic cells shape innate and adaptive immunity.

PubMed

Brilot, Fabienne; Strowig, Till; Munz, Christian

2008-05-01

While natural killer (NK) cells received their name from their ability to mediate spontaneous cytotoxicity, it has recently become clear that they require activation to target most transformed and infected cells. Dendritic cells (DCs) have been shown to mediate NK cell activation during innate immune responses. Surprisingly, this interaction was recently reported to be required to restrict infections by NK cells, and to take place in secondary lymphoid organs. Here we review these recent studies on NK cell interactions with DCs, discuss the molecular mechanisms underlying the cross-talk between these two innate lymphocyte populations, and out-line how DCs and NK cells synergize to enhance innate immunity against microbes and tumors as well as shape the adaptive immune system. Based on this better understanding, we propose that NK cells should be targeted for their protective functions and as an adjuvant during immunotherapy development.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A)-Mediated changes in Fas Expression and Fas-Dependent Apoptosis: Role of Lyn/Syk activation

PubMed Central

Incrocci, Ryan; Hussain, Samira; Stone, Amanda; Bieging, Kathryn; Alt, Lauren A.C.; Fay, Michael J.; Swanson-Mungerson, Michelle

2015-01-01

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, −3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies. PMID:26255694

Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies.

PubMed

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-06-01

The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy. PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R. Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance. (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors.

In Vitro Analysis of Breast Cancer Cell Line Tumourspheres and Primary Human Breast Epithelia Mammospheres Demonstrates Inter- and Intrasphere Heterogeneity

PubMed Central

Vargas, Ana Cristina; Keith, Patricia; Reid, Lynne; Wockner, Leesa; Amiri, Marjan Askarian; Sarkar, Debina; Simpson, Peter T.; Clarke, Catherine; Schmidt, Chris W.; Reynolds, Brent A.

2013-01-01

Mammosphere and breast tumoursphere culture have gained popularity as in vitro assays for propagating and analysing normal and cancer stem cells. Whether the spheres derived from different sources or parent cultures themselves are indeed single entities enriched in stem/progenitor cells compared to other culture formats has not been fully determined. We surveyed sphere-forming capacity across 26 breast cell lines, immunophenotyped spheres from six luminal- and basal-like lines by immunohistochemistry and flow cytometry and compared clonogenicity between sphere, adherent and matrigel culture formats using in vitro functional assays. Analyses revealed morphological and molecular intra- and inter-sphere heterogeneity, consistent with adherent parental cell line phenotypes. Flow cytometry showed sphere culture does not universally enrich for markers previously associated with stem cell phenotypes, although we found some cell-line specific changes between sphere and adherent formats. Sphere-forming efficiency was significantly lower than adherent or matrigel clonogenicity and constant over serial passage. Surprisingly, self-renewal capacity of sphere-derived cells was similar/lower than other culture formats. We observed significant correlation between long-term-proliferating-cell symmetric division rates in sphere and adherent cultures, suggesting functional overlap between the compartments sustaining them. Experiments with normal primary human mammary epithelia, including sorted luminal (MUC1+) and basal/myoepithelial (CD10+) cells revealed distinct luminal-like, basal-like and mesenchymal entities amongst primary mammospheres. Morphological and colony-forming-cell assay data suggested mammosphere culture may enrich for a luminal progenitor phenotype, or induce reversion/relaxation of the basal/mesenchymal in vitro selection occurring with adherent culture. Overall, cell line tumourspheres and primary mammospheres are not homogenous entities enriched for stem cells, suggesting a more cautious approach to interpreting data from these assays and careful consideration of its limitations. Sphere culture may represent an alternative 3-dimensional culture system which rather than universally ‘enriching’ for stem cells, has utility as one of a suite of functional assays that provide a read-out of progenitor activity. PMID:23750209

A Multi-Omics Analysis of Recombinant Protein Production in Hek293 Cells

PubMed Central

Dietmair, Stefanie; Hodson, Mark P.; Quek, Lake-Ee; Timmins, Nicholas E.; Gray, Peter; Nielsen, Lars K.

2012-01-01

Hek293 cells are the predominant hosts for transient expression of recombinant proteins and are used for stable expression of proteins where post-translational modifications performed by CHO cells are inadequate. Nevertheless, there is little information available on the key cellular features underpinning recombinant protein production in Hek293 cells. To improve our understanding of recombinant protein production in Hek293 cells and identify targets for the engineering of an improved host cell line, we have compared a stable, recombinant protein producing Hek293 cell line and its parental cell line using a combination of transcriptomics, metabolomics and fluxomics. Producer cultures consumed less glucose than non-producer cultures while achieving the same growth rate, despite the additional burden of recombinant protein production. Surprisingly, there was no indication that producer cultures compensated for the reduction in glycolytic energy by increasing the efficiency of glucose utilization or increasing glutamine consumption. In contrast, glutamine consumption was lower and the majority of genes involved in oxidative phosphorylation were downregulated in producer cultures. We observed an overall downregulation of a large number of genes associated with broad cellular functions (e.g., cell growth and proliferation) in producer cultures, and therefore speculate that a broad adaptation of the cellular network freed up resources for recombinant protein production while maintaining the same growth rate. Increased abundance of genes associated with endoplasmic reticulum stress indicated a possible bottleneck at the point of protein folding and assembly. PMID:22937046

Dual effect of F-actin targeted carrier combined with antimitotic drug on aggressive colorectal cancer cytoskeleton: Allying dissimilar cell cytoskeleton disrupting mechanisms.

PubMed

Taranejoo, Shahrouz; Janmaleki, Mohsen; Pachenari, Mohammad; Seyedpour, Seyed Morteza; Chandrasekaran, Ramya; Cheng, Wenlong; Hourigan, Kerry

2016-11-20

A recent approach to colon cancer therapy is to employ selective drugs with specific extra/intracellular sites of action. Alteration of cytoskeletal protein reorganization and, subsequently, to cellular biomechanical behaviour during cancer progression highly affects the cancer cell progress. Hence, cytoskeleton targeted drugs are an important class of cancer therapy agents. We have studied viscoelastic alteration of the human colon adenocarcinoma cell line, SW48, after treatment with a drug delivery system comprising chitosan as the carrier and albendazole as the microtubule-targeting agent (MTA). For the first time, we have evaluated the biomechanical characteristics of the cell line, using the micropipette aspiration (MA) method after treatment with drug delivery systems. Surprisingly, employing a chitosan-albendazole pair, in comparison with both neat materials, resulted in more significant change in the viscoelastic parameters of cells, including the elastic constants (K 1 and K 2 ) and the coefficient of viscosity (μ). This difference was more pronounced for cancer cells after 48h of the treatment. Microtubule and actin microfilament (F-actin) contents in the cell line were studied by immunofluorescent staining. Good agreement was observed between the mechanical characteristics results and microtubule/F-actin contents of the treated SW48 cell line, which declined after treatment. The results showed that chitosan affected F-actin more, while MTA was more effective for microtubules. Toxicity studies were performed against two cancer cell lines (SW48 and MCF10CA1h) and compared to normal cells, MCF10A. The results showed cancer selectiveness, safety of formulation, and enhanced anticancer efficacy of the CS/ABZ conjugate. This study suggests that employing such a suitable pair of drug-carriers with dissimilar sites of action, thus allying the different cell cytoskeleton disrupting mechanisms, may provide a more efficient cancer therapy approach. Copyright © 2016 Elsevier B.V. All rights reserved.

Surprise but not coherence: sensitivity to verbal humor in right-hemisphere patients.

PubMed

Brownell, H H; Michel, D; Powelson, J; Gardner, H

1983-01-01

Verbal humor deficits were investigated in right-hemisphere-damaged patients. It was hypothesized that the appreciation of jokes presupposes two elements: sensitivity to the surprise element entailed in the punch line of a joke and apprehension of the coherence which results when the punch line has been integrated with the body of the joke. The possible dissociation between these elements was tested by asking subjects to select from four alternatives the appropriate ending to a joke. Right-hemisphere patients exhibited a selective attraction to endings which contained an element of surprise but which were not otherwise coherent with the body of the joke. This finding suggests that right-hemisphere patients have difficulty in integrating content across parts of a narrative and confirms the psychological reality of the proposed distinction between the surprise and coherence elements of humor processing.

Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines.

PubMed

Al-Asmari, Abdulrahman Khazim; Albalawi, Sulaiman Mansour; Athar, Md Tanwir; Khan, Abdul Quaiyoom; Al-Shahrani, Hamoud; Islam, Mozaffarul

2015-01-01

In this study we investigated the anti-cancer effect of Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70-90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2-3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast and colorectal cancers.

How do heterogeneities in single cell rigidity influence the mechanical behavior at the tissue level?

NASA Astrophysics Data System (ADS)

Bi, Dapeng; Wetzel, Franziska; Fritsch, Anatol; Marchetti, M. Cristina; Manning, M. Lisa; Kaes, Josef

It has been long recognized that solid tumor tissues are mechanically more rigid than surrounding healthy tissues. However recent experiments have shown that in primary tumor samples from patients with mammary and cervix carcinomas, cells exhibit a broad distribution of rigidities, with a higher fraction of softer and more contractile cells compared to normal tissues. This gives rise to a paradox: does softness emerge from adaptation to mechanical and chemical cues in the external microenvironment, or are soft cells already present inside a primary solid tumor? Motivated by these observations, we study a model of dense tissues that incorporates the experimental data for cell stiffness variations to reveal that, surprisingly, tumors with a significant fraction of very soft cells can still remain rigid. Moreover, in tissues with the observed distributions of cell stiffnesses, softer cells spontaneously self-organize into lines or streams, possibly facilitating cancer metastasis.

Human T Cell Leukemia Virus Type 1 Tax Inhibits Innate Antiviral Signaling via NF-κB-Dependent Induction of SOCS1▿

PubMed Central

Charoenthongtrakul, Soratree; Zhou, Qinjie; Shembade, Noula; Harhaj, Nicole S.; Harhaj, Edward W.

2011-01-01

Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-β expression accompanied by reduced HTLV-1 replication and p19Gag levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein. PMID:21593151

Modification of the estrogenic properties of diphenols by the incorporation of ferrocene. Generation of antiproliferative effects in vitro.

PubMed

Vessières, Anne; Top, Siden; Pigeon, Pascal; Hillard, Elizabeth; Boubeker, Leila; Spera, Daniela; Jaouen, Gérard

2005-06-16

We report here the synthesis and the strong and unexpected antiproliferative effect of the organometallic diphenolic compound 1,1-bis(4'-hydroxyphenyl)-2-ferrocenyl-but-1-ene (4) on both hormone-dependent (MCF7) and -independent (MDA-MB231) breast cancer cells (IC(50) = 0.7 and 0.6 microM). Surprisingly, 6 [1,2-bis(4'-hydroxyphenyl)-2-ferrocenyl-but-1-ene], the regioisomer of 4, shows only a modest effect on these cell lines. This pertinent organometallic modification seems to trigger an intracellular oxidation of the structurally favorable compound 4, leading to the generation of a potent cytotoxic compound.

The Human Cytomegalovirus Lytic Cycle Is Induced by 1,25-Dihydroxyvitamin D3 in Peripheral Blood Monocytes and in the THP-1 Monocytic Cell Line

PubMed Central

Wu, Shu-En; Miller, William E.

2015-01-01

Human cytomegalovirus (HCMV) resides in a latent form in hematopoietic progenitors and undifferentiated cells within the myeloid lineage. Maturation and differentiation along the myeloid lineage triggers lytic replication. Here, we used peripheral blood monocytes and the monocytic cell line THP-1 to investigate the effects of 1,25-dihydroxyvitamin D3 on HCMV replication. Interestingly, 1,25-dihydroxyvitamin D3 induces lytic replication marked by upregulation of HCMV gene expression and production of infectious virus. Moreover, we demonstrate that the effects of 1,25-dihydroxyvitamin D3 correlate with maturation/differentiation of the monocytes and not by directly stimulating the MIEP. These results are somewhat surprising as 1,25-dihydroxyvitamin D3 typically boosts immunity to bacteria and viruses rather than driving the infectious life cycle as it does for HCMV. Defining the signaling pathways kindled by 1,25-dihydroxyvitamin D3 will lead to a better understanding of the underlying molecular mechanisms that determine the fate of HCMV once it infects cells in the myeloid lineage. PMID:25965798

Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance

PubMed Central

Meirelles, Katia; Benedict, Leo Andrew; Dombkowski, David; Pepin, David; Preffer, Frederic I.; Teixeira, Jose; Tanwar, Pradeep Singh; Young, Robert H.; MacLaughlin, David T.; Donahoe, Patricia K.; Wei, Xiaolong

2012-01-01

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad−) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad− cells. Similarly, proliferation of the 3+Ecad− cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3−Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad− subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad− cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics. PMID:22308459

Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance.

PubMed

Meirelles, Katia; Benedict, Leo Andrew; Dombkowski, David; Pepin, David; Preffer, Frederic I; Teixeira, Jose; Tanwar, Pradeep Singh; Young, Robert H; MacLaughlin, David T; Donahoe, Patricia K; Wei, Xiaolong

2012-02-14

Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad-) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad- cells. Similarly, proliferation of the 3+Ecad- cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3-Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad- subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad- cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics.

The chemokine receptor CXCR6 and its ligand CXCL16 are expressed in carcinomas and inhibit proliferation.

PubMed

Meijer, Joost; Ogink, Janneke; Kreike, Bas; Nuyten, Dimitry; de Visser, Karin E; Roos, Ed

2008-06-15

The chemokine receptor CXCR6 and its ligand CXCL16 are involved in inflammation. Thus far, they were known to be expressed mainly by T cells and macrophages, respectively. However, we detected both in all of 170 human primary mammary carcinomas and at similar levels in all 8 human mammary carcinoma cell lines tested by microarray analysis. Expression was confirmed by reverse transcription-PCR and for the cell lines also by fluorescence-activated cell sorting analysis. CXCR6 and CXCL16 were also detected in several mouse and human mammary, colon, and pancreatic carcinoma cell lines. CXCL16 is a transmembrane protein from which the soluble chemokine can be cleaved off. The transmembrane form is present on the surface of the carcinoma cells. Surprisingly, suppression of either CXCR6 or CXCL16 led to greatly enhanced proliferation in vitro as well as in vivo, indicating that their interaction inhibits proliferation. This notion was verified using inhibitory antibodies and by introduction of CXCL16 into a rare CXCL16-negative cell line. The effect was mediated by the G protein-coupled receptor CXCR6 because it was blocked by the G(i) protein inhibitor pertussis toxin. In contrast, the soluble CXCL16 chemokine enhanced proliferation, and this was also mediated by CXCR6 but not via G(i) protein. It is remarkable that both CXCR6 and CXCL16 are expressed by all mammary carcinomas because cells that lose either acquire a growth advantage and should be selected during tumor progression. This suggests an unknown important role in tumor formation. Proteases, possibly macrophage derived, might convert inhibitory transmembrane CXCL16 into the stimulatory chemokine.

Long interspersed element-1 protein expression is a hallmark of many human cancers.

PubMed

Rodić, Nemanja; Sharma, Reema; Sharma, Rajni; Zampella, John; Dai, Lixin; Taylor, Martin S; Hruban, Ralph H; Iacobuzio-Donahue, Christine A; Maitra, Anirban; Torbenson, Michael S; Goggins, Michael; Shih, Ie-Ming; Duffield, Amy S; Montgomery, Elizabeth A; Gabrielson, Edward; Netto, George J; Lotan, Tamara L; De Marzo, Angelo M; Westra, William; Binder, Zev A; Orr, Brent A; Gallia, Gary L; Eberhart, Charles G; Boeke, Jef D; Harris, Chris R; Burns, Kathleen H

2014-05-01

Cancers comprise a heterogeneous group of human diseases. Unifying characteristics include unchecked abilities of tumor cells to proliferate and spread anatomically, and the presence of clonal advantageous genetic changes. However, universal and highly specific tumor markers are unknown. Herein, we report widespread long interspersed element-1 (LINE-1) repeat expression in human cancers. We show that nearly half of all human cancers are immunoreactive for a LINE-1-encoded protein. LINE-1 protein expression is a common feature of many types of high-grade malignant cancers, is rarely detected in early stages of tumorigenesis, and is absent from normal somatic tissues. Studies have shown that LINE-1 contributes to genetic changes in cancers, with somatic LINE-1 insertions seen in selected types of human cancers, particularly colon cancer. We sought to correlate this observation with expression of the LINE-1-encoded protein, open reading frame 1 protein, and found that LINE-1 open reading frame 1 protein is a surprisingly broad, yet highly tumor-specific, antigen. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Heat-shock protein 27 (HSP27, HSPB1) is synthetic lethal to cells with oncogenic activation of MET, EGFR and BRAF.

PubMed

Konda, John D; Olivero, Martina; Musiani, Daniele; Lamba, Simona; Di Renzo, Maria F

2017-06-01

The small heat-shock protein of 27 kDa (HSP27) is highly expressed in many cancers and is associated with aggressive tumour behaviour, metastasis, poor prognosis and resistance to chemotherapy. We aimed at assessing the role of HSP27 in modulating responses to target therapies. We selected several oncogene-addicted cancer cell lines, which undergo either cell cycle blockade or cell death in response to agents that target the specific oncogene. Surprisingly, HSP27 suppression alone resulted in the apoptotic death of MET-addicted EBC-1 lung cancer cells, epidermal growth factor receptor (EGFR)-addicted colorectal carcinoma (CRC) DiFi cells and BRAF-addicted CRC COLO205 and OXCO-1 and melanoma COLO741 cells, all of which also undergo death when treated with the specific targeted agent. In other cell lines, such as MET-addicted gastric carcinoma MKN45 and EGFR-addicted CRC SW48 lines, where oncogene inhibition only blocked proliferation, HSP27 knockdown made targeted agents switch from cytostatic to cytotoxic activity. Mechanistically, the more the cells were susceptible to HSP27 suppression, the more they were primed for death, as demonstrated by increased levels of mitochondrial outer membrane permeabilization. Priming for death was accompanied by the increase in pro-apoptotic proteins of the BCL2 family and of active caspase-3 and lamin B. Together, these data suggest that oncogene-addicted cells require HSP27 for survival and that HSP27 might interfere with the effectiveness of targeted agents. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

PubMed Central

Goodhardt, M; Babinet, C; Lutfalla, G; Kallenbach, S; Cavelier, P; Rougeon, F

1989-01-01

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo. Images PMID:2508061

Loss of insulin-like growth factor II imprinting is a hallmark associated with enhanced chemo/radiotherapy resistance in cancer stem cells.

PubMed

Zhao, Xin; Liu, Xiaoliang; Wang, Guanjun; Wen, Xue; Zhang, Xiaoying; Hoffman, Andrew R; Li, Wei; Hu, Ji-Fan; Cui, Jiuwei

2016-08-09

Insulin-like growth factor II (IGF2) is maternally imprinted in most tissues, but the epigenetic regulation of the gene in cancer stem cells (CSCs) has not been defined. To study the epigenetic mechanisms underlying self-renewal, we isolated CSCs and non-CSCs from colon cancer (HT29, HRT18, HCT116), hepatoma (Hep3B), breast cancer (MCF7) and prostate cancer (ASPC) cell lines. In HT29 and HRT18 cells that show loss of IGF2 imprinting (LOI), IGF2 was biallelically expressed in the isolated CSCs. Surprisingly, we also found loss of IGF2 imprinting in CSCs derived from cell lines HCT116 and ASPC that overall demonstrate maintenance of IGF2 imprinting. Using chromatin conformation capture (3C), we found that intrachromosomal looping between the IGF2 promoters and the imprinting control region (ICR) was abrogated in CSCs, in parallel with loss of IGF2 imprinting in these CSCs. Loss of imprinting led to increased IGF2 expression in CSCs, which have a higher rate of colony formation and greater resistance to chemotherapy and radiotherapy in vitro. These studies demonstrate that IGF2 LOI is a common feature in CSCs, even when the stem cells are derived from a cell line in which the general population of cells maintain IGF2 imprinting. This finding suggests that aberrant IGF2 imprinting may be an intrinsic epigenetic control mechanism that enhances stemness, self-renewal and chemo/radiotherapy resistance in cancer stem cells.

Fibroblast growth factor 2 restrains Ras-driven proliferation of malignant cells by triggering RhoA-mediated senescence.

PubMed

Costa, Erico T; Forti, Fábio L; Matos, Tatiana G F; Dermargos, Alexandre; Nakano, Fábio; Salotti, Jacqueline; Rocha, Kátia M; Asprino, Paula F; Yoshihara, Celina K; Koga, Marianna M; Armelin, Hugo A

2008-08-01

Fibroblast growth factor 2 (FGF2) is considered to be a bona fide oncogenic factor, although results from our group and others call this into question. Here, we report that exogenous recombinant FGF2 irreversibly inhibits proliferation by inducing senescence in Ras-dependent malignant mouse cells, but not in immortalized nontumorigenic cell lines. We report the following findings in K-Ras-dependent malignant Y1 adrenocortical cells and H-Ras V12-transformed BALB-3T3 fibroblasts: (a) FGF2 inhibits clonal growth and tumor onset in nude and immunocompetent BALB/c mice, (b) FGF2 irreversibly blocks the cell cycle, and (c) FGF2 induces the senescence-associated beta-galactosidase with no accompanying signs of apoptosis or necrosis. The tyrosine kinase inhibitor PD173074 completely protected malignant cells from FGF2. In Y1 adrenal cells, reducing the constitutively high levels of K-Ras-GTP using the dominant-negative RasN17 mutant made cells resistant to FGF2 cytotoxicity. In addition, transfection of the dominant-negative RhoA-N19 into either Y1 or 3T3-B61 malignant cell lines yielded stable clonal transfectants that were unable to activate RhoA and were resistant to the FGF2 stress response. We conclude that in Ras-dependent malignant cells, FGF2 interacts with its cognate receptors to trigger a senescence-like process involving RhoA-GTP. Surprisingly, attempts to select FGF2-resistant cells from the Y1 and 3T3-B61 cell lines yielded only rare clones that (a) had lost the overexpressed ras oncogene, (b) were dependent on FGF2 for proliferation, and (c) were poorly tumorigenic. Thus, FGF2 exerted a strong negative selection that Ras-dependent malignant cells could rarely overcome.

Response of Head and Neck Squamous Cell Carcinoma Cells Carrying PIK3CA Mutations to Select Targeted Therapies

PubMed Central

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-01-01

Importance The PIK3CA mutation is one of the most common mutations in Head and Neck Squamous Cell Carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. Objectives 1) To determine the role of oncogene dependence on one of the more common and targetable oncogenes in HNSCC – PIK3CA; 2) To evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. Study Design In vitro study. Setting Academic research laboratory. Participants Cell culture based study assessing the viability of PIK3CA mutated head and neck cell lines when treated with targeted therapy. Exposures PIK3CA mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Main Outcome and Measures Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R Results Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred decreased, rather than increased, sensitivity as measured by IC50 when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared to the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared to control while those expressing E545K showed slightly increased sensitivity of unclear significance. Conclusions and Relevance 1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared to control with dual PI3K/mTOR inhibition. 2) Oncogene addiction to PIK3CA hot spot mutations, if it occurs, is likely to evolve in vivo molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors. PMID:25855885

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility

PubMed Central

2011-01-01

Background Increased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231). Methods Stable cell lines with TFPI (both α and β) and only TFPIβ downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion. Results Downregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIβ was downregulated, revealing a novel function of this isoform in cancer metastasis. Conclusions Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer. PMID:21849050

Splicing factors PTBP1 and PTBP2 promote proliferation and migration of glioma cell lines

PubMed Central

Cheung, Hannah C.; Hai, Tao; Zhu, Wen; Baggerly, Keith A.; Tsavachidis, Spiridon; Krahe, Ralf

2009-01-01

Polypyrimidine tract-binding protein 1 (PTBP1) is a multi-functional RNA-binding protein that is aberrantly overexpressed in glioma. PTBP1 and its brain-specific homologue polypyrimidine tract-binding protein 2 (PTBP2) regulate neural precursor cell differentiation. However, the overlapping and non-overlapping target transcripts involved in this process are still unclear. To determine why PTBP1 and not PTBP2 would promote glial cell-derived tumours, both PTBP1 and PTBP2 were knocked down in the human glioma cell lines U251 and LN229 to determine the role of these proteins in cell proliferation, migration, and adhesion. Surprisingly, removal of both PTBP1 and PTBP2 slowed cell proliferation, with the double knockdown having no additive effects. Decreased expression of both proteins individually and in combination inhibited cell migration and increased adhesion of cells to fibronectin and vitronectin. A global survey of differential exon expression was performed following PTBP1 knockdown in U251 cells using the Affymetrix Exon Array to identify PTBP1-specific splicing targets that enhance gliomagenesis. In the PTBP1 knockdown, previously determined targets were unaltered in their splicing patterns. A single gene, RTN4 (Nogo) had significantly enhanced inclusion of exon 3 when PTBP1 was removed. Overexpression of the splice isoform containing exon 3 decreased cell proliferation to a similar degree as the removal of PTBP1. These results provide the first evidence that RNA-binding proteins affect the invasive and rapid growth characteristics of glioma cell lines. Its actions on proliferation appear to be mediated, in part, through alternative splicing of RTN4. PMID:19506066

Paucity of PD-L1 expression in prostate cancer: innate and adaptive immune resistance.

PubMed

Martin, A M; Nirschl, T R; Nirschl, C J; Francica, B J; Kochel, C M; van Bokhoven, A; Meeker, A K; Lucia, M S; Anders, R A; DeMarzo, A M; Drake, C G

2015-12-01

Primary prostate cancers are infiltrated with programmed death-1 (PD-1) expressing CD8+ T-cells. However, in early clinical trials, men with metastatic castrate-resistant prostate cancer did not respond to PD-1 blockade as a monotherapy. One explanation for this unresponsiveness could be that prostate tumors generally do not express programmed death ligand-1 (PD-L1), the primary ligand for PD-1. However, lack of PD-L1 expression in prostate cancer would be surprising, given that phosphatase and tensin homolog (PTEN) loss is relatively common in prostate cancer and several studies have shown that PTEN loss correlates with PD-L1 upregulation--constituting a mechanism of innate immune resistance. This study tested whether prostate cancer cells were capable of expressing PD-L1, and whether the rare PD-L1 expression that occurs in human specimens correlates with PTEN loss. Human prostate cancer cell lines were evaluated for PD-L1 expression and loss of PTEN by flow cytometry and western blotting, respectively. Immunohistochemical (IHC) staining for PTEN was correlated with PD-L1 IHC using a series of resected human prostate cancer samples. In vitro, many prostate cancer cell lines upregulated PD-L1 expression in response to inflammatory cytokines, consistent with adaptive immune resistance. In these cell lines, no association between PTEN loss and PD-L1 expression was apparent. In primary prostate tumors, PD-L1 expression was rare, and was not associated with PTEN loss. These studies show that some prostate cancer cell lines are capable of expressing PD-L1. However, in human prostate cancer, PTEN loss is not associated with PD-L1 expression, arguing against innate immune resistance as a mechanism that mitigates antitumor immune responses in this disease.

In vitro downregulated hypoxia transcriptome is associated with poor prognosis in breast cancer.

PubMed

Abu-Jamous, Basel; Buffa, Francesca M; Harris, Adrian L; Nandi, Asoke K

2017-06-15

Hypoxia is a characteristic of breast tumours indicating poor prognosis. Based on the assumption that those genes which are up-regulated under hypoxia in cell-lines are expected to be predictors of poor prognosis in clinical data, many signatures of poor prognosis were identified. However, it was observed that cell line data do not always concur with clinical data, and therefore conclusions from cell line analysis should be considered with caution. As many transcriptomic cell-line datasets from hypoxia related contexts are available, integrative approaches which investigate these datasets collectively, while not ignoring clinical data, are required. We analyse sixteen heterogeneous breast cancer cell-line transcriptomic datasets in hypoxia-related conditions collectively by employing the unique capabilities of the method, UNCLES, which integrates clustering results from multiple datasets and can address questions that cannot be answered by existing methods. This has been demonstrated by comparison with the state-of-the-art iCluster method. From this collection of genome-wide datasets include 15,588 genes, UNCLES identified a relatively high number of genes (>1000 overall) which are consistently co-regulated over all of the datasets, and some of which are still poorly understood and represent new potential HIF targets, such as RSBN1 and KIAA0195. Two main, anti-correlated, clusters were identified; the first is enriched with MYC targets participating in growth and proliferation, while the other is enriched with HIF targets directly participating in the hypoxia response. Surprisingly, in six clinical datasets, some sub-clusters of growth genes are found consistently positively correlated with hypoxia response genes, unlike the observation in cell lines. Moreover, the ability to predict bad prognosis by a combined signature of one sub-cluster of growth genes and one sub-cluster of hypoxia-induced genes appears to be comparable and perhaps greater than that of known hypoxia signatures. We present a clustering approach suitable to integrate data from diverse experimental set-ups. Its application to breast cancer cell line datasets reveals new hypoxia-regulated signatures of genes which behave differently when in vitro (cell-line) data is compared with in vivo (clinical) data, and are of a prognostic value comparable or exceeding the state-of-the-art hypoxia signatures.

The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

PubMed

Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

2017-04-06

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (EpCAM) identification of fibroblasts from breast and prostate tumor tissues is advised. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Solvent-Free Addition of Indole to Aldehydes: Unexpected Synthesis of Novel 1-[1-(1H-Indol-3-yl) Alkyl]-1H-Indoles and Preliminary Evaluation of Their Cytotoxicity in Hepatocarcinoma Cells.

PubMed

Tocco, Graziella; Zedda, Gloria; Casu, Mariano; Simbula, Gabriella; Begala, Michela

2017-10-17

New 1-[1-(1 H -indol-3-yl) alkyl]-1 H -indoles, surprisingly, have been obtained from the addition of indole to a variety of aldehydes under neat conditions. CaO, present in excess, was fundamental for carrying out the reaction with paraformaldehyde. Under the same reaction conditions, aromatic and heteroaromatic aldehydes afforded only classical bis (indolyl) aryl indoles. In this paper, the role of CaO, together with the regiochemistry and the mechanism of the reaction, are discussed in detail. The effect of some selected 3,3'- and 1,3'-diindolyl methane derivatives on cell proliferation of the hepatoma cell line FaO was also evaluated.

Engineered LINE-1 retrotransposition in nondividing human neurons

PubMed Central

Macia, Angela; Widmann, Thomas J.; Heras, Sara R.; Ayllon, Veronica; Sanchez, Laura; Benkaddour-Boumzaouad, Meriem; Muñoz-Lopez, Martin; Rubio, Alejandro; Amador-Cubero, Suyapa; Blanco-Jimenez, Eva; Garcia-Castro, Javier; Menendez, Pablo; Ng, Philip; Muotri, Alysson R.; Goodier, John L.; Garcia-Perez, Jose L.

2017-01-01

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80–100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought. PMID:27965292

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

PubMed Central

Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

2006-01-01

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

The regrowth kinetic of the surviving population is independent of acute and chronic responses to temozolomide in glioblastoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silva, Andrew Oliveira, E-mail: andrewbiomed@gmail.com; Dalsin, Eloisa, E-mail: dalsineloisa@gmail.com; Onzi, Giovana Ravizzoni, E-mail: gioonzi@gmail.com

Chemotherapy acts on cancer cells by producing multiple effects on a cell population including cell cycle arrest, necrosis, apoptosis and senescence. However, often a subpopulation of cells survives and the behavior of this subpopulation, which is responsible for cancer recurrence, remains obscure. Here we investigated the in vitro short- and long-term responses of six glioblastoma cell lines to clinically relevant doses of temozolomide for 5 days followed by 23 days of recovery, mimicking the standard schedule used in glioblastoma patient for this drug. These cells presented different profiles of sensitivity to temozolomide with varying levels of cell cycle arrest, autophagymore » and senescence, followed by a regrowth of the surviving cells. The initial reduction in cell number and the subsequent regrowth was analyzed with four new parameters applied to Cumulative Population Doubling (CPD) curves that describe the overall sensitivity of the population and the characteristic of the regrowth: the relative end point CPD (RendCPD); the relative Area Under Curve (rAUC); the Relative Time to Cross a Threshold (RTCT); and the Relative Proliferation Rate (RPR). Surprisingly, the kinetics of regrowth were not predicted by the mechanisms activated after treatment nor by the acute or overall sensitivity. With this study we added new parameters that describe key responses of glioblastoma cell populations to temozolomide treatment. These parameters can also be applied to other cell types and treatments and will help to understand the behavior of the surviving cancer cells after treatment and shed light on studies of cancer resistance and recurrence. - Highlights: • Little is known about the behavior of the glioma cells surviving to TMZ. • The short- and long-term response of six glioma cells lines to TMZ varies considerably. • These glioma cells lines recovered proliferation after therapeutic levels of TMZ. • The growth velocity of the surviving cells was different from the untreated cells. • The kinetic of regrowth was not predicted by any TMZ-triggered mechanism.« less

Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines

PubMed Central

Al-Asmari, Abdulrahman Khazim; Albalawi, Sulaiman Mansour; Athar, Md Tanwir; Khan, Abdul Quaiyoom; Al-Shahrani, Hamoud; Islam, Mozaffarul

2015-01-01

In this study we investigated the anti-cancer effect of Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70–90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2–3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast and colorectal cancers. PMID:26288313

Agronomic performance of Populus deltoides trees engineered for biofuel production

DOE PAGES

Macaya-Sanz, David; Chen, Jin?Gui; Kalluri, Udaya C.; ...

2017-11-30

Background: One of the major barriers to the development of lignocellulosic feedstocks is the recalcitrance of plant cell walls to deconstruction and saccharification. Recalcitrance can be reduced by targeting genes involved in cell wall biosynthesis, but this can have unintended consequences that compromise the agronomic performance of the trees under field conditions. Here we report the results of a field trial of fourteen distinct transgenic Populus deltoides lines that had previously demonstrated reduced recalcitrance without yield penalties under greenhouse conditions.Results: Survival and productivity of the trial were excellent in the first year, and there was little evidence for reduced performancemore » of the transgenic lines with modified target gene expression. Surprisingly, the most striking phenotypic effects in this trial were for two empty-vector control lines that had modified bud set and bud flush. This is most likely due to somaclonal variation or insertional mutagenesis. Traits related to yield, crown architecture, herbivory, pathogen response, and frost damage showed few significant differences between target gene transgenics and empty vector controls. However, there were a few interesting exceptions. Lines overexpressing the DUF231 gene, a putative O-acetyltransferase, showed early bud flush and marginally increased height growth. Lines overexpressing the DUF266 gene, a putative glycosyltransferase, had significantly decreased stem internode length and slightly higher volume index. Finally, lines overexpressing the PFD2 gene, a putative member of the prefoldin complex, had a slightly reduced volume index.Conclusions: This field trial demonstrates that these cell wall modifications, which decreased cell wall recalcitrance under laboratory conditions, did not seriously compromise first-year performance in the field, despite substantial challenges, including an outbreak of a stem boring insect (Gypsonoma haimbachiana), attack by a leaf rust pathogen (Melampsora spp.), and a late frost event. This bodes well for the potential utility of these lines as advanced biofuels feedstocks.« less

Agronomic performance of Populus deltoides trees engineered for biofuel production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Macaya-Sanz, David; Chen, Jin?Gui; Kalluri, Udaya C.

Background: One of the major barriers to the development of lignocellulosic feedstocks is the recalcitrance of plant cell walls to deconstruction and saccharification. Recalcitrance can be reduced by targeting genes involved in cell wall biosynthesis, but this can have unintended consequences that compromise the agronomic performance of the trees under field conditions. Here we report the results of a field trial of fourteen distinct transgenic Populus deltoides lines that had previously demonstrated reduced recalcitrance without yield penalties under greenhouse conditions.Results: Survival and productivity of the trial were excellent in the first year, and there was little evidence for reduced performancemore » of the transgenic lines with modified target gene expression. Surprisingly, the most striking phenotypic effects in this trial were for two empty-vector control lines that had modified bud set and bud flush. This is most likely due to somaclonal variation or insertional mutagenesis. Traits related to yield, crown architecture, herbivory, pathogen response, and frost damage showed few significant differences between target gene transgenics and empty vector controls. However, there were a few interesting exceptions. Lines overexpressing the DUF231 gene, a putative O-acetyltransferase, showed early bud flush and marginally increased height growth. Lines overexpressing the DUF266 gene, a putative glycosyltransferase, had significantly decreased stem internode length and slightly higher volume index. Finally, lines overexpressing the PFD2 gene, a putative member of the prefoldin complex, had a slightly reduced volume index.Conclusions: This field trial demonstrates that these cell wall modifications, which decreased cell wall recalcitrance under laboratory conditions, did not seriously compromise first-year performance in the field, despite substantial challenges, including an outbreak of a stem boring insect (Gypsonoma haimbachiana), attack by a leaf rust pathogen (Melampsora spp.), and a late frost event. This bodes well for the potential utility of these lines as advanced biofuels feedstocks.« less

Combined radiation and p53 gene therapy of malignant glioma cells.

PubMed

Badie, B; Goh, C S; Klaver, J; Herweijer, H; Boothman, D A

1999-01-01

More than half of malignant gliomas reportedly have alterations in the p53 tumor suppressor gene. Because p53 plays a key role in the cellular response to DNA-damaging agents, we investigated the role of p53 gene therapy before ionizing radiation in cultured human glioma cells containing normal or mutated p53. Three established human glioma cell lines expressing the wild-type (U87 MG, p53wt) or mutant (A172 and U373 MG, p53mut) p53 gene were transduced by recombinant adenoviral vectors bearing human p53 (Adp53) and Escherichia coli beta-galactosidase genes (AdLacZ, control virus) before radiation (0-20 Gy). Changes in p53, p21, and Bax expression were studied by Western immunoblotting, whereas cell cycle alterations and apoptosis were investigated by flow cytometry and nuclear staining. Survival was assessed by clonogenic assays. Within 48 hours of Adp53 exposure, all three cell lines demonstrated p53 expression at a viral multiplicity of infection of 100. p21, which is a p53-inducible downstream effector gene, was overexpressed, and cells were arrested in the G1 phase. Bax expression, which is thought to play a role in p53-induced apoptosis, did not change with either radiation or Adp53. Apoptosis and survival after p53 gene therapy varied. U87 MG (p53wt) cells showed minimal apoptosis after Adp53, irradiation, or combined treatments. U373 MG (p53mut) cells underwent massive apoptosis and died within 48 hours of Adp53 treatment, independent of irradiation. Surprisingly, A172 (p53mut) cells demonstrated minimal apoptosis after Adp53 exposure; however, unlike U373 MG cells, apoptosis increased with radiation dose. Survival of all three cell lines was reduced dramatically after >10 Gy. Although Adp53 transduction significantly reduced the survival of U373 MG cells and inhibited A172 growth, it had no effect on the U87 MG cell line. Transduction with AdLacZ did not affect apoptosis or cell cycle progression and only minimally affected survival in all cell lines. We conclude that responses to p53 gene therapy are variable among gliomas and most likely depend upon both cellular p53 status and as yet ill-defined downstream pathways involving activation of cell cycle regulatory and apoptotic genes.

Aggravation of cold-induced injury in Vero-B4 cells by RPMI 1640 medium - identification of the responsible medium components.

PubMed

Pless-Petig, Gesine; Metzenmacher, Martin; Türk, Tobias R; Rauen, Ursula

2012-10-10

In modern biotechnology, there is a need for pausing cell lines by cold storage to adapt large-scale cell cultures to the variable demand for their products. We compared various cell culture media/solutions for cold storage of Vero-B4 kidney cells, a cell line widely used in biotechnology. Cold storage in RPMI 1640 medium, a recommended cell culture medium for Vero-B4 cells, surprisingly, strongly enhanced cold-induced cell injury in these cells in comparison to cold storage in Krebs-Henseleit buffer or other cell culture media (DMEM, L-15 and M199). Manufacturer, batch, medium supplements and the most likely components with concentrations outside the range of the other media/solutions (vitamin B12, inositol, biotin, p-aminobenzoic acid) did not cause this aggravation of cold-induced injury in RPMI 1640. However, a modified Krebs-Henseleit buffer with a low calcium concentration (0.42 mM), a high concentration of inorganic phosphate (5.6 mM), and glucose (11.1 mM; i.e. concentrations as in RPMI 1640) evoked a cell injury and loss of metabolic function corresponding to that observed in RPMI 1640. Deferoxamine improved cell survival and preserved metabolic function in modified Krebs-Henseleit buffer as well as in RPMI 1640. Similar Ca2+ and phosphate concentrations did not increase cold-induced cell injury in the kidney cell line LLC-PK1, porcine aortic endothelial cells or rat hepatocytes. However, more extreme conditions (Ca2+ was nominally absent and phosphate concentration raised to 25 mM as in the organ preservation solution University of Wisconsin solution) also increased cold-induced injury in rat hepatocytes and porcine aortic endothelial cells. These data suggest that the combination of low calcium and high phosphate concentrations in the presence of glucose enhances cold-induced, iron-dependent injury drastically in Vero-B4 cells, and that a tendency for this pathomechanism also exists in other cell types.

Functional significance of SPINK1 promoter variants in chronic pancreatitis.

PubMed

Derikx, Monique H M; Geisz, Andrea; Kereszturi, Éva; Sahin-Tóth, Miklós

2015-05-01

Chronic pancreatitis is a progressive inflammatory disorder of the pancreas, which often develops as a result of genetic predisposition. Some of the most frequently identified risk factors affect the serine protease inhibitor Kazal type 1 (SPINK1) gene, which encodes a trypsin inhibitor responsible for protecting the pancreas from premature trypsinogen activation. Recent genetic and functional studies indicated that promoter variants in the SPINK1 gene might contribute to disease risk in carriers. Here, we investigated the functional effects of 17 SPINK1 promoter variants using luciferase reporter gene expression assay in four different cell lines, including three pancreatic acinar cell lines (rat AR42J with or without dexamethasone-induced differentiation and mouse 266-6) and human embryonic kidney 293T cells. We found that most variants caused relatively small changes in promoter activity. Surprisingly, however, we observed significant variations in the effects of the promoter variants in the different cell lines. Only four variants exhibited consistently reduced promoter activity in all acinar cell lines, confirming previous reports that variants c.-108G>T, c.-142T>C, and c.-147A>G are risk factors for chronic pancreatitis and identifying c.-52G>T as a novel risk variant. In contrast, variant c.-215G>A, which is linked with the disease-associated splice-site mutation c.194 + 2T>C, caused increased promoter activity, which may mitigate the overall effect of the pathogenic haplotype. Our study lends further support to the notion that sequence evaluation of the SPINK1 promoter region in patients with chronic pancreatitis is justified as part of the etiological investigation. Copyright © 2015 the American Physiological Society.

MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

PubMed

Abbas, Hussein H K; Alhamoudi, Kheloud M H; Evans, Mark D; Jones, George D D; Foster, Steven S

2018-04-16

Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.

Rhabdovirus-like endogenous viral elements in the genome of Spodoptera frugiperda insect cells are actively transcribed: Implications for adventitious virus detection.

PubMed

Geisler, Christoph; Jarvis, Donald L

2016-07-01

Spodoptera frugiperda (Sf) cell lines are used to produce several biologicals for human and veterinary use. Recently, it was discovered that all tested Sf cell lines are persistently infected with Sf-rhabdovirus, a novel rhabdovirus. As part of an effort to search for other adventitious viruses, we searched the Sf cell genome and transcriptome for sequences related to Sf-rhabdovirus. To our surprise, we found intact Sf-rhabdovirus N- and P-like ORFs, and partial Sf-rhabdovirus G- and L-like ORFs. The transcribed and genomic sequences matched, indicating the transcripts were derived from the genomic sequences. These appear to be endogenous viral elements (EVEs), which result from the integration of partial viral genetic material into the host cell genome. It is theoretically impossible for the Sf-rhabdovirus-like EVEs to produce infectious virus particles as 1) they are disseminated across 4 genomic loci, 2) the G and L ORFs are incomplete, and 3) the M ORF is missing. Our finding of transcribed virus-like sequences in Sf cells underscores that MPS-based searches for adventitious viruses in cell substrates used to manufacture biologics should take into account both genomic and transcribed sequences to facilitate the identification of transcribed EVE's, and to avoid false positive detection of replication-competent adventitious viruses. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Rhabdovirus-like endogenous viral elements in the genome of Spodoptera frugiperda insect cells are actively transcribed: implications for adventitious virus detection

PubMed Central

Geisler, Christoph; Jarvis, Donald L.

2016-01-01

Spodoptera frugiperda (Sf) cell lines are used to produce several biologicals for human and veterinary use. Recently, it was discovered that all tested Sf cell lines are persistently infected with Sf-rhabdovirus, a novel rhabdovirus. As part of an effort to search for other adventitious viruses, we searched the Sf cell genome and transcriptome for sequences related to Sf-rhabdovirus. To our surprise, we found intact Sf-rhabdovirus N- and P-like ORFs, and partial Sf-rhabdovirus G- and L-like ORFs. The transcribed and genomic sequences matched, indicating the transcripts were derived from the genomic sequences. These appear to be endogenous viral elements (EVEs), which result from the integration of partial viral genetic material into the host cell genome. It is theoretically impossible for the Sf-rhabdovirus-like EVEs to produce infectious virus particles as 1) they are disseminated across 4 genomic loci, 2) the G and L ORFs are incomplete, and 3) the M ORF is missing. Our finding of transcribed virus-like sequences in Sf cells underscores that MPS-based searches for adventitious viruses in cell substrates used to manufacture biologics should take into account both genomic and transcribed sequences to facilitate the identification of transcribed EVE's, and to avoid false positive detection of replication-competent adventitious viruses. PMID:27236849

Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene.

PubMed

Stolić, Ivana; Mišković, Katarina; Piantanida, Ivo; Lončar, Mirela Baus; Glavaš-Obrovac, Ljubica; Bajić, Miroslav

2011-02-01

A series of novel 2,5-bis(amidinophenyl)-3,4-ethylenedioxythiophenes (5-10 and 15) has been synthesized. Compounds 5-10 bind to the DNA minor groove as the dominant binding site and strongly stabilize the double helix of ct-DNA. Surprisingly, the same compounds also thermally stabilize ds-RNA, whereby most of them form stacked dimers along the RNA double helix. The only exception is compound 15 which, due to its structural features, showed no interaction with DNA or RNA. Compounds 5-10 have shown a moderate to strong cytotoxic effect (GI50=1.5-9.0 μM) on a panel of seven tumour cell lines. The diimidazoline derivative 9, due to its highest inhibitory potential on the growth of all tested tumour cell lines, was investigated in more detail by testing its ability to enter into cells and influence the cell cycle. Compound 9 (5 μM) was internalized successfully in cell cytoplasm during a 30-min incubation period, followed by nuclear localization upon 90-min incubation. Significant arrest in HeLa cells in the G2/M phase, shown by cell cycle analysis at an equitoxic (50 μM) concentration, suggests interaction of a studied compound with cellular DNA as the main mode of biological action. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Brain Tumor’s Radioresistance: The Neighborhood Helps | Center for Cancer Research

Cancer.gov

Glioblastoma (GBM) is the most common and most aggressive form of brain cancer. The primary treatment for GBM is radiation therapy. Unfortunately, while some patients initially respond, the vast majority of GBM patients fail radiotherapy, and the tumor usually grows back within two years. To gain a better understanding of the biological basis for GBM resistance to radiation, researchers initially studied GBM cell lines in vitro. In recent years, the focus has been on so-called tumor stem-like cells (TSCs), which are thought to be responsible for driving and maintaining tumor growth. To the researchers’ surprise, TSCs grown in vitro did not have the same ability to resist radiation as TSCs in the GBM tumors.

Pancreatic cancer cell detection by targeted lipid microbubbles and multiphoton imaging

NASA Astrophysics Data System (ADS)

Cromey, Benjamin; McDaniel, Ashley; Matsunaga, Terry; Vagner, Josef; Kieu, Khanh Quoc; Banerjee, Bhaskar

2018-04-01

Surgical resection of pancreatic cancer represents the only chance of cure and long-term survival in this common disease. Unfortunately, determination of a cancer-free margin at surgery is based on one or two tiny frozen section biopsies, which is far from ideal. Not surprisingly, cancer is usually left behind and is responsible for metastatic disease. We demonstrate a method of receptor-targeted imaging using peptide ligands, lipid microbubbles, and multiphoton microscopy that could lead to a fast and accurate way of examining the entire cut surface during surgery. Using a plectin-targeted microbubble, we performed a blinded in-vitro study to demonstrate avid binding of targeted microbubbles to pancreatic cancer cells but not noncancerous cell lines. Further work should lead to a much-needed point-of-care diagnostic test for determining clean margins in oncologic surgery.

PI3K and Inhibitor of Apoptosis Proteins Modulate Gentamicin- Induced Hair Cell Death in the Zebrafish Lateral Line

PubMed Central

Wiedenhoft, Heather; Hayashi, Lauren; Coffin, Allison B.

2017-01-01

Inner ear hair cell death leads to sensorineural hearing loss and can be a direct consequence of aminoglycoside antibiotic treatment. Aminoglycosides such as gentamicin are effective therapy for serious Gram-negative bacterial infections such as some forms of meningitis, pneumonia, and sepsis. Aminoglycosides enter hair cells through mechanotransduction channels at the apical end of hair bundles and initiate intrinsic cell death cascades, but the precise cell signaling that leads to hair cell death is incompletely understood. Here, we examine the cell death pathways involved in aminoglycoside damage using the zebrafish (Danio rerio). The zebrafish lateral line contains hair cell-bearing organs called neuromasts that are homologous to hair cells of the mammalian inner ear and represents an excellent model to study ototoxicity. Based on previous research demonstrating a role for p53, Bcl2 signaling, autophagy, and proteasomal degradation in aminoglycoside-damaged hair cells, we used the Cytoscape GeneMANIA Database to identify additional proteins that might play a role in neomycin or gentamicin ototoxicity. Our bioinformatics analysis identified the pro-survival proteins phosphoinositide-dependent kinase-1 (PDK1) and X-linked inhibitor of apoptosis protein (Xiap) as potential mediators of gentamicin-induced hair cell damage. Pharmacological inhibition of PDK1 or its downstream mediator protein kinase C facilitated gentamicin toxicity, as did Xiap mutation, suggesting that both PI3K and endogenous Xiap confer protection. Surprisingly, aminoglycoside-induced hair cell death was highly attenuated in wild type Tupfel long-fin (TL fish; the background strain for the Xiap mutant line) compared to wild type ∗AB zebrafish. Pharmacologic manipulation of p53 suggested that the strain difference might result from decreased p53 in TL hair cells, allowing for increased hair cell survival. Overall, our studies identified additional steps in the cell death cascade triggered by aminoglycoside damage, suggesting possible drug targets to combat hearing loss resulting from aminoglycoside exposure. PMID:29093665

Discordant sex in monozygotic XXY/XX twins: a case report.

PubMed

Tachon, G; Lefort, G; Puechberty, J; Schneider, A; Jeandel, C; Boulot, P; Prodhomme, O; Meyer, P; Taviaux, S; Touitou, I; Pellestor, F; Geneviève, D; Gatinois, V

2014-12-01

We report a case of discordant phenotypic sex in monozygotic twins mosaic 47,XXY/46,XX: monozygotic heterokaryotypic twins. The twins presented with cognitive and comprehension delay, behavioural and language disorders, all symptoms frequently reported in Klinefelter syndrome. Molecular zygosity analysis with several markers confirmed that the twins are in effect monozygotic (MZ). Array comparative genomic hybridization found no evidence for the implication of copy number variation in the phenotypes. Ultrasound scans of the reproductive organs revealed no abnormalities. Endocrine tests showed a low testosterone level in Twin 1 (male phenotype) and a low gonadotrophin level in Twin 2 (female phenotype) which, combined with the results from ultrasound examination, provided useful information for potentially predicting the future fertility potential of the twins. Blood karyotypes revealed the presence of a normal 46,XX cell line and an aneuploïd 47,XXY cell line in both patients. Examination of the chromosome constitutions of various tissues such as blood, buccal smear and urinary sediment not surprisingly showed different proportions for the 46,XX and 47,XXY cell lines, which most likely explains the discordant phenotypic sex and mild Klinefelter features. The most plausible underlying biological mechanism is a post-zygotic loss of the Y chromosome in an initially 47,XXY zygote. This would result in an embryo with both 46,XX and 47,XXY cells lines which could subsequently divide into two monozygotic embryos through a twinning process. The two cell lines would then be distributed differently between tissues which could result in phenotypic discordances in the twins. These observations emphasize the importance of regular paediatric evaluations to determine the optimal timing for fertility preservation measures and to detect new Klinefelter features which could appear throughout childhood in the two subjects. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells.

PubMed

Meadows, Adam L; Kong, Becky; Berdichevsky, Marina; Roy, Siddhartha; Rosiva, Rosiva; Blanch, Harvey W; Clark, Douglas S

2008-01-01

The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.

Engineered LINE-1 retrotransposition in nondividing human neurons.

PubMed

Macia, Angela; Widmann, Thomas J; Heras, Sara R; Ayllon, Veronica; Sanchez, Laura; Benkaddour-Boumzaouad, Meriem; Muñoz-Lopez, Martin; Rubio, Alejandro; Amador-Cubero, Suyapa; Blanco-Jimenez, Eva; Garcia-Castro, Javier; Menendez, Pablo; Ng, Philip; Muotri, Alysson R; Goodier, John L; Garcia-Perez, Jose L

2017-03-01

Half the human genome is made of transposable elements (TEs), whose ongoing activity continues to impact our genome. LINE-1 (or L1) is an autonomous non-LTR retrotransposon in the human genome, comprising 17% of its genomic mass and containing an average of 80-100 active L1s per average genome that provide a source of inter-individual variation. New LINE-1 insertions are thought to accumulate mostly during human embryogenesis. Surprisingly, the activity of L1s can further impact the somatic human brain genome. However, it is currently unknown whether L1 can retrotranspose in other somatic healthy tissues or if L1 mobilization is restricted to neuronal precursor cells (NPCs) in the human brain. Here, we took advantage of an engineered L1 retrotransposition assay to analyze L1 mobilization rates in human mesenchymal (MSCs) and hematopoietic (HSCs) somatic stem cells. Notably, we have observed that L1 expression and engineered retrotransposition is much lower in both MSCs and HSCs when compared to NPCs. Remarkably, we have further demonstrated for the first time that engineered L1s can retrotranspose efficiently in mature nondividing neuronal cells. Thus, these findings suggest that the degree of somatic mosaicism and the impact of L1 retrotransposition in the human brain is likely much higher than previously thought. © 2017 Macia et al.; Published by Cold Spring Harbor Laboratory Press.

Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

PubMed Central

Deplus, Rachel; Lampe, Xavier; Krusy, Nathalie; Calonne, Emilie; Delbecque, Katty; Kridelka, Frederic; Fuks, François; Ennaji, My Mustapha; Delvenne, Philippe

2012-01-01

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. PMID:22880087

Diverse matrix metalloproteinase functions regulate cancer amoeboid migration

PubMed Central

Orgaz, Jose L.; Pandya, Pahini; Viros, Amaya; Albrengues, Jean; Nestle, Frank O.; Ridley, Anne J.; Gaggioli, Cedric; Marais, Richard; Karagiannis, Sophia N.; Sanz-Moreno, Victoria

2014-01-01

Rounded-amoeboid cancer cells use actomyosin contractility driven by Rho-ROCK and JAK-STAT3 to migrate efficiently. It has been suggested that rounded-amoeboid cancer cells do not require matrix metalloproteinases (MMPs) to invade. Here we compare MMP levels in rounded-amoeboid and elongated-mesenchymal melanoma cells. Surprisingly, we find that rounded-amoeboid melanoma cells secrete higher levels of several MMPs, including collagenase MMP-13 and gelatinase MMP-9. As a result, rounded-amoeboid melanoma cells degrade collagen I more efficiently than elongated-mesenchymal cells. Furthermore, using a non-catalytic mechanism, MMP-9 promotes rounded-amoeboid 3D migration through regulation of actomyosin contractility via CD44 receptor. MMP-9 is upregulated in a panel of rounded-amoeboid compared with elongated-mesenchymal melanoma cell lines and its levels are controlled by ROCK-JAK-STAT3 signalling. MMP-9 expression increases during melanoma progression and it is particularly prominent in the invasive fronts of lesions, correlating with cell roundness. Therefore, rounded-amoeboid cells use both catalytic and non-catalytic activities of MMPs for invasion. PMID:24963846

Enhanced degradation of p53 protein in HPV-6 and BPV-1 E6-immortalized human mammary epithelial cells.

PubMed Central

Band, V; Dalal, S; Delmolino, L; Androphy, E J

1993-01-01

Normal mammary epithelial cells are efficiently immortalized by the E6 gene of human papillomavirus (HPV)-16, a virus commonly associated with cervical cancers. Surprisingly, introduction of the E6 gene from HPV-6, which is rarely found in cervical cancer, or bovine papillomavirus (BPV)-1, into normal mammary cells resulted in the generation of immortal cell lines. The establishment of HPV-6 and BPV-1 E6-immortalized cells was less efficient and required a longer period in comparison to HPV-16 E6. These HPV-6- and BPV-1 E6-immortalized cells demonstrated dramatically reduced levels of p53 protein by immunoprecipitation. While the half-life of p53 protein in normal mammary epithelial cells was approximately 3 h, it was reduced to approximately 15 min in all the E6-immortalized cells. These results demonstrate that the E6 genes of both high-risk and low-risk papilloma viruses immortalize human mammary epithelial cells and induce a marked degradation of p53 protein in vivo. Images PMID:8387914

Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines.

PubMed

Hearst, Scoty M; Gilder, Andrew S; Negi, Sandeep S; Davis, Misty D; George, Eric M; Whittom, Angela A; Toyota, Cory G; Husedzinovic, Alma; Gruss, Oliver J; Hebert, Michael D

2009-06-01

Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells.

The cornucopia of meaningful leads: Applying deep adversarial autoencoders for new molecule development in oncology

PubMed Central

Kadurin, Artur; Aliper, Alexander; Kazennov, Andrey; Mamoshina, Polina; Vanhaelen, Quentin; Khrabrov, Kuzma; Zhavoronkov, Alex

2017-01-01

Recent advances in deep learning and specifically in generative adversarial networks have demonstrated surprising results in generating new images and videos upon request even using natural language as input. In this paper we present the first application of generative adversarial autoencoders (AAE) for generating novel molecular fingerprints with a defined set of parameters. We developed a 7-layer AAE architecture with the latent middle layer serving as a discriminator. As an input and output the AAE uses a vector of binary fingerprints and concentration of the molecule. In the latent layer we also introduced a neuron responsible for growth inhibition percentage, which when negative indicates the reduction in the number of tumor cells after the treatment. To train the AAE we used the NCI-60 cell line assay data for 6252 compounds profiled on MCF-7 cell line. The output of the AAE was used to screen 72 million compounds in PubChem and select candidate molecules with potential anti-cancer properties. This approach is a proof of concept of an artificially-intelligent drug discovery engine, where AAEs are used to generate new molecular fingerprints with the desired molecular properties. PMID:28029644

Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

PubMed

Szulc-Dabrowska, Lidia; Gregorczyk, Karolina P; Struzik, Justyna; Boratynska-Jasinska, Anna; Szczepanowska, Joanna; Wyzewski, Zbigniew; Toka, Felix N; Gierynska, Malgorzata; Ostrowska, Agnieszka; Niemialtowski, Marek G

2016-08-01

Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells.

PubMed

Boonkaew, Benjawan; Kempf, Margit; Kimble, Roy; Cuttle, Leila

2014-12-01

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver(®) and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver(®) showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Cilia distribution and polarity in the epithelial lining of the mouse middle ear cavity

PubMed Central

Luo, Wenwei; Yi, Hong; Taylor, Jeannette; Li, Jian-dong; Chi, Fanglu; Todd, N. Wendell; Lin, Xi; Ren, Dongdong; Chen, Ping

2017-01-01

The middle ear conducts sound to the cochlea for hearing. Otitis media (OM) is the most common illness in childhood. Moreover, chronic OM with effusion (COME) is the leading cause of conductive hearing loss. Clinically, COME is highly associated with Primary Ciliary Dyskinesia, implicating significant contributions of cilia dysfunction to COME. The understanding of middle ear cilia properties that are critical to OM susceptibility, however, is limited. Here, we confirmed the presence of a ciliated region near the Eustachian tube orifice at the ventral region of the middle ear cavity, consisting mostly of a lumen layer of multi-ciliated and a layer of Keratin-5-positive basal cells. We also found that the motile cilia are polarized coordinately and display a planar cell polarity. Surprisingly, we also found a region of multi-ciliated cells that line the posterior dorsal pole of the middle ear cavity which was previously thought to contain only non-ciliated cells. Our study provided a more complete understanding of cilia distribution and revealed for the first time coordinated polarity of cilia in the epithelium of the mammalian middle ear, thus illustrating novel structural features that are likely critical for middle ear functions and related to OM susceptibility. PMID:28358397

Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves.

PubMed

Nicolas, William J; Grison, Magali S; Trépout, Sylvain; Gaston, Amélia; Fouché, Mathieu; Cordelières, Fabrice P; Oparka, Karl; Tilsner, Jens; Brocard, Lysiane; Bayer, Emmanuelle M

2017-06-12

Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.

Cell-cycle dependent expression of a translocation-mediated fusion oncogene mediates checkpoint adaptation in rhabdomyosarcoma.

PubMed

Kikuchi, Ken; Hettmer, Simone; Aslam, M Imran; Michalek, Joel E; Laub, Wolfram; Wilky, Breelyn A; Loeb, David M; Rubin, Brian P; Wagers, Amy J; Keller, Charles

2014-01-01

Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells.

The putative G-protein coupled estrogen receptor agonist G-1 suppresses proliferation of ovarian and breast cancer cells in a GPER-independent manner

PubMed Central

Wang, Cheng; Lv, Xiangmin; Jiang, Chao; Davis, John S

2012-01-01

G-protein coupled estrogen receptor 1 (GPER) plays an important role in mediating estrogen action in many different tissues under both physiological and pathological conditions. G-1 (1-[4-(6-bromobenzo[1,3]dioxol-5yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta [c]quinolin-8-yl]-ethanone) has been developed as a selective GPER agonist to distinguish estrogen actions mediated by GPER from those mediated by classic estrogen receptors. In the present study, we surprisingly found that G-1 suppressed proliferation and induced apoptosis of KGN cells (a human ovarian granulosa cell tumor cell line), actions that were not blocked by a selective GPER antagonist G15 or siRNA knockdown of GPER. G-1 also suppressed proliferation and induced cell apoptosis in GPER-negative HEK-293 cells and MDA-MB 231 breast cancer cells. Our results demonstrate that G-1 suppresses proliferation of ovarian and breast cancer cells in a GPER-independent manner. G-1 may be a candidate for the development of drugs against ovarian and breast cancer. PMID:23145207

Dexamethasone enhances agonist induction of tissue factor in monocytes but not in endothelial cells.

PubMed

Bottles, K D; Morrissey, J H

1993-06-01

Stimulation of monocytic cells by inflammatory agents such as bacterial lipopolysaccharide or tumour necrosis factor-alpha leads to the rapid and transient expression of tissue factor, the major cellular initiator of the extrinsic coagulation cascade in both haemostasis and tissue inflammation. In this study we investigated whether the synthetic anti-inflammatory glucocorticoid, dexamethasone, would inhibit agonist induction of tissue factor expression in both monocytes and endothelial cells. Surprisingly, dexamethasone significantly enhanced the induction of tissue factor expression by peripheral blood mononuclear cells and an established monocytic cell line, THP-1, in response to lipopolysaccharide or tumour necrosis factor-alpha. However, unlike monocytic cells, dexamethasone did not enhance agonist induction of tissue factor in endothelial cells. Synergistic enhancement of tissue factor expression by dexamethasone was also reflected in tissue factor mRNA levels in THP-1 cells, but was not the result of improved TF mRNA stability. Synergism between bacterial lipopolysaccharide and glucocorticoid in the induction of monocyte effector function is extremely unusual and may help to explain the variable outcome of glucocorticoid treatment of septic shock.

NONO couples the circadian clock to the cell cycle.

PubMed

Kowalska, Elzbieta; Ripperger, Juergen A; Hoegger, Dominik C; Bruegger, Pascal; Buch, Thorsten; Birchler, Thomas; Mueller, Anke; Albrecht, Urs; Contaldo, Claudio; Brown, Steven A

2013-01-29

Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.

Identification of Mutation Accumulation as Resistance Mechanism Emerging in First-Line Osimertinib Treatment.

PubMed

Uchibori, Ken; Inase, Naohiko; Nishio, Makoto; Fujita, Naoya; Katayama, Ryohei

2018-04-24

The survival of patients with EGFR mutation-positive lung cancer has dramatically improved since the introduction of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Recently, osimertinib showed significantly prolonged progression-free survival than first-generation EGFR-TKI in first-line treatment, suggesting that a paradigm change that would move osimetinib to first-line treatment is indicated. We performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening to uncover the resistant mechanism in first- and second-line osimertinib treatment. Ba/F3 cells harboring EGFR activating-mutation with or without secondary resistant mutation were exposed to ENU for 24 hours to introduce random mutations and selected with gefitinib, afatinib, or osimertinib. Mutations of emerging resistant cells were assessed. The resistance of T790M and C797S to gefitinib and osimertinib, respectively, was prevalent in the mutagenesis screening with the Ba/F3 cells harboring activating-mutation alone. From C797S/activating-mutation expressing Ba/F3, the additional T790M was a major resistant mechanism in gefitinib and afatinib selection and the additional T854A and L792H were minor resistance mechanisms only in afatinib selection. However, the additional T854A or L792H mediated resistance to all classes of EGFR-TKI. Surprisingly, no resistant clone due to secondary mutation emerged from activating-mutation alone in the gefitinib + osimertinib selection. We showed the resistance mechanism to EGFR-TKI focusing on first- and second-line osimertinib using ENU mutagenesis screening. Additional T854A and L792H on C797S/activating-mutation were found as afatinib resistance and not as gefitinib resistance. Thus, compared to afatinib, the first-generation EGFR-TKI might be preferable as second-line treatment to C797S/activating-mutation emerging after first-line osimertinib treatment. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Pancreatic cancer cell detection by targeted lipid microbubbles and multiphoton imaging.

PubMed

Cromey, Benjamin; McDaniel, Ashley; Matsunaga, Terry; Vagner, Josef; Kieu, Khanh Quoc; Banerjee, Bhaskar

2018-04-01

Surgical resection of pancreatic cancer represents the only chance of cure and long-term survival in this common disease. Unfortunately, determination of a cancer-free margin at surgery is based on one or two tiny frozen section biopsies, which is far from ideal. Not surprisingly, cancer is usually left behind and is responsible for metastatic disease. We demonstrate a method of receptor-targeted imaging using peptide ligands, lipid microbubbles, and multiphoton microscopy that could lead to a fast and accurate way of examining the entire cut surface during surgery. Using a plectin-targeted microbubble, we performed a blinded in-vitro study to demonstrate avid binding of targeted microbubbles to pancreatic cancer cells but not noncancerous cell lines. Further work should lead to a much-needed point-of-care diagnostic test for determining clean margins in oncologic surgery. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

IFIT1 Expression Patterns Induced by H9N2 Virus and Inactivated Viral Particle in Human Umbilical Vein Endothelial Cells and Bronchus Epithelial Cells.

PubMed

Feng, Bo; Zhang, Qian; Wang, Jianfang; Dong, Hong; Mu, Xiang; Hu, Ge; Zhang, Tao

2018-04-30

IFIT1 (also known as ISG56) is a member of the interferon-inducible protein with tetratricopeptide repeats (IFITs) family. IFITs are strongly induced by type I interferon (IFN), double-stranded RNA and virus infection. Here, we investigated IFIT1 expression in human umbilical vein endothelial cells (HUVECs) and in human bronchus epithelial cells (BEAS-2Bs) induced by the H9N2 virus and inactivated viral particle at different time points. We also investigated the effect of H9N2 virus and viral particle infection on IFN-α/β production, and assessed whether hemagglutinin or neuraminidase protein induced IFIT1 expression. Results showed that both H9N2 virus infection and viral particle inoculation induced the expression of IFIT1 at mRNA and protein levels in the two cell lines. Hemagglutinin or neuraminidase protein binding alone is not sufficient to induce IFIT1 expression. Surprisingly, the expression patterns of IFIT1 in response to H9N2 virus and viral particles in the two cell lines were opposite, and production kinetics of IFN-α/β also differed. An additional finding was that induction of IFIT1 in response to H9N2 virus infection or viral particle inoculation was more sensitive in HUVECs than in BEAS-2Bs. Our data offers new insight into the innate immune response of endothelial cells to H9N2 virus infection.

IFIT1 Expression Patterns Induced by H9N2 Virus and Inactivated Viral Particle in Human Umbilical Vein Endothelial Cells and Bronchus Epithelial Cells

PubMed Central

Feng, Bo; Zhang, Qian; Wang, Jianfang; Dong, Hong; Mu, Xiang; Hu, Ge; Zhang, Tao

2018-01-01

IFIT1 (also known as ISG56) is a member of the interferon-inducible protein with tetratricopeptide repeats (IFITs) family. IFITs are strongly induced by type I interferon (IFN), double-stranded RNA and virus infection. Here, we investigated IFIT1 expression in human umbilical vein endothelial cells (HUVECs) and in human bronchus epithelial cells (BEAS-2Bs) induced by the H9N2 virus and inactivated viral particle at different time points. We also investigated the effect of H9N2 virus and viral particle infection on IFN-α/β production, and assessed whether hemagglutinin or neuraminidase protein induced IFIT1 expression. Results showed that both H9N2 virus infection and viral particle inoculation induced the expression of IFIT1 at mRNA and protein levels in the two cell lines. Hemagglutinin or neuraminidase protein binding alone is not sufficient to induce IFIT1 expression. Surprisingly, the expression patterns of IFIT1 in response to H9N2 virus and viral particles in the two cell lines were opposite, and production kinetics of IFN-α/β also differed. An additional finding was that induction of IFIT1 in response to H9N2 virus infection or viral particle inoculation was more sensitive in HUVECs than in BEAS-2Bs. Our data offers new insight into the innate immune response of endothelial cells to H9N2 virus infection. PMID:29629559

A herpes simplex virus 2 glycoprotein D mutant generated by bacterial artificial chromosome mutagenesis is severely impaired for infecting neuronal cells and infects only Vero cells expressing exogenous HVEM.

PubMed

Wang, Kening; Kappel, Justin D; Canders, Caleb; Davila, Wilmer F; Sayre, Dean; Chavez, Mayra; Pesnicak, Lesley; Cohen, Jeffrey I

2012-12-01

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine.

A Herpes Simplex Virus 2 Glycoprotein D Mutant Generated by Bacterial Artificial Chromosome Mutagenesis Is Severely Impaired for Infecting Neuronal Cells and Infects Only Vero Cells Expressing Exogenous HVEM

PubMed Central

Kappel, Justin D.; Canders, Caleb; Davila, Wilmer F.; Sayre, Dean; Chavez, Mayra; Pesnicak, Lesley; Cohen, Jeffrey I.

2012-01-01

We constructed a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) clone, bHSV2-BAC38, which contains full-length HSV-2 inserted into a BAC vector. Unlike previously reported HSV-2 BAC clones, the virus genome inserted into this BAC clone has no known gene disruptions. Virus derived from the BAC clone had a wild-type phenotype for growth in vitro and for acute infection, latency, and reactivation in mice. HVEM, expressed on epithelial cells and lymphocytes, and nectin-1, expressed on neurons and epithelial cells, are the two principal receptors used by HSV to enter cells. We used the HSV-2 BAC clone to construct an HSV-2 glycoprotein D mutant (HSV2-gD27) with point mutations in amino acids 215, 222, and 223, which are critical for the interaction of gD with nectin-1. HSV2-gD27 infected cells expressing HVEM, including a human epithelial cell line. However, the virus lost the ability to infect cells expressing only nectin-1, including neuronal cell lines, and did not infect ganglia in mice. Surprisingly, we found that HSV2-gD27 could not infect Vero cells unless we transduced the cells with a retrovirus expressing HVEM. High-level expression of HVEM in Vero cells also resulted in increased syncytia and enhanced cell-to-cell spread in cells infected with wild-type HSV-2. The inability of the HSV2-gD27 mutant to infect neuronal cells in vitro or sensory ganglia in mice after intramuscular inoculation suggests that this HSV-2 mutant might be an attractive candidate for a live attenuated HSV-2 vaccine. PMID:22993162

Spine-bellied sea snake (Hydrophis curtus) venom shows greater skeletal myotoxicity compared with cardiac myotoxicity.

PubMed

Neale, Vanessa; Smout, Michael J; Seymour, Jamie E

2018-03-01

For the first time the impedance-based xCELLigence real-time cell analysis system was used to measure the myotoxicity of sea snake venom. With a focus on the spine-bellied sea snake (Hydrophis curtus), the venom of four sea snake species and three terrestrial snake species were compared for myotoxicity against a human skeletal muscle cell line (HSkMC). Hydrophis curtus venom was also tested on a human cardiac muscle cell line (HCM). Surprisingly, all four sea snake venoms tested on HSkMC produced an initial 100-280% rise in xCELLigence cell index that peaked within the first two hours before falling. The cell index rise of H. curtus venom was correlated with the WST-1 cell proliferation assay, which demonstrated an increase in mitochondrial metabolism. The myotoxicity of H. curtus was 4.7-8.2 fold less potent than the other sea snakes tested, the Australian beaked sea snake (Hydrophis zweifeli), the elegant sea snake (Hydrophis elegans) and the olive sea snake (Aipysurus laevis). If our cell-based results translate to H. curtus envenomations, this implies that H. curtus would be less myotoxic than the other three. Yet the myotoxicity of H. curtus venom to cardiac muscle cells was nine times weaker than for skeletal muscle cells, providing evidence that the venom has a selective effect on skeletal muscle cells. This evidence, combined with the slow-acting nature of the venom, supports a digestive role for sea snake myotoxins. Copyright © 2018 Elsevier Ltd. All rights reserved.

Neurogenic radial glia in the outer subventricular zone of human neocortex.

PubMed

Hansen, David V; Lui, Jan H; Parker, Philip R L; Kriegstein, Arnold R

2010-03-25

Neurons in the developing rodent cortex are generated from radial glial cells that function as neural stem cells. These epithelial cells line the cerebral ventricles and generate intermediate progenitor cells that migrate into the subventricular zone (SVZ) and proliferate to increase neuronal number. The developing human SVZ has a massively expanded outer region (OSVZ) thought to contribute to cortical size and complexity. However, OSVZ progenitor cell types and their contribution to neurogenesis are not well understood. Here we show that large numbers of radial glia-like cells and intermediate progenitor cells populate the human OSVZ. We find that OSVZ radial glia-like cells have a long basal process but, surprisingly, are non-epithelial as they lack contact with the ventricular surface. Using real-time imaging and clonal analysis, we demonstrate that these cells can undergo proliferative divisions and self-renewing asymmetric divisions to generate neuronal progenitor cells that can proliferate further. We also show that inhibition of Notch signalling in OSVZ progenitor cells induces their neuronal differentiation. The establishment of non-ventricular radial glia-like cells may have been a critical evolutionary advance underlying increased cortical size and complexity in the human brain.

Revisit: A Surprising Demonstration of Total Internal Reflection

ERIC Educational Resources Information Center

Lee, Jiwon; Cha, Yu Wha; Jung, Yeon Su; Oh, Eun Ju; Moon, Ye Lin; Kim, Jung Bog

2016-01-01

Melton demonstrated a surprising disappearance using total internal reflection. When he put a Florence flask filled with marbles into a water tank and looked straight down from directly above the flask, he was only able to see marbles above a certain water level. When he added more water into the tank above the top line of the marbles, all of the…

Surprisal analysis characterizes the free energy time course of cancer cells undergoing epithelial-to-mesenchymal transition.

PubMed

Zadran, Sohila; Arumugam, Rameshkumar; Herschman, Harvey; Phelps, Michael E; Levine, R D

2014-09-09

The epithelial-to-mesenchymal transition (EMT) initiates the invasive and metastatic behavior of many epithelial cancers. Mechanisms underlying EMT are not fully known. Surprisal analysis of mRNA time course data from lung and pancreatic cancer cells stimulated to undergo TGF-β1-induced EMT identifies two phenotypes. Examination of the time course for these phenotypes reveals that EMT reprogramming is a multistep process characterized by initiation, maturation, and stabilization stages that correlate with changes in cell metabolism. Surprisal analysis characterizes the free energy time course of the expression levels throughout the transition in terms of two state variables. The landscape of the free energy changes during the EMT for the lung cancer cells shows a stable intermediate state. Existing data suggest this is the previously proposed maturation stage. Using a single-cell ATP assay, we demonstrate that the TGF-β1-induced EMT for lung cancer cells, particularly during the maturation stage, coincides with a metabolic shift resulting in increased cytosolic ATP levels. Surprisal analysis also characterizes the absolute expression levels of the mRNAs and thereby examines the homeostasis of the transcription system during EMT.

Acquisition of relative interstrand crosslinker resistance and PARP inhibitor sensitivity in Fanconi anemia head and neck cancers

PubMed Central

Lombardi, Anne J.; Hoskins, Elizabeth E.; Foglesong, Grant D.; Wikenheiser-Brokamp, Kathryn A.; Wiesmüller, Lisa; Hanenberg, Helmut; Andreassen, Paul R.; Jacobs, Allison J.; Olson, Susan B.; Keeble, Winifred W.; Hays, Laura E.; Wells, Susanne I.

2015-01-01

Purpose Fanconi anemia (FA) is an inherited disorder associated with a constitutional defect in the FA DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with FA are predisposed to formation of head and neck squamous cell carcinomas (HNSCCs) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. Experimental Design Using HNSCC cell lines derived from the tumors of FA patients, and murine HNSCC cell lines derived from the tumors of wild type and Fancc−/− mice, we sought to define FA-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of FA HNSCC cells for non-homologous end joining (NHEJ). Results Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily FA-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in FA cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by poly(ADP-ribose) polymerase (PARP) in FA-deficient cells. Moreover, human and murine FA HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by FA gene complementation. Conclusions The observed reliance upon PARP-mediated mechanisms reveals a means by which FA HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual. PMID:25609062

Activity and Ca2+ regulate the mobility of TRPV1 channels in the plasma membrane of sensory neurons

PubMed Central

Senning, Eric N; Gordon, Sharona E

2015-01-01

TRPV1 channels are gated by a variety of thermal, chemical, and mechanical stimuli. We used optical recording of Ca2+ influx through TRPV1 to measure activity and mobility of single TRPV1 molecules in isolated dorsal root ganglion neurons and cell lines. The opening of single TRPV1 channels produced sparklets, representing localized regions of elevated Ca2+. Unlike sparklets reported for L-type Ca2+ channels, TRPV4 channels, and AchR channels, TRPV1 channels diffused laterally in the plasma membrane as they gated. Mobility was highly variable from channel-to-channel and, to a smaller extent, from cell to cell. Most surprisingly, we found that mobility decreased upon channel activation by capsaicin, but only in the presence of extracellular Ca2+. We propose that decreased mobility of open TRPV1 could act as a diffusion trap to concentrate channels in cell regions with high activity. DOI: http://dx.doi.org/10.7554/eLife.03819.001 PMID:25569155

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

PubMed

Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

2016-08-01

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

The CXCR5 chemokine receptor is expressed by carcinoma cells and promotes growth of colon carcinoma in the liver.

PubMed

Meijer, Joost; Zeelenberg, Ingrid S; Sipos, Bence; Roos, Ed

2006-10-01

The chemokine receptor CXCR5 is expressed by B cells and certain T cells and controls their migration into and within lymph nodes. Its ligand BCA-1/CXCL13 is present in lymph nodes and spleen and also in the liver. Surprisingly, we detected CXCR5 in several mouse and human carcinoma cell lines. CXCR5 was particularly prominent in pancreatic carcinoma cell lines and was also detected by immunohistochemistry in 7 of 18 human pancreatic carcinoma tissues. Expression in CT26 colon carcinoma was low in vitro, up-regulated in vivo, and rapidly lost when cells were explanted in vitro. CXCL13 strongly promoted proliferation of CXCR5-transfected CT26 cells in vitro. In the liver, after intrasplenic injection, these CXCR5 transfectants initially grew faster than controls, but the growth rate of control tumors accelerated later to become similar to the transfectants, likely due to the up-regulation of CXCR5. Inhibition of CXCR5 function, by trapping CXCR5 in the endoplasmic reticulum using a CXCL13-KDEL "intrakine," had no effect on initial growth of liver foci but later caused a prolonged growth arrest. In contrast, s.c. and lung tumors of CXCR5- and intrakine-transfected cells grew at similar rates as controls. We conclude that expression of CXCR5 on tumor cells promotes the growth of tumor cells in the liver and, at least for CT26 cells, seems to be required for outgrowth to large liver tumors. Given the limited expression on normal cells, CXCR5 may constitute an attractive target for therapy, particularly for pancreatic carcinoma.

Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize (Zea mays L.) lines

PubMed Central

Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D.; Williams, Daniel L.; Magee, Timothy D.; Kaeppler, Shawn M.; de Leon, Natalia; Hodge, David B.

2015-01-01

A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA) content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. This indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment. PMID:25871649

Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines

PubMed Central

Hearst, Scoty M.; Gilder, Andrew S.; Negi, Sandeep S.; Davis, Misty D.; George, Eric M.; Whittom, Angela A.; Toyota, Cory G.; Husedzinovic, Alma; Gruss, Oliver J.; Hebert, Michael D.

2009-01-01

Summary Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells. PMID:19435804

Pyrazolo[1,5-a]-1,3,5-triazine as a purine bioisostere: access to potent cyclin-dependent kinase inhibitor (R)-roscovitine analogue.

PubMed

Popowycz, Florence; Fournet, Guy; Schneider, Cédric; Bettayeb, Karima; Ferandin, Yoan; Lamigeon, Cyrile; Tirado, Oscar M; Mateo-Lozano, Silvia; Notario, Vicente; Colas, Pierre; Bernard, Philippe; Meijer, Laurent; Joseph, Benoît

2009-02-12

Pharmacological inhibitors of cyclin-dependent kinases (CDKs) have a wide therapeutic potential. Among the CDK inhibitors currently under clinical trials, the 2,6,9-trisubstituted purine (R)-roscovitine displays rather high selectivity, low toxicity, and promising antitumor activity. In an effort to improve this structure, we synthesized several bioisosteres of roscovitine. Surprisingly, one of them, pyrazolo[1,5-a]-1,3,5-triazine 7a (N-&-N1, GP0210), displayed significantly higher potency, compared to (R)-roscovitine and imidazo[2,1-f]-1,2,4-triazine 13 (N-&-N2, GP0212), at inhibiting various CDKs and at inducing cell death in a wide variety of human tumor cell lines. This approach may thus provide second generation analogues with enhanced biomedical potential.

PLC-γ1 is involved in the inflammatory response induced by influenza A virus H1N1 infection.

PubMed

Zhu, Liqian; Yuan, Chen; Ding, Xiuyan; Xu, Shuai; Yang, Jiayun; Liang, Yuying; Zhu, Qiyun

2016-09-01

We have previously reported that phosphoinositide-specific phospholipase γ1 (PLC-γ1) signaling is activated by influenza virus H1N1 infection and mediates efficient viral entry in human epithelial cells. In this study, we show that H1N1 also activates PLCγ-1 signaling in human promonocytic cell line -derived macrophages. Surprisingly, the activated PLCγ-1 signaling is not important for viral replication in macrophages, but is involved in the virus-induced inflammatory responses. PLC-γ1-specific inhibitor U73122 strongly inhibits the H1N1 virus-induced NF-κB signaling, blocking the up-regulation of TNF-α, IL-6, MIP-1α, and reactive oxidative species. In a positive feedback loop, IL-1β and TNF-α activate the PLCγ-1 signaling in both epithelial and macrophage cell lines. In summary, we have shown for the first time that the PLCγ-1 signaling plays an important role in the H1N1-induced inflammatory responses. Our study suggests that targeting the PLCγ-1 signaling is a potential antiviral therapy against H1N1 by inhibiting both viral replication and excessive inflammation. Copyright © 2016 Elsevier Inc. All rights reserved.

O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C.

PubMed

Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

2006-03-01

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica).

PubMed

Dare, Andrew P; Tomes, Sumathi; Jones, Midori; McGhie, Tony K; Stevenson, David E; Johnson, Ross A; Greenwood, David R; Hellens, Roger P

2013-05-01

We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down-regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down-regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS-silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS-silenced line was smaller than the 'Royal Gala' controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS-silenced line compared with the 'Royal Gala' control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Follicle-stimulating hormone synthesis and fertility are intact in mice lacking SMAD3 DNA binding activity and SMAD2 in gonadotrope cells

PubMed Central

Fortin, Jérôme; Boehm, Ulrich; Weinstein, Michael B.; Graff, Jonathan M.; Bernard, Daniel J.

2014-01-01

The activin/inhibin system regulates follicle-stimulating hormone (FSH) synthesis and release by pituitary gonadotrope cells in mammals. In vitro cell line data suggest that activins stimulate FSH β-subunit (Fshb) transcription via complexes containing the receptor-regulated SMAD proteins SMAD2 and SMAD3. Here, we used a Cre-loxP approach to determine the necessity for SMAD2 and/or SMAD3 in FSH synthesis in vivo. Surprisingly, mice with conditional mutations in both Smad2 and Smad3 specifically in gonadotrope cells are fertile and produce FSH at quantitatively normal levels. Notably, however, we discovered that the recombined Smad3 allele produces a transcript that encodes the entirety of the SMAD3 C-terminal Mad homology 2 (MH2) domain. This protein behaves similarly to full-length SMAD3 in Fshb transcriptional assays. As the truncated protein lacks the N-terminal Mad homology 1 (MH1) domain, these results show that SMAD3 DNA-binding activity as well as SMAD2 are dispensable for normal FSH synthesis in vivo. Furthermore, the observation that deletion of proximal exons does not remove all SMAD3 function may facilitate interpretation of divergent phenotypes previously described in different Smad3 knockout mouse lines.—Fortin, J., Boehm, U., Weinstein, M. B., Graff, J. M., Bernard, D. J. Follicle-stimulating hormone synthesis and fertility are intact in mice lacking SMAD3 DNA binding activity and SMAD2 in gonadotrope cells. PMID:24308975

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

PubMed Central

Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

2014-01-01

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary. PMID:24658126

Acquisition of Relative Interstrand Crosslinker Resistance and PARP Inhibitor Sensitivity in Fanconi Anemia Head and Neck Cancers.

PubMed

Lombardi, Anne J; Hoskins, Elizabeth E; Foglesong, Grant D; Wikenheiser-Brokamp, Kathryn A; Wiesmüller, Lisa; Hanenberg, Helmut; Andreassen, Paul R; Jacobs, Allison J; Olson, Susan B; Keeble, Winifred W; Hays, Laura E; Wells, Susanne I

2015-04-15

Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc(-/-) mice, we sought to define Fanconi anemia-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ). Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia-deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation. The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual. ©2015 American Association for Cancer Research.

Quantitative analysis of antigen for the induction of tolerance in carcinoembryonic antigen transgenic mice.

PubMed Central

Hasegawa, T; Isobe, K; Nakashima, I; Shimokata, K

1992-01-01

In order to analyse the amounts of antigen in the thymus for the induction of tolerance, several carcinoembryonic antigen (CEA) transgenic lines were established which expressed human CEA antigen with different amounts. The chimeric KSN nude mice transplanted with the thymus of the B601 line (in which CEA mRNA and CEA protein could be detected in various tissues) to kidney capsule showed tolerance to human CEA. On the other hand, the chimeric KSN nude mice transplanted with the thymus of the B602 or BC60 line (in which neither CEA mRNA nor CEA protein could be detected by Northern blot analysis and flow cytometry analysis) or normal C57BL/6 (B6) did not develop the tolerance to human CEA. However, the chimeric KSN nude mice transplanted simultaneously with thymus of the B6 and spleen of the B601 line became tolerant to human CEA antigen. In the case of systemic immunization with cells which had CEA antigen, the B601 line was tolerant to human CEA. Surprisingly, the B602 and BC60 lines were also tolerant to CEA molecule. These results indicate that not only the antigen present in the thymus but also the antigen which flows from the peripheral organs to the thymus may be necessary for the induction of CEA tolerance. Images Figure 1 PMID:1493931

Stem cell marker prominin-1/AC133 is expressed in duct cells of the adult human pancreas.

PubMed

Lardon, Jessy; Corbeil, Denis; Huttner, Wieland B; Ling, Zhidong; Bouwens, Luc

2008-01-01

Many efforts are spent in identifying stem cells in adult pancreas because these could provide a source of beta cells for cell-based therapy of type 1 diabetes. Prominin-1, particularly its specific glycosylation-dependent AC133 epitope, is expressed on stem/progenitor cells of various human tissues and can be used to isolate them. We, therefore, examined its expression in adult human pancreas. To detect prominin-1 protein, monoclonal antibody CD133/1 (AC133 clone), which recognizes the AC133 epitope, and the alphahE2 antiserum, which is directed against the human prominin-1 polypeptide, were used. Prominin-1 RNA expression was analyzed by real-time polymerase chain reaction. We report that all duct-lining cells of the pancreas express prominin-1. Most notably, the cells that react with the alphahE2 antiserum also react with the AC133 antibody. After isolation and culture of human exocrine cells, we found a relative increase in prominin-1 expression both at protein and RNA expression level, which can be explained by an enrichment of cells with ductal phenotype in these cultures. Our data show that pancreatic duct cells express prominin-1 and surprisingly reveal that its particular AC133 epitope is not an exclusive stem and progenitor cell marker.

A PHGDH inhibitor reveals coordination of serine synthesis and 1-carbon unit fate

PubMed Central

Pacold, Michael E.; Brimacombe, Kyle R.; Chan, Sze Ham; Rohde, Jason M.; Lewis, Caroline A.; Swier, Lotteke J.Y.M.; Possemato, Richard; Chen, Walter W.; Sullivan, Lucas B.; Fiske, Brian P.; Cho, Sung Won; Freinkman, Elizaveta; Birsoy, Kıvanç; Abu-Remaileh, Monther; Shaul, Yoav D.; Liu, Chieh Min; Zhou, Minerva; Koh, Min Jung; Chung, Haeyoon; Davidson, Shawn M.; Luengo, Alba; Wang, Amy Q.; Xu, Xin; Yasgar, Adam; Liu, Li; Rai, Ganesha; Westover, Kenneth D.; Vander Heiden, Matthew G.; Shen, Min; Gray, Nathanael S.; Boxer, Matthew B.; Sabatini, David M.

2016-01-01

Serine is a both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical glucose-derived serine synthesis pathway, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic towards PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we use a quantitative high-throughput screen to identify small molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and suggest that one-carbon unit wasting may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo. PMID:27110680

Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering

NASA Astrophysics Data System (ADS)

Fu, Dan; Zhou, Jing; Zhu, Wenjing Suzanne; Manley, Paul W.; Wang, Y. Karen; Hood, Tami; Wylie, Andrew; Xie, X. Sunney

2014-07-01

ABL1 tyrosine-kinase inhibitors (TKI) are front-line therapy for chronic myelogenous leukaemia and are among the best-known examples of targeted cancer therapeutics. However, the dynamic uptake into cells of TKIs of low molecular weight and their intracellular behaviour is unknown because of the difficulty of observing non-fluorescent small molecules at subcellular resolution. Here we report the direct label-free visualization and quantification of two TKI drugs (imatinib and nilotinib) inside living cells using hyperspectral stimulated Raman scattering imaging. Concentrations of both drugs were enriched over 1,000-fold in lysosomes as a result of their lysosomotropic properties. In addition, low solubility appeared to contribute significantly to the surprisingly large accumulation of nilotinib. We further show that the lysosomal trapping of imatinib was reduced more than tenfold when chloroquine is used simultaneously, which suggests that chloroquine may increase the efficacy of TKIs through lysosome-mediated drug-drug interaction in addition to the commonly proposed autophagy-inhibition mechanism.

Estrogen-related receptor alpha is critical for the growth of estrogen receptor-negative breast cancer

PubMed Central

Stein, Rebecca A.; Chang, Ching-yi; Kazmin, Dmitri A.; Way, James; Schroeder, Thies; Wergin, Melanie; Dewhirst, Mark W.; McDonnell, Donald P.

2009-01-01

Expression of estrogen-related receptor alpha (ERRα) has recently been shown to carry negative prognostic significance in breast and ovarian cancers. The specific role of this orphan nuclear receptor in tumor growth and progression, however, is yet to be fully understood. The significant homology between estrogen receptor alpha (ERα) and ERRα initially suggested that these receptors may have similar transcriptional targets. Using the well-characterized ERα-positive MCF-7 breast cancer cell line, we sought to gain a genome-wide picture of ERα-ERRα cross-talk using an unbiased microarray approach. In addition to generating a host of novel ERRα target genes, this study yielded the surprising result that most ERRα-regulated genes are unrelated to estrogen-signaling. The relatively small number of genes regulated by both ERα and ERRα led us to expand our study to the more aggressive and less clinically treatable ERα-negative class of breast cancers. In this setting we found that ERRα expression is required for the basal level of expression of many known and novel ERRα target genes. Introduction of an siRNA directed to ERRα into the highly aggressive breast carcinoma MDA-MB-231 cell line dramatically reduced the migratory potential of these cells. Although stable knockdown of ERRα expression in MDA-MB-231 cells had no impact on in vitro cell proliferation, a significant reduction of tumor growth rate was observed when these cells were implanted as xenografts. Our results confirm a role for ERRα in breast cancer growth and highlight it as a potential therapeutic target for estrogen receptor-negative breast cancer. PMID:18974123

Regulation of expression of Na+,K+-ATPase in androgen-dependent and androgen-independent prostate cancer

PubMed Central

Blok, L J; Chang, G T G; Steenbeek-Slotboom, M; Weerden, W M van; Swarts, H G P; Pont, J J H H M De; Steenbrugge, G J van; Brinkmann, A O

1999-01-01

The β1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of androgens. Down-regulation of the β1-subunit was initiated at concentrations between 0.01 nM and 0.03 nM of the synthetic androgen R1881 after relatively long incubation times (> 24 h). Using polyclonal antibodies, the concentration of β1-subunit protein, but not of the α1-subunit protein, was markedly reduced in androgen-dependent human prostate cancer cells (LNCaP-FGC) cultured in the presence of androgens. In line with these observations it was found that the protein expression of total Na+,K+-ATPase in the membrane (measured by 3H-ouabain binding) was also markedly decreased. The main function of Na+,K+-ATPase is to maintain sodium and potassium homeostasis in animal cells. The resulting electrochemical gradient is facilitative for transport of several compounds over the cell membrane (for example cisplatin, a chemotherapeutic agent experimentally used in the treatment of hormone-refractory prostate cancer). Here we observed that a ouabain-induced decrease of Na+,K+-ATPase activity in LNCaP-FGC cells results in reduced sensitivity of these cells to cisplatin-treatment. Surprisingly, androgen-induced decrease of Na+,K+-ATPase expression, did not result in significant protection against the chemotherapeutic agent. © 1999 Cancer Research Campaign PMID:10487609

Use of LysoTracker dyes: a flow cytometric study of autophagy.

PubMed

Chikte, Shaheen; Panchal, Neelam; Warnes, Gary

2014-02-01

The flow cytometric use of LysoTracker dyes was employed to investigate the autophagic process and to compare this with the upregulation of autophagy marker, the microtubule-associated protein LC3B. Although the mechanism of action of LysoTracker dyes is not fully understood, they have been used in microscopy to image acidic spherical organelles, and their use in flow cytometry has not been thoroughly investigated in the study of autophagy. This investigation uses numerous autophagy-inducing agents including chloroquine (CQ), rapamycin, low serum (<1%) RPMI, and nutrient starvation to induce autophagy in Jurkat T-cell leukemia and K562 erythromyeloid cell lines. LC3B showed an increase with CQ treatment although this was different to LysoTracker signals in terms of dose and time. Rapamycin, low serum (<1%) RPMI, and nutrient starvation induction of autophagy also induced an increase in LysoTracker and LC3B signals. CQ also induced apoptosis in cell lines, which was blocked by pan-caspase inhibitor z-VAD resulting in a reduction in cells undergoing apoptosis and a subsequent upregulation of autophagic markers LC3B and lysosomal dye signals. Given that LC3B and LysoTracker are measuring different biological events in the autophagic process, they surprisingly both upregulated during autophagic process. This study, however, shows that although LysoTracker dyes do not specifically label lysosomes or autophagosomes within the cell, they allow the simultaneous measurement of an autophagy-related process and other live-cell functions, which are not possible with the standard LC3B antibody-labeling technique. This method has the advantage of other live-cell LCB-GFP-tagged experiments in that be used to analyze patient cells as well as easier to use and significantly less costly. Copyright © 2013 International Society for Advancement of Cytometry.

Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize (Zea mays L.) lines.

PubMed

Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D; Williams, Daniel L; Magee, Timothy D; Kaeppler, Shawn M; de Leon, Natalia; Hodge, David B

2015-07-01

A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA) content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. This indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize ( Zea mays L.) lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D.

A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA)more » content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Also, another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. In conclusion, this indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment« less

Cell-wall properties contributing to improved deconstruction by alkaline pre-treatment and enzymatic hydrolysis in diverse maize ( Zea mays L.) lines

DOE PAGES

Li, Muyang; Heckwolf, Marlies; Crowe, Jacob D.; ...

2015-02-20

A maize (Zea mays L. subsp. mays) diversity panel consisting of 26 maize lines exhibiting a wide range of cell-wall properties and responses to hydrolysis by cellulolytic enzymes was employed to investigate the relationship between cell-wall properties, cell-wall responses to mild NaOH pre-treatment, and enzymatic hydrolysis yields. Enzymatic hydrolysis of the cellulose in the untreated maize was found to be positively correlated with the water retention value, which is a measure of cell-wall susceptibility to swelling. It was also positively correlated with the lignin syringyl/guaiacyl ratio and negatively correlated with the initial cell-wall lignin, xylan, acetate, and p-coumaric acid (pCA)more » content, as well as pCA released from the cell wall by pre-treatment. The hydrolysis yield following pre-treatment exhibited statistically significant negative correlations to the lignin content after pre-treatment and positive correlations to the solubilized ferulic acid and pCA. Several unanticipated results were observed, including a positive correlation between initial lignin and acetate content, lack of correlation between acetate content and initial xylan content, and negative correlation between each of these three variables to the hydrolysis yields for untreated maize. Also, another surprising result was that pCA release was negatively correlated with hydrolysis yields for untreated maize and, along with ferulic acid release, was positively correlated with the pre-treated maize hydrolysis yields. In conclusion, this indicates that these properties that may negatively contribute to the recalcitrance in untreated cell walls may positively contribute to their deconstruction by alkaline pre-treatment« less

Antagonism between curcumin and the topoisomerase II inhibitor etoposide

PubMed Central

Saleh, Ekram M.; El-awady, Raafat A; Eissa, Nadia A.; Abdel-Rahman, Wael M.

2012-01-01

The use of combinations of chemotherapy and natural products has recently emerged as a new method of cancer therapy, relying on the capacity of certain natural compounds to trigger cell death with low doses of chemotherapeutic agents and few side effects. The current study aims to evaluate the modulatory effects of curcumin (CUR), Nigella sativa (NS) and taurine on etoposide (ETP) cytotoxicity in a panel of cancer cell lines and to identify their underlying mechanisms. CUR alone showed potent antitumor activity, but surprisingly, its interaction with ETP was antagonistic in four out of five cancer cell lines. Neither taurine nor Nigella sativa affect the sensitivity of cancer cells to ETP. Examination of the DNA damage response machinery (DDR) showed that both ETP and CUR elicited DNA double-strand breaks (DSB) and evoked γ-H2AX foci formation at doses as low as 1 µg/ml. Cell cycle analysis revealed S phase arrest after ETP or CUR application, whereas co-treatment with ETP and CUR led to increased arrest of the cell cycle in S phase (MCF-7 cells) or the accumulation of cells in G2/M phases (HCT116, and HeLa cells). Furthermore, cotreatment with ETP and CUR resulted in modulation of the level of DNA damage induction and repair compared with either agent alone. Electron microscopic examination demonstrated that different modalities of cell death occurred with each treatment. CUR alone induced autophagy, apoptosis and necrosis, whereas ETP alone or in combination with CUR led to apoptosis and necrosis. Conclusions: Cotreatment with ETP and CUR resulted in an antagonistic interaction. This antagonism is related, in part, to the enhanced arrest of tumor cells in both S and G2/M phases, which prevents the cells from entering M-phase with damaged DNA and, consequently, prevents cell death from occurring. This arrest allows time for the cells to repair DNA damage so that cell cycle -arrested cells can eventually resume cell cycle progression and continue their physiological program. PMID:22895066

The Intergenerational Transmission of Low Self-Control

ERIC Educational Resources Information Center

Boutwell, Brian B.; Beaver, Kevin M.

2010-01-01

There is a vast line of literature showing that antisocial behaviors and personality traits are transmitted across generational lines. Given the ascendancy of Gottfredson and Hirschi's general theory of crime, it is somewhat surprising that no research has examined whether levels of self-control are passed from parent to child. The authors examine…

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor-stroma interaction.

PubMed

Milia-Argeiti, Eleni; Mourah, Samia; Vallée, Benoit; Huet, Eric; Karamanos, Nikos K; Theocharis, Achilleas D; Menashi, Suzanne

2014-08-01

Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines. EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR. The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells. The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. Copyright © 2014 Elsevier B.V. All rights reserved.

Bruton's tyrosine kinase inhibitors in B-cell lymphoma: current experience and future perspectives.

PubMed

Seiler, T; Dreyling, M

2017-08-01

The Bruton tyrosine kinase (BTK) is a central hub in the B cell receptor (BCR) pathway and strongly influences B cell maturation, differentiation and proliferation. Not surprisingly, BTK plays an essential role in the pathogenesis of various B cell lymphomas. Inhibitors of BTK have broadened our therapeutic options in several B cell lymphomas and already are an integral element in the treatment of Mantle Cell Lymphoma (MCL), chronic lymphocytic leukemia (CLL) and Waldenström's marcoglobulinemia. Several second generation BTK inhibitors are in clinical development and might further improve tolerability and efficacy of therapy in advanced stage CLL and MCL. Areas covered: This review illustrates the mechanism of action of BTK inhibitors and provides a comprehensive summary of key clinical trials in the development of BTK inhibitors. Characteristics of second generation BTK-inhibitors are described. Expert opinion: With accumulation of clinical experience after drug approval, longer patient follow-up and larger numbers of treated patients, future development will focus on the identification of intelligent treatment combinations. Individual selection of patients with distinct biologically properties might guide treatment decisions. While BTK inhibitors are moving to earlier treatment lines, the incorporation of these drugs into a comprehensive therapeutic strategy is still difficult to date.

Let-7 represses Nr6a1 and a mid-gestation developmental program in adult fibroblasts

PubMed Central

Gurtan, Allan M.; Ravi, Arvind; Rahl, Peter B.; Bosson, Andrew D.; JnBaptiste, Courtney K.; Bhutkar, Arjun; Whittaker, Charles A.; Young, Richard A.; Sharp, Phillip A.

2013-01-01

MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function. PMID:23630078

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakaeda, Yoshiichi; Hiroi, Miki; Shimojima, Takahiro

Sulindac, a non-steroidal anti-inflammatory drug, has been shown to exert an anti-tumor effect on several types of cancer. To determine the effect of sulindac on intracellular signaling pathways in host immune cells such as macrophages, we investigated the effect of the drug on interferon gamma (IFN{gamma})-induced expression of signal transducer and activator of transcription 1 (STAT1) and other genes in mouse macrophage-like cell line RAW264.7 cells. Sulindac, but not aspirin or sodium salicylate, inhibited IFN{gamma}-induced expression of the CXC ligand 9 (CXCL9) mRNA, a chemokine for activated T cells, whereas the interferon-induced expression of CXCL10 or IFN regulatory factor-1 wasmore » not affected by sulindac. Luciferase reporter assay demonstrated that sulindac inhibited IFN{gamma}-induced promoter activity of the CXCL9 gene. Surprisingly, sulindac had no inhibitory effect on IFN{gamma}-induced STAT1 activation; however, constitutive nuclear factor {kappa}B activity was suppressed by the drug. These results indicate that sulindac selectively inhibited IFN{gamma}-inducible gene expression without inhibiting STAT1 activation.« less

Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells.

PubMed

Yang, Min; Ma, Bo; Shao, Hanshuang; Clark, Amanda M; Wells, Alan

2016-07-07

Metastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment. Here we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages. We found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells. Our findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due to malignant metastases in breast cancer.

Defined plant extracts can protect human cells against combined xenobiotic effects

PubMed Central

2011-01-01

Background Pollutants representative of common environmental contaminants induce intracellular toxicity in human cells, which is generally amplified in combinations. We wanted to test the common pathways of intoxication and detoxification in human embryonic and liver cell lines. We used various pollutants such as Roundup residues, Bisphenol-A and Atrazine, and five precise medicinal plant extracts called Circ1, Dig1, Dig2, Sp1, and Uro1 in order to understand whether specific molecular actions took place or not. Methods Kidney and liver are major detoxification organs. We have studied embryonic kidney and hepatic human cell lines E293 and HepG2. The intoxication was induced on the one hand by a formulation of one of the most common herbicides worldwide, Roundup 450 GT+ (glyphosate and specific adjuvants), and on the other hand by a mixture of Bisphenol-A and Atrazine, all found in surface waters, feed and food. The prevention and curative effects of plant extracts were also measured on mitochondrial succinate dehydrogenase activity, on the entry of radiolabelled glyphosate (in Roundup) in cells, and on cytochromes P450 1A2 and 3A4 as well as glutathione-S-transferase. Results Clear toxicities of pollutants were observed on both cell lines at very low sub-agricultural dilutions. The prevention of such phenomena took place within 48 h with the plant extracts tested, with success rates ranging between 25-34% for the E293 intoxicated by Roundup, and surprisingly up to 71% for the HepG2. By contrast, after intoxication, no plant extract was capable of restoring E293 viability within 48 h, however, two medicinal plant combinations did restore the Bisphenol-A/Atrazine intoxicated HepG2 up to 24-28%. The analysis of underlying mechanisms revealed that plant extracts were not capable of preventing radiolabelled glyphosate from entering cells; however Dig2 did restore the CYP1A2 activity disrupted by Roundup, and had only a mild preventive effect on the CYP3A4, and no effect on the glutathione S-transferase. Conclusions Environmental pollutants have intracellular effects that can be prevented, or cured in part, by precise medicinal plant extracts in two human cell lines. This appears to be mediated at least in part by the cytochromes P450 modulation. PMID:21251308

Maize early endosperm growth and development: from fertilization through cell type differentiation.

PubMed

Leroux, Brian M; Goodyke, Austin J; Schumacher, Katelyn I; Abbott, Chelsi P; Clore, Amy M; Yadegari, Ramin; Larkins, Brian A; Dannenhoffer, Joanne M

2014-08-01

• Given the worldwide economic importance of maize endosperm, it is surprising that its development is not the most comprehensively studied of the cereals. We present detailed morphometric and cytological descriptions of endosperm development in the maize inbred line B73, for which the genome has been sequenced, and compare its growth with four diverse Nested Association Mapping (NAM) founder lines.• The first 12 d of B73 endosperm development were described using semithin sections of plastic-embedded kernels and confocal microscopy. Longitudinal sections were used to compare endosperm length, thickness, and area.• Morphometric comparison between Arizona- and Michigan-grown B73 showed a common pattern. Early endosperm development was divided into four stages: coenocytic, cellularization through alveolation, cellularization through partitioning, and differentiation. We observed tightly synchronous nuclear divisions in the coenocyte, elucidated that the onset of cellularization was coincident with endosperm size, and identified a previously undefined cell type (basal intermediate zone, BIZ). NAM founders with small mature kernels had larger endosperms (0-6 d after pollination) than lines with large mature kernels.• Our B73-specific model of early endosperm growth links developmental events to relative endosperm size, while accounting for diverse growing conditions. Maize endosperm cellularizes through alveolation, then random partitioning of the central vacuole. This unique cellularization feature of maize contrasts with the smaller endosperms of Arabidopsis, barley, and rice that strictly cellularize through repeated alveolation. NAM analysis revealed differences in endosperm size during early development, which potentially relates to differences in timing of cellularization across diverse lines of maize. © 2014 Botanical Society of America, Inc.

Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Railo, Antti; Pajunen, Antti; Itaeranta, Petri

2009-10-01

Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-{beta}-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of {beta}-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays ofmore » gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.« less

Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids.

PubMed

Seiffert, M; Stilgenbauer, S; Döhner, H; Lichter, P

2007-09-01

Accumulation of neoplastic cells in B-cell chronic lymphocytic leukemia (B-CLL) is thought to be due to intrinsic defects in the apoptotic machinery of the leukemic cells or to an altered, survival-stimulating microenvironment in vivo. Despite their long survival in vivo, B-CLL cells undergo rapid spontaneous apoptosis ex vivo. To maintain survival in vitro, we established a coculture system using the human bone marrow-derived stromal cell line HS-5. The microenvironment in these cocultures lead to B-CLL cell survival for at least several months and therefore provided a tool for valid in vitro analysis, mimicking the in vivo situation. Although primary B lymphocytes are notoriously resistant to most gene transfer techniques, we achieved high transfection efficiency and cell viability in this coculture system by using a nucleofection-based strategy. Surprisingly, the introduction of circular plasmid DNA into B cells and B-CLL cells induced rapid apoptosis, which was independent of the type of transgene used, but dependent on the DNA concentration. However, transfection of these cells with mRNA was highly efficient and resulted in sustained cell viability and potent transgene expression. The described procedure represents a new approach to study gene function in primary B cells and B-CLL cells.

Selective impact of CDK4/6 suppression on patient-derived models of pancreatic cancer.

PubMed

Witkiewicz, Agnieszka K; Borja, Nicholas A; Franco, Jorge; Brody, Jonathan R; Yeo, Charles J; Mansour, John; Choti, Michael A; McCue, Peter; Knudsen, Erik S

2015-06-30

Pancreatic ductal adenocarcinoma (PDA) harbors an exceedingly poor prognosis, and is generally considered a therapy-recalcitrant disease due to poor response to conventional chemotherapy coupled with non-actionable genetic drivers (e.g. KRAS mutations). However, PDA frequently loses p16ink4a, thereby leading to deregulation of CDK4/6. Surprisingly, in established cell models and xenografts, CDK4/6 inhibition has a modest effect on proliferation and resistance develops rapidly. To determine if such weak response was an intrinsic feature of PDA, we developed primary tumor explants that maintain the tumor environment and recapitulate feuture of primary PDA. The CDK4/6 inhibitor PD-0332991 was highly efficient at suppressing proliferation in 14 of the 15 explants. In the single resistant explant, we identified the rare loss of the RB tumor suppressor as the basis for resistance. Patient-derived xenografts (PDXs) were developed in parallel, and unlike the xenografts emerging from established cell lines, the PDXs maintained the histoarchitecture of the primary tumor. These PDXs were highly sensitive to CDK4/6 inhibition, yielding a complete suppression of PDA proliferation. Together, these data indicate that primary PDA is sensitive to CDK4/6 inhibition, that specific biomarkers can delineate intrinsic resistance, and that established cell line models may not represent an adequate means for evaluating therapeutic sensitivities.

Adults with autism overestimate the volatility of the sensory environment.

PubMed

Lawson, Rebecca P; Mathys, Christoph; Rees, Geraint

2017-09-01

Insistence on sameness and intolerance of change are among the diagnostic criteria for autism spectrum disorder (ASD), but little research has addressed how people with ASD represent and respond to environmental change. Here, behavioral and pupillometric measurements indicated that adults with ASD are less surprised than neurotypical adults when their expectations are violated, and decreased surprise is predictive of greater symptom severity. A hierarchical Bayesian model of learning suggested that in ASD, a tendency to overlearn about volatility in the face of environmental change drives a corresponding reduction in learning about probabilistically aberrant events, thus putatively rendering these events less surprising. Participant-specific modeled estimates of surprise about environmental conditions were linked to pupil size in the ASD group, thus suggesting heightened noradrenergic responsivity in line with compromised neural gain. This study offers insights into the behavioral, algorithmic and physiological mechanisms underlying responses to environmental volatility in ASD.

Adults with autism over-estimate the volatility of the sensory environment

PubMed Central

Mathys, Christoph; Rees, Geraint

2017-01-01

Insistence on sameness and intolerance of change are part of the diagnostic criteria for Autism Spectrum Disorder (ASD) but there is little research addressing how people with ASD represent and respond to environmental change. Here, we find that behavioural and pupillometric measurements show adults with ASD are less surprised than neurotypical adults when expectations are violated, with reduced surprise predicting greater symptom severity. A hierarchical Bayesian model of learning suggests that in ASD a tendency to over-learn about volatility in the face of environmental change drives a corresponding reduction in learning about probabilistically aberrant events – putatively rendering them less surprising. Participant-specific modelled estimates of surprise about environmental conditions are linked to pupil size in the ASD group, suggesting heightened phasic noradrenergic responsivity in line with neural gain impairments. This study offers novel insight into the behavioural, algorithmic and physiological mechanisms that underlie responses to environmental volatility in ASD. PMID:28758996

Novel peptides from the RAS-p21 and p53 proteins for the treatment of cancer

PubMed Central

Bowne, Wilbur B.; Michl, Josef; Bluth, Martin H.; Zenilman, Michael E.; Pincus, Matthew R.

2007-01-01

Summary We have employed a novel computer-based molecular modeling method to design peptides from the ras-p21 and p53 proteins that block proliferation of cancer cells. The rationale of our approach is to identify peptide domains from each protein that alter conformation in response to oncogenic amino acid substitutions in their polypeptide chain. We accomplish this by first generating and comparing low energy average structures for oncogenic and wild-type proteins using conformational energy calculations. Peptides are then synthesized corresponding to these domains. These domains are then linked to a trans-membrane-penetrating sequence (called penetratin) and tested against cancer and untransformed cell lines. Remarkably, we have found that two ras-p21 peptides, 35–47 and 96–110, called PNC-7 and PNC-2, respectively, can induce phenotypic reversion of ras-transformed TUC-3 pancreatic cancer cells and ras-transformed HT1080 human fibrosarcoma cells to their untransformed phenotypes. Moreover, both peptides were found to be cytotoxic to ras-transformed human MIA-PaCa-2 pancreatic carcinoma cells and human U-251 astrocytoma cells. Importantly, these peptides have no effect on the growth of their normal cellular counterparts. We have also synthesized peptides from the p53 protein corresponding to its hdm-2-binding domain sequences (residues 12–26), also linked to the penetratin sequence. Surprisingly, we have found that these peptides induce 100 percent tumor cell necrosis, not apoptosis, in 13 different human cancer cell lines but have no effect on normal pancreatic acinar cells, breast epithelial cells, and human stem cells. Moreover, these peptides are cytotoxic to TUC-3 pancreatic tumor cells in nude mice plus eradicate these tumor cells when administered at sites near these tumors. These novel peptides appear to hold much promise as new, non-toxic anti-cancer agents. PMID:18007958

BOK promotes chemical-induced hepatocarcinogenesis in mice.

PubMed

Rabachini, Tatiana; Fernandez-Marrero, Yuniel; Montani, Matteo; Loforese, Giulio; Sladky, Valentina; He, Zhaoyue; Bachmann, Daniel; Wicki, Simone; Villunger, Andreas; Stroka, Deborah; Kaufmann, Thomas

2018-03-01

BCL-2-related ovarian killer (BOK) is a conserved and widely expressed BCL-2 family member with sequence homology to pro-apoptotic BAX and BAK, but with poorly understood pathophysiological function. Since several members of the BCL-2 family are critically involved in the regulation of hepatocellular apoptosis and carcinogenesis we aimed to establish whether loss of BOK affects diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. Short-term exposure to DEN lead to upregulation of BOK mRNA and protein in the liver. Of note, induction of CHOP and the pro-apoptotic BH3-only proteins PUMA and BIM by DEN was strongly reduced in the absence of BOK. Accordingly, Bok -/- mice were significantly protected from DEN-induced acute hepatocellular apoptosis and associated inflammation. As a consequence, Bok -/- animals were partially protected against chemical-induced hepatocarcinogenesis showing fewer and, surprisingly, also smaller tumors than WT controls. Gene expression profiling revealed that downregulation of BOK results in upregulation of genes involved in cell cycle arrest. Bok -/- hepatocellular carcinoma (HCC) displayed higher expression levels of the cyclin kinase inhibitors p19 INK4d and p21 cip1 . Accordingly, hepatocellular carcinoma in Bok -/- animals, BOK-deficient human HCC cell lines, as well as non-transformed cells, showed significantly less proliferation than BOK-proficient controls. We conclude that BOK is induced by DEN, contributes to DEN-induced hepatocellular apoptosis and resulting hepatocarcinogenesis. In line with its previously reported predominant localization at the endoplasmic reticulum, our findings support a role of BOK that links the cell cycle and cell death machineries upstream of mitochondrial damage.

Interaction of Bacterial Phenazines with Colistimethate in Bronchial Epithelial Cells.

PubMed

Mossine, Valeri V; Chance, Deborah L; Waters, James K; Mawhinney, Thomas P

2018-05-21

Multidrug-resistant bacterial infections are being increasingly treated in clinics with polymyxins, a class of antibiotics associated with adverse effects in the kidney, nervous system, or airways of a significant proportion of human and animal patients. Although many of the resistant pathogens display enhanced virulence, a hazard of cytotoxic interactions between polymyxin antibiotics and bacterial virulence factors (VFs) has not been assessed, to date. We report here on testing paired combinations of four Pseudomonas aeruginosa VF phenazine toxins, pyocyanin (PYO), 1-hydroxyphenazine (1-HP), phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and two commonly prescribed polymyxin drugs, colistimethate (CMS)/colistin and polymyxin B, in three human airway cell lines, BEAS-2B, HBE-1, and CFT-1. Cytotoxicities of individual antibiotics, toxins, and their combinations were evaluated by simultaneous measurement of mitochondrial metabolic, total transcriptional/translational, and the Nrf2 stress response regulator activities in treated cells. Two phenazines, PYO and 1-HP, were cytotoxic at clinically relevant concentrations (100-150 μM) and prompted a significant increase in the oxidative stress-induced transcriptional activity in surviving cells. The polymyxin antibiotics arrested the cell proliferation at clinically achievable (< 1 mM) concentrations, as well, with CMS displaying a surprisingly high cytotoxicity (ED 50 = 180 μM) in BEAS-2B. The dose-response curves were probed by the median-effect analysis which established a synergistically enhanced cytotoxicity of the PYO/CMS combination in all three airway cell lines; a particularly strong effect was observed in the BEAS-2B cells, with the combination index (CI) = 0.27 at ED 50 PCA, PCN, and 1-HP potentiated CMS cytotoxicity to a smaller extent. The cytotoxicity of CMS could be reduced with 10 mM N -acetyl-cysteine. Iron chelators, while ineffective against the polymyxins, could rescue all three bronchial epithelial cell lines treated with lethal PYO or CMS/PYO doses. These findings suggest further evaluations of CMS safety are needed, along with a search for means to moderate the potentially cytotoxic interactions. Copyright © 2018 Mossine et al.

The structure of the electron diffusion region during asymmetric anti-parallel magnetic reconnection

NASA Astrophysics Data System (ADS)

Swisdak, M.; Drake, J. F.; Price, L.; Burch, J. L.; Cassak, P.

2017-12-01

The structure of the electron diffusion region during asymmetric magnetic reconnection is ex- plored with high-resolution particle-in-cell simulations that focus on an magnetopause event ob- served by the Magnetospheric Multiscale Mission (MMS). A major surprise is the development of a standing, oblique whistler-like structure with regions of intense positive and negative dissipation. This structure arises from high-speed electrons that flow along the magnetosheath magnetic sepa- ratrices, converge in the dissipation region and jet across the x-line into the magnetosphere. The jet produces a region of negative charge and generates intense parallel electric fields that eject the electrons downstream along the magnetospheric separatrices. The ejected electrons produce the parallel velocity-space crescents documented by MMS.

Fish Rhabdovirus Cell Entry Is Mediated by Fibronectin

PubMed Central

Bearzotti, Monique; Delmas, Bernard; Lamoureux, Annie; Loustau, Anne-Marie; Chilmonczyk, Stefan; Bremont, Michel

1999-01-01

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to the Rhabdoviridae family. PMID:10438860

Surprises from a Deep ASCA Spectrum of the Broad Absorption Line Quasar PHL 5200

NASA Technical Reports Server (NTRS)

Mathur, Smita; Matt, G.; Green, P. J.; Elvis, M.; Singh, K. P.

2002-01-01

We present a deep (approx. 85 ks) ASCA observation of the prototype broad absorption line quasar (BALQSO) PHL 5200. This is the best X-ray spectrum of a BALQSO yet. We find the following: (1) The source is not intrinsically X-ray weak. (2) The line-of-sight absorption is very strong, with N(sub H) = 5 x 10(exp 23)/sq cm. (3) The absorber does not cover the source completely; the covering fraction is approx. 90%. This is consistent with the large optical polarization observed in this source, implying multiple lines of sight. The most surprising result of this observation is that (4) the spectrum of this BALQSO is not exactly similar to other radio-quiet quasars. The hard X-ray spectrum of PHL 5200 is steep, with the power-law spectral index alpha approx. 1.5. This is similar to the steepest hard X-ray slopes observed so far. At low redshifts, such steep slopes are observed in narrow-line Seyfert 1 (NLS1) galaxies, believed to be accreting at a high Eddington rate. This observation strengthens the analogy between BALQSOs and NLS1 galaxies and supports the hypothesis that BALQSOs represent an early evolutionary state of quasars. It is well accepted that the orientation to the line of sight determines the appearance of a quasar: age seems to play a significant role as well.

19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines.

PubMed

Asou, H; Koike, M; Elstner, E; Cambell, M; Le, J; Uskokovic, M R; Kamada, N; Koeffler, H P

1998-10-01

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.

Genome-wide localization of exosome components to active promoters and chromatin insulators in Drosophila

PubMed Central

Lim, Su Jun; Boyle, Patrick J.; Chinen, Madoka; Dale, Ryan K.; Lei, Elissa P.

2013-01-01

Chromatin insulators are functionally conserved DNA–protein complexes situated throughout the genome that organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the precise mechanisms are not yet understood. Here we identify the exosome, the highly conserved major cellular 3′ to 5′ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. Genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32 and CTCF insulator proteins. Colocalization occurs mainly at promoters but also boundary elements such as Mcp, Fab-8, scs and scs′, which overlaps with a promoter. Surprisingly, exosome associates primarily with promoters but not gene bodies of active genes, arguing against simple cotranscriptional recruitment to RNA substrates. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is required specifically for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel positive relationship between exosome and chromatin insulators throughout the genome. PMID:23358822

Cell Wall Structure in Cells Adapted to Growth on the Cellulose-Synthesis Inhibitor 2,6-Dichlorobenzonitrile 1

PubMed Central

Shedletzky, Esther; Shmuel, Miri; Trainin, Tali; Kalman, Sara; Delmer, Deborah

1992-01-01

Our previous work (E. Shedletzky, M. Shmuel, D.P. Delmer, D.T.A. Lamport [1990] Plant Physiol 94:980-987) showed that suspension-cultured tomato cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a markedly altered cell wall composition, most notably a markedly reduced level of the cellulose-xyloglucan network. This study compares the adaptation to DCB of two cell lines from dicots (tomato [Lycopersicon esculentum] and tobacco [Nicotiana tabacum]) and a Graminaceous monocot (barley [Hordeum bulbosum] endosperm). The difference in wall structures between the dicots and the monocot is reflected in the very different types of wall modifications induced by growth on DCB. The dicots, having reduced levels of cellulose and xyloglucan, possess walls the major integrity of which is provided by Ca2+-bridged pectates because protoplasts can be prepared from these cells simply by treatment with divalent cation chelator and a purified endopolygalacturonase. The tensile strength of these walls is considerably less than walls from nonadapted cells, but wall porosity is not altered. In contrast, walls from adapted barley cells contain very little pectic material and normal to elevated levels of noncellulosic polysaccharides compared with walls from nonadapted cells. Surprisingly, they have tensile strengths higher than their nonadapted counterpart, although cellulose levels are reduced by 70%. Evidence is presented that these walls obtain their additional strength by an altered pattern of cross-linking of polymers involving phenolic components. Such cross-linking may also explain the observation that the porosity of these walls is also considerably reduced. Cells of adapted lines of both the dicots and barley are resistant to plasmolysis, suggesting that they possess very strong connections between the wall and the plasma membrane. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16652933

Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin.

PubMed

Pruett, W; Yuan, Y; Rose, E; Batzer, A G; Harada, N; Skolnik, E Y

1995-03-01

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.

Vibrio parahaemolyticus strengthens their virulence through modulation of cellular reactive oxygen species in vitro

PubMed Central

El-Malah, Shimaa S.; Yang, Zhenquan; Hu, Maozhi; Li, Qiuchun; Pan, Zhiming; Jiao, Xinan

2014-01-01

Vibrio parahaemolyticus (Vp) is one of the emergent food-borne pathogens that are commensally associated with various shellfish species throughout the world. It is strictly environmental and many strains are pathogenic to humans. The virulent strains cause distinct diseases, including wound infections, septicemia, and most commonly, acute gastroenteritis, which is acquired through the consumption of raw or undercooked seafood, especially shellfish. Vp has two type three secretion systems (T3SSs), which triggering its cytotoxicity and enterotoxicity via their effectors. To better understand the pathogenesis of Vp, we established a cell infection model in vitro using a non-phagocytic cell line. Caco-2 cells were infected with different strains of Vp (pandemic and non-pandemic strains) and several parameters of cytotoxicity were measured together with adhesion and invasion indices, which reflect the pathogen's virulence. Our results show that Vp adheres to cell monolayers and can invade non-phagocytic cells. It also survives and persists in non-phagocytic cells by modulating reactive oxygen species (ROS), allowing its replication, and resulting in complete cellular destruction. We conclude that the pathogenicity of Vp is based on its capacities for adhesion and invasion. Surprisingly's; enhanced of ROS resistance period could promote the survival of Vp inside the intestinal tract, facilitating tissue infection by repressing the host's oxidative stress response. PMID:25566508

Live attenuated yellow fever 17D infects human DCs and allows for presentation of endogenous and recombinant T cell epitopes.

PubMed

Barba-Spaeth, Giovanna; Longman, Randy S; Albert, Matthew L; Rice, Charles M

2005-11-07

The yellow fever (YF) 17D vaccine is one of the most successful live attenuated vaccines available. A single immunization induces both long-lasting neutralizing antibody and YF-specific T cell responses. Surprisingly, the mechanism for this robust immunity has not been addressed. In light of several recent reports suggesting flavivirus interaction with dendritic cells (DCs), we investigated the mechanism of YF17D interaction with DCs and the importance of this interaction in generating T cell immunity. Our results show that YF17D can infect immature and mature human DCs. Viral entry is Ca(2+) dependent, but it is independent of DC-SIGN as well as multiple integrins expressed on the DC surface. Similar to infection of cell lines, YF infection of immature DCs is cytopathic. Although infection itself does not induce DC maturation in vitro, TNF-alpha-induced maturation protects DCs from YF-induced cytopathogenicity. Furthermore, we show that DCs infected with YF17D or YF17D carrying a recombinant epitope can process and present antigens for CD8(+) T cell stimulation. These findings offer insight into the immunologic mechanisms associated with the highly capable YF17D vaccine that may guide effective vaccine design.

Lipid and cationic polymer based transduction of botulinum holotoxin, or toxin protease alone, extends the target cell range and improves the efficiency of intoxication.

PubMed

Kuo, Chueh-Ling; Oyler, George; Shoemaker, Charles B

2010-01-01

Botulinum neurotoxin (BoNT) heavy chain (Hc) facilitates receptor-mediated endocytosis into neuronal cells and transport of the light chain (Lc) protease to the cytosol where neurotransmission is inhibited as a result of SNARE protein cleavage. Here we show that the role of BoNT Hc in cell intoxication can be replaced by commercial lipid-based and polycationic polymer DNA transfection reagents. BoNT "transduction" by these reagents permits efficient intoxication of neuronal cells as well as some non-neuronal cell lines normally refractory to BoNT. Surprisingly, the reagents facilitate delivery of recombinant BoNT Lc protease to the cytosol of both neuronal and non-neuronal cells in the absence of BoNT Hc, and with sensitivities approaching that of BoNT holotoxin. Transduction of BoNT, as with natural intoxication, is inhibited by bafilomycin A1, methylamine and ammonium chloride indicating that both pathways require endosome acidification. DNA transfection reagents facilitate intoxication by holotoxins, or isolated Lc proteases, of all three BoNT serotypes tested (A, B, E). These results suggest that lipid and cationic polymer transfection reagents facilitate cytosolic delivery of BoNT holotoxins and isolated Lc proteases by an endosomal uptake pathway.

Lipid and cationic polymer based transduction of botulinum holotoxin, or toxin protease alone, extends the target cell range and improves the efficiency of intoxication

PubMed Central

Kuo, Chueh-Ling; Oyler, George; Shoemaker, Charles B.

2009-01-01

Botulinum neurotoxin (BoNT) heavy chain (Hc) facilitates receptor-mediated endocytosis into neuronal cells and transport of the light chain (Lc) protease to the cytosol where neurotransmission is inhibited as a result of SNARE protein cleavage. Here we show that the role of BoNT Hc in cell intoxication can be replaced by commercial lipid-based and polycationic polymer DNA transfection reagents. BoNT “transduction” by these reagents permits efficient intoxication of neuronal cells as well as some non-neuronal cell lines normally refractory to BoNT. Surprisingly, the reagents facilitate delivery of recombinant BoNT Lc protease to the cytosol of both neuronal and non-neuronal cells in the absence of BoNT Hc, and with sensitivities approaching that of BoNT holotoxin. Transduction of BoNT, as with natural intoxication, is inhibited by bafilomycin A1, methylamine and ammonium chloride indicating that both pathways require endosome acidification. DNA transfection reagents facilitate intoxication by holotoxins, or isolated Lc proteases, of all three BoNT serotypes tested (A, B, E). These results suggest that lipid and cationic polymer transfection reagents facilitate cytosolic delivery of BoNT holotoxins and isolated Lc proteases by an endosomal uptake pathway. PMID:19852976

GT-094, a NO-NSAID, inhibits colon cancer cell growth by activation of a reactive oxygen species-microRNA-27a: ZBTB10-specificity protein pathway.

PubMed

Pathi, Satya S; Jutooru, Indira; Chadalapaka, Gayathri; Sreevalsan, Sandeep; Anand, S; Thatcher, Gregory Rj; Safe, Stephen

2011-02-01

Ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)benzoate (GT-094) is a novel nitric oxide (NO) chimera containing an nonsteroidal anti-inflammatory drug (NSAID) and NO moieties and also a disulfide pharmacophore that in itself exhibits cancer chemopreventive activity. In this study, the effects and mechanism of action of GT-094 were investigated in RKO and SW480 colon cancer cells. GT-094 inhibited cell proliferation and induced apoptosis in both cell lines and this was accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and these responses were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated genes associated with cell growth [cyclin D1, hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor (EGFR)], survival (bcl-2, survivin), and angiogenesis [VEGF and its receptors (VEGFR1 and VEGFR2)]. Results of previous RNA interference studies in this laboratory has shown that these genes are regulated, in part, by specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 that are overexpressed in colon and other cancer cell lines and not surprisingly, GT-094 also decreased Sp1, Sp3, and Sp4 in colon cancer cells. GT-094-mediated repression of Sp and Sp-regulated gene products was due to downregulation of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp repressor that is regulated by miR-27a in colon cancer cells. Moreover, the effects of GT-094 on Sp1, Sp3, Sp4, miR-27a, and ZBTB10 were also inhibited by glutathione suggesting that the anticancer activity of GT-094 in colon cancer cells is due, in part, to activation of an ROS-miR-27a:ZBTB10-Sp transcription factor pathway.

Identification of the zinc finger 216 (ZNF216) in human carcinoma cells: a potential regulator of EGFR activity

PubMed Central

Mincione, Gabriella; Di Marcantonio, Maria Carmela; Tarantelli, Chiara; Savino, Luca; Ponti, Donatella; Marchisio, Marco; Lanuti, Paola; Sancilio, Silvia; Calogero, Antonella; Di Pietro, Roberta; Muraro, Raffaella

2016-01-01

Epidermal Growth Factor Receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase (RTK) proteins, is aberrantly expressed or deregulated in tumors and plays pivotal roles in cancer onset and metastatic progression. ZNF216 gene has been identified as one of Immediate Early Genes (IEGs) induced by RTKs. Overexpression of ZNF216 protein sensitizes 293 cell line to TNF-α induced apoptosis. However, ZNF216 overexpression has been reported in medulloblastomas and metastatic nasopharyngeal carcinomas. Thus, the role of this protein is still not clearly understood. In this study, the inverse correlation between EGFR and ZNF216 expression was confirmed in various human cancer cell lines differently expressing EGFR. EGF treatment of NIH3T3 cells overexpressing both EGFR and ZNF216 (NIH3T3-EGFR/ZNF216), induced a long lasting activation of EGFR in the cytosolic fraction and an accumulation of phosphorylated EGFR (pEGFR) more in the nuclear than in the cytosolic fraction compared to NIH3T3-EGFR cells. Moreover, EGF was able to stimulate an increased expression of ZNF216 in the cytosolic compartment and its nuclear translocation in a time-dependent manner in NIH3T3-EGFR/ZNF216. A similar trend was observed in A431 cells endogenously expressing the EGFR and transfected with Znf216. The increased levels of pEGFR and ZNF216 in the nuclear fraction of NIH3T3-EGFR/ZNF216 cells were paralleled by increased levels of phospho-MAPK and phospho-Akt. Surprisingly, EGF treatment of NIH3T3-EGFR/ZNF216 cells induced a significant increase of apoptosis thus indicating that ZNF216 could sensitize cells to EGF-induced apoptosis and suggesting that it may be involved in the regulation and effects of EGFR signaling. PMID:27732953

Chronic myeloid leukemia progenitor cells require autophagy when leaving hypoxia-induced quiescence

PubMed Central

Ianniciello, Angela; Dumas, Pierre-Yves; Drullion, Claire; Guitart, Amélie; Villacreces, Arnaud; Peytour, Yan; Chevaleyre, Jean; Brunet de la Grange, Philippe; Vigon, Isabelle; Desplat, Vanessa; Priault, Muriel; Sbarba, Persio Dello; Ivanovic, Zoran; Mahon, François-Xavier; Pasquet, Jean-Max

2017-01-01

Albeit tyrosine kinase inhibitors anti-Abl used in Chronic Myeloid Leukemia (CML) block the deregulated activity of the Bcr-Abl tyrosine kinase and induce remission in 90% of patients, they do not eradicate immature hematopoietic compartments of leukemic stem cells. To elucidate if autophagy is important for stem cell survival and/or proliferation, we used culture in low oxygen concentration (0.1% O2 for 7 days) followed back by non-restricted O2 supply (normoxic culture) to mimic stem cell proliferation and commitment. Knockdown of Atg7 expression, a key player in autophagy, in K562 cell line inhibited autophagy compared to control cells. Upon 7 days at 0.1% O2 both K562 and K562 shATG7 cells stopped to proliferate and a similar amount of viable cells remained. Back to non-restricted O2 supply K562 cells proliferate whereas K562 shATG7 cells exhibited strong apoptosis. Using immunomagnetic sorted normal and CML CD34+ cells, we inhibited the autophagic process by lentiviral infection expressing shATG7 or using a Vps34 inhibitor. Both, normal and CML CD34+ cells either competent or deficient for autophagy stopped to proliferate in hypoxia. Surprisingly, while normal CD34+ cells proliferate back to non restricted O2 supply, the CML CD34+ cells deficient for autophagy failed to proliferate. All together, these results suggest that autophagy is required for CML CD34+ commitment while it is dispensable for normal CD34 cells. PMID:29228587

Differential cytotoxicity induced by the Titanium(IV)Salan complex Tc52 in G2-phase independent of DNA damage.

PubMed

Pesch, Theresa; Schuhwerk, Harald; Wyrsch, Philippe; Immel, Timo; Dirks, Wilhelm; Bürkle, Alexander; Huhn, Thomas; Beneke, Sascha

2016-07-13

Chemotherapy is one of the major treatment modalities for cancer. Metal-based compounds such as derivatives of cisplatin are in the front line of therapy against a subset of cancers, but their use is restricted by severe side-effects and the induction of resistance in treated tumors. Subsequent research focused on development of cytotoxic metal-complexes without cross-resistance to cisplatin and reduced side-effects. This led to the discovery of first-generation titanium(IV)salan complexes, which reached clinical trials but lacked efficacy. New-generation titanium (IV)salan-complexes show promising anti-tumor activity in mice, but their molecular mechanism of cytotoxicity is completely unknown. Four different human cell lines were analyzed in their responses to a toxic (Tc52) and a structurally highly related but non-toxic (Tc53) titanium(IV)salan complex. Viability assays were used to reveal a suitable treatment range, flow-cytometry analysis was performed to monitor the impact of dosage and treatment time on cell-cycle distribution and cell death. Potential DNA strand break induction and crosslinking was investigated by immunostaining of damage markers as well as automated fluorometric analysis of DNA unwinding. Changes in nuclear morphology were analyzed by DAPI staining. Acidic beta-galactosidase activity together with morphological changes was monitored to detect cellular senescence. Western blotting was used to analyze induction of pro-apoptotic markers such as activated caspase7 and cleavage of PARP1, and general stress kinase p38. Here we show that the titanium(IV)salan Tc52 is effective in inducing cell death in the lower micromolar range. Surprisingly, Tc52 does not target DNA contrary to expectations deduced from the reported activity of other titanium complexes. Instead, Tc52 application interferes with progression from G2-phase into mitosis and induces apoptotic cell death in tested tumor cells. Contrarily, human fibroblasts undergo senescence in a time and dose-dependent manner. As deduced from fluorescence studies, the potential cellular target seems to be the cytoskeleton. In summary, we could demonstrate in four different human cell lines that tumor cells were specifically killed without induction of major cytotoxicity in non-tumorigenic cells. Absence of DNA damaging activity and the cell-cycle block in G2 instead of mitosis makes Tc52 an attractive compound for further investigations in cancer treatment.

The search for the elusive companion of EG andromedae

NASA Technical Reports Server (NTRS)

Pesce, Joseph E.; Stencel, Robert E.; Oliversen, Nancy A.

1987-01-01

Observations are reported at opposite quadratures of the interacting symbiotic binary EG And (HD-4174, Period = 470 d). After correcting for absolute motion at the system, it appears, surprisingly, that many of the nebular lines arise from material that moves with the red giant star. This fact is used to interpret the observed complex line profiles of C IV and He II in the object.

Theories and models on the biological of cells in space

NASA Technical Reports Server (NTRS)

Todd, P.; Klaus, D. M.

1996-01-01

A wide variety of observations on cells in space, admittedly made under constraining and unnatural conditions in may cases, have led to experimental results that were surprising or unexpected. Reproducibility, freedom from artifacts, and plausibility must be considered in all cases, even when results are not surprising. The papers in symposium on 'Theories and Models on the Biology of Cells in Space' are dedicated to the subject of the plausibility of cellular responses to gravity -- inertial accelerations between 0 and 9.8 m/sq s and higher. The mechanical phenomena inside the cell, the gravitactic locomotion of single eukaryotic and prokaryotic cells, and the effects of inertial unloading on cellular physiology are addressed in theoretical and experimental studies.

pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana

PubMed Central

2017-01-01

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is widely used as a tool for genome engineering in various organisms. A complex consisting of Cas9 and single guide RNA (sgRNA) induces a DNA double-strand break in a sequence-specific manner, resulting in knockout. Some binary vectors for CRISPR/Cas9 in plants have been reported, but there is a problem with low efficiency. Here, we present a newly developed, highly efficient CRISPR/Cas9 vector for Arabidopsis thaliana, pKAMA-ITACHI Red (pKIR), harboring the RIBOSOMAL PROTEIN S5 A (RPS5A) promoter to drive Cas9. The RPS5A promoter maintains high constitutive expression at all developmental stages starting from the egg cell and including meristematic cells. Even in the T1 generation, pKIR induced null phenotypes in some genes: PHYTOENE DESATURASE 3 (PDS3), AGAMOUS (AG) and DUO POLLEN 1 (DUO1). Mutations induced by pKIR were carried in the germ cell line of the T1 generation. Surprisingly, in some lines, 100% of the T2 plants had the adh1 (ALCOHOL DEHYDROGENASE 1) null phenotype, indicating that pKIR strongly induced heritable mutations. Cas9-free T2 mutant plants were obtained by removing T2 seeds expressing a fluorescent marker in pKIR. Our results suggest that the pKIR system is a powerful molecular tool for genome engineering in Arabidopsis. PMID:27856772

Context-Dependent Antagonism between Akt Inhibitors and Topoisomerase Poisons

PubMed Central

Gálvez-Peralta, Marina; Flatten, Karen S.; Loegering, David A.; Peterson, Kevin L.; Schneider, Paula A.; Erlichman, Charles

2014-01-01

Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more complicated than previously recognized. PMID:24569089

Context-dependent antagonism between Akt inhibitors and topoisomerase poisons.

PubMed

Gálvez-Peralta, Marina; Flatten, Karen S; Loegering, David A; Peterson, Kevin L; Schneider, Paula A; Erlichman, Charles; Kaufmann, Scott H

2014-05-01

Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more complicated than previously recognized.

NPM and BRG1 mediate transcriptional resistance to retinoic acid in Acute Promyelocytic Leukemia

PubMed Central

Nichol, Jessica N.; Galbraith, Matthew D.; Kleinman, Claudia L.; Espinosa, Joaquín M.; Miller, Wilson H.

2016-01-01

Summary Perturbation in the transcriptional control of genes driving differentiation is an established paradigm whereby oncogenic fusion proteins promote leukemia. From a retinoic acid (RA) sensitive Acute Promyelocytic Leukemia (APL) cell line, we derived an RA-resistant clone characterized by a block in transcription initiation, despite maintaining wild-type PML/RARA expression. We uncovered an aberrant interaction between PML/RARA, Nucleophosmin (NPM) and Topoisomerase II Beta (TOP2B). Surprisingly, RA stimulation in these cells results in enhanced chromatin association of the nucleosome remodeler BRG1. Inhibition of NPM or TOP2B abrogated BRG1 recruitment. Furthermore, NPM inhibition and targeting BRG1 restored differentiation when combined with RA. Here, we demonstrate a role for NPM and BRG1 in obstructing RA-differentiation and implicate chromatin remodeling in mediating therapeutic resistance in malignancies. NPM mutations are the most common genetic change in patients with acute leukemia (AML) therefore, our model may be applicable to other more common leukemias driven by NPM. PMID:26997274

Basic anatomy and tumor biology of the RPS6KA6 gene that encodes the p90 ribosomal S6 kinase-4

PubMed Central

Sun, Yuan; Cao, Shousong; Yang, Min; Wu, Sihong; Wang, Zhe; Lin, Xiukun; Song, Xiangrang; Liao, D.J.

2012-01-01

The RPS6KA6 gene encodes the p90 ribosomal S6 kinase-4 (RSK4) that is still largely uncharacterized. In this study we identified a new RSK4 transcription initiation site and several alternative splice sites with a 5’RACE approach. The resulting mRNA variants encompass four possible first start codons. The first 15 nucleotides (nt) of exon 22 in mouse and the penultimate exon in both human (exon 21) and mouse (exon 24) RSK4 underwent alternative splicing, although the penultimate exon deleted variant appeared mainly in cell clines, but not in most normal tissues. Demethylation agent 5-azacytidine inhibited the deletion of the penultimate exon whereas two indolocarbazole-derived inhibitors of cyclin dependent kinase 4 or 6 induced deletion of the first 39 nt from exon 21 of human RSK4. In all human cancer cell lines studied, the 90-kD wild type RSK4 was sparse but, surprisingly, several isoforms at or smaller than 72-kD were expressed as detected by seven different antibodies. On immunoblots, each of these smaller isoforms often appeared as a duplet or triplet and the levels of these isoforms varied greatly among different cell lines and culture conditions. Cyclin D1 inhibited RSK4 expression and serum starvation enhanced the inhibition, whereas c-Myc and RSK4 inhibited cyclin D1. The effects of RSK4 on cell growth, cell death and chemoresponse depended on the mRNA variant or the protein isoform expressed, on the specificity of the cell lines, as well as on the anchorage-dependent or -independent growth conditions and the in vivo situation. Moreover, we also observed that even a given cDNA might be expressed to multiple proteins; therefore, when using a cDNA, one needs to exclude this possibility before attribution of the biological results from the cDNA to the anticipated protein. Collectively, our results suggest that whether RSK4 is oncogenic or tumor suppressive depends on many factors. PMID:22614021

Persistent 3'-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3'-phosphatase.

PubMed

Chalasani, Sri Lakshmi; Kawale, Ajinkya S; Akopiants, Konstantin; Yu, Yaping; Fanta, Mesfin; Weinfeld, Michael; Povirk, Lawrence F

2018-05-25

Polynucleotide kinase/phosphatase (PNKP) has been implicated in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). To assess the consequences of PNKP deficiency for NHEJ of 3'-phosphate-ended DSBs, PNKP-deficient derivatives of HCT116 and of HeLa cells were generated using CRISPR/CAS9. For both cell lines, PNKP deficiency conferred sensitivity to ionizing radiation as well as to neocarzinostatin (NCS), which specifically induces DSBs bearing protruding 3'-phosphate termini. Moreover, NCS-induced DSBs, detected as 53BP1 foci, were more persistent in PNKP -/- HCT116 cells compared to their wild-type (WT) counterparts. Surprisingly, PNKP-deficient whole-cell and nuclear extracts were biochemically competent in removing both protruding and recessed 3'-phosphates from synthetic DSB substrates, albeit much less efficiently than WT extracts, suggesting an alternative 3'-phosphatase. Measurements by ligation-mediated PCR showed that PNKP-deficient HeLa cells contained significantly more 3'-phosphate-terminated and fewer 3'-hydroxyl-terminated DSBs than parental cells 5-15 min after NCS treatment, but this difference disappeared by 1 h. These results suggest that, despite presence of an alternative 3'-phosphatase, loss of PNKP significantly sensitizes cells to 3'-phosphate-terminated DSBs, due to a 3'-dephosphorylation defect. Copyright © 2018 Elsevier B.V. All rights reserved.

Assessment of the potential activity of major dietary compounds as selective estrogen receptor modulators in two distinct cell models for proliferation and differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lecomte, Sylvain; Lelong, Marie; Bourgine, Gaëlle

Estrogen receptors (ERs) α and β are distributed in most tissues of women and men. ERs are bound by estradiol (E2), a natural hormone, and mediate the pleiotropic and tissue-specific effects of E2, such as proliferation of breast epithelial cells or protection and differentiation of neuronal cells. Numerous environmental molecules, called endocrine disrupting compounds, also interact with ERs. Phytoestrogens belong to this large family and are considered potent therapeutic molecules that act through their selective estrogen receptor modulator (SERM) activity. Using breast cancer cell lines as a model of estrogen-dependent proliferation and a stably ER-expressing PC12 cell line as amore » model of neuronal differentiating cells, we studied the SERM activity of major dietary compounds, such as apigenin, liquiritigenin, daidzein, genistein, coumestrol, resveratrol and zearalenone. The ability of these compounds to induce ER-transactivation and breast cancer cell proliferation and enhance Nerve Growth Factor (NGF) -induced neuritogenesis was assessed. Surprisingly, although all compounds were able to activate the ER through an estrogen responsive element reporter gene, they showed differential activity toward proliferation or differentiation. Apigenin and resveratrol showed a partial or no proliferative effect on breast cancer cells but fully contributed to the neuritogenesis effect of NGF. However, daidzein and zearalenone showed full effects on cellular proliferation but did not induce cellular differentiation. In summary, our results suggest that the therapeutic potential of phytoestrogens can diverge depending on the molecule and the phenotype considered. Hence, apigenin and resveratrol might be used in the development of therapeutics for breast cancer and brain diseases. - Highlights: • SERM activity of dietary compounds on proliferation and differentiation is studied. • All the dietary compounds tested transactivate estrogen receptors. • Apigenin and resveratrol could be good candidates for future therapeutics. • Daidzein and zearalenone are to be avoided to maintain human health.« less

Guided genetic screen to identify genes essential in the regeneration of hair cells and other tissues.

PubMed

Pei, Wuhong; Xu, Lisha; Huang, Sunny C; Pettie, Kade; Idol, Jennifer; Rissone, Alberto; Jimenez, Erin; Sinclair, Jason W; Slevin, Claire; Varshney, Gaurav K; Jones, MaryPat; Carrington, Blake; Bishop, Kevin; Huang, Haigen; Sood, Raman; Lin, Shuo; Burgess, Shawn M

2018-01-01

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration.

A Proposed Molecular Mechanism of High-Dose Vitamin D3 Supplementation in Prevention and Treatment of Preeclampsia.

PubMed

Zabul, Piotr; Wozniak, Michal; Slominski, Andrzej T; Preis, Krzysztof; Gorska, Magdalena; Korozan, Marek; Wieruszewski, Jan; Zmijewski, Michal A; Zabul, Ewa; Tuckey, Robert; Kuban-Jankowska, Alicja; Mickiewicz, Wieslawa; Knap, Narcyz

2015-06-09

A randomized prospective clinical study performed on a group of 74 pregnant women (43 presenting with severe preeclampsia) proved that urinary levels of 15-F(2t)-isoprostane were significantly higher in preeclamptic patients relative to the control (3.05 vs. 2.00 ng/mg creatinine). Surprisingly enough, plasma levels of 25-hydroxyvitamin D3 in both study groups were below the clinical reference range with no significant difference between the groups. In vitro study performed on isolated placental mitochondria and placental cell line showed that suicidal self-oxidation of cytochrome P450scc may lead to structural disintegration of heme, potentially contributing to enhancement of oxidative stress phenomena in the course of preeclampsia. As placental cytochrome P450scc pleiotropic activity is implicated in the metabolism of free radical mediated arachidonic acid derivatives as well as multiple Vitamin D3 hydroxylations and progesterone synthesis, we propose that Vitamin D3 might act as a competitive inhibitor of placental cytochrome P450scc preventing the production of lipid peroxides or excess progesterone synthesis, both of which may contribute to the etiopathogenesis of preeclampsia. The proposed molecular mechanism is in accord with the preliminary clinical observations on the surprisingly high efficacy of high-dose Vitamin D3 supplementation in prevention and treatment of preeclampsia.

Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues

NASA Technical Reports Server (NTRS)

Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

Low-dose retinoic acid enhances in vitro invasiveness of human oral squamous-cell-carcinoma cell lines

PubMed Central

Uchida, D; Kawamata, H; Nakashiro, K; Omotehara, F; Hino, S; Hoque, M O; Begum, N-M; Yoshida, H; Sato, M; Fujimori, T

2001-01-01

Retinoids inhibit the proliferation of several types of tumour cells, and are used for patients with several malignant tumours. In this study, we examined the effect of retinoic acids (RAs) on the invasive potentials of the oral squamous cell carcinoma (SCC) cells, BHY and HNt. BHY cells expressed all of retinoid nuclear receptors (RARα, β, γ, and RXRα) and cytoplasmic retinoic acid binding proteins (CRABP1 and CRABP2). HNt cells lacked the expression of RARβ, but expressed other nuclear receptors and CRABPs. All-trans retinoic acid (ATRA) and 13-cis retinoic acid (13-cisRA) (10−6and 10−7M) inhibited the growth of the cells, but low-dose ATRA and 13-cisRA (10−8M) marginally affected the growth of the cells. Surprisingly, low-dose RAs enhanced the activity of tissue-type plasminogen activator (tPA), and activated pro-matrix metalloproteinases (proMMP2 and proMMP9). Activation of proMMP2 and proMMP9 was inhibited by aprotinin, a serine-proteinase, tPA inhibitor. Furthermore, low-dose RAs enhanced the in vitro invasiveness of BHY cells. These results indicate that low-dose RAs enhances the in vitro invasiveness of oral SCC cells via an activation of proMMP2 and proMMP9 probably mediated by the induction of tPA. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11437413

Magnetite Biomineralization: Fifty years of progress, from beach-combing to the SQUID microscope

NASA Astrophysics Data System (ADS)

Kirschvink, J. L.; Dixson, A. D.; Raub, T.

2012-12-01

Magnetite biomineralization was first discovered 50 years ago as a hardening agent in the teeth of the Polyplacophoran molluscs (chitons) by the late Prof. Heinz A. Lowenstam of Caltech, when he noticed unusual erosional effects produced by their grazing in the intertidal zones of Palau (Lowenstam, 1962). Since then, biogenic magnetite has been detected in a broad range of organisms, including magnetotactic bacteria, protists, insects, fish, amphibians, reptiles, birds, and mammals including humans. In many species, the role of ferromagnetic material as a neurophysiological transducer is demonstrated clearly through the effects of pulse-remagnetization on behavior. A brief (1 uS), properly configured magnetic discharge from a rectified LC circuit, tailored to exceed the coercivity of the magnetite, will often abolish a magnetic behavioral response, or in some cases make the organism go the wrong way. This is a unique ferromagnetic effect. The genes controlling magnetite biomineralization are well characterized in several species of bacteria, and the ability of some of these bacterial genes to initiate magnetite precipitation in mammalian cell lines argues for a common descent, probably via a magnetotactic mitochondrial ancestor. Previous studies in fish reported the presence of single-domain magnetite crystals in cells near projections of the trigeminal nerve, co-located in the olfactory epithelium. Although the cells are rare, the recent development of a spinning magnetic field technique allows easy identification and isolation of these cells for individual study (Eder et al., 2012). The cells are surprisingly magnetic, with moments hundreds of times larger than typical magnetotactic bacteria. Subsequent efforts to identify the anatomical seat of magnetoreceptors have focused on the same locations in new organisms, excluding other areas. Using SQUID moment magnetometry and SQUID scanning microscopy, we report here the unexpected presence of biogenic magnetite in the lateral line region of the zebrafish, Danio rerio. We suspect that the magnetic field receptor cells of the trigeminal system in animals may be co-located within a variety of other sensory tissues (olfaction, lateral line, vision, hearing, taste, etc.) as a means of spatially dispersing cells with large magnetic moments to prevent magnetostatic interactions between them. References: Eder et al., Magnetic characterization of isolated candidate vertebrate magnetoreceptor cells. Proc. Natl. Acad. Sci. USA 2012; 109:12022-12027. Lowenstam, H.A., 1962. Magnetite in denticle capping in recent chitons (Polyplacophora). Bulletin of the Geological Society of America 73, 435-438.

Overexpression of poplar xylem sucrose synthase in tobacco leads to a thickened cell wall and increased height.

PubMed

Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong

2015-01-01

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue.

Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

PubMed Central

Wei, Zhigang; Qu, Zanshuang; Zhang, Lijie; Zhao, Shuanjing; Bi, Zhihong; Ji, Xiaohui; Wang, Xiaowen; Wei, Hairong

2015-01-01

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue. PMID:25807295

Salience and Attention in Surprisal-Based Accounts of Language Processing.

PubMed

Zarcone, Alessandra; van Schijndel, Marten; Vogels, Jorrig; Demberg, Vera

2016-01-01

The notion of salience has been singled out as the explanatory factor for a diverse range of linguistic phenomena. In particular, perceptual salience (e.g., visual salience of objects in the world, acoustic prominence of linguistic sounds) and semantic-pragmatic salience (e.g., prominence of recently mentioned or topical referents) have been shown to influence language comprehension and production. A different line of research has sought to account for behavioral correlates of cognitive load during comprehension as well as for certain patterns in language usage using information-theoretic notions, such as surprisal. Surprisal and salience both affect language processing at different levels, but the relationship between the two has not been adequately elucidated, and the question of whether salience can be reduced to surprisal / predictability is still open. Our review identifies two main challenges in addressing this question: terminological inconsistency and lack of integration between high and low levels of representations in salience-based accounts and surprisal-based accounts. We capitalize upon work in visual cognition in order to orient ourselves in surveying the different facets of the notion of salience in linguistics and their relation with models of surprisal. We find that work on salience highlights aspects of linguistic communication that models of surprisal tend to overlook, namely the role of attention and relevance to current goals, and we argue that the Predictive Coding framework provides a unified view which can account for the role played by attention and predictability at different levels of processing and which can clarify the interplay between low and high levels of processes and between predictability-driven expectation and attention-driven focus.

Role of Cytochrome c in Apoptosis: Increased Sensitivity to Tumor Necrosis Factor Alpha Is Associated with Respiratory Defects but Not with Lack of Cytochrome c Release▿

PubMed Central

Vempati, Uma D.; Diaz, Francisca; Barrientos, Antoni; Narisawa, Sonoko; Mian, Abdul M.; Millán, José Luis; Boise, Lawrence H.; Moraes, Carlos T.

2007-01-01

Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-α)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, we found that this cell line (CRL 2613) respired at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, was able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, we developed a mouse knockout for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-α-induced apoptosis, but this process was still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways. PMID:17210651

A Genome-Wide siRNA Screen in Mammalian Cells for Regulators of S6 Phosphorylation

PubMed Central

Papageorgiou, Angela; Rapley, Joseph; Mesirov, Jill P.; Tamayo, Pablo; Avruch, Joseph

2015-01-01

mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms. PMID:25790369

Flexible adaptation of male germ cells from female iPSCs of endangered Tokudaia osimensis.

PubMed

Honda, Arata; Choijookhuu, Narantsog; Izu, Haruna; Kawano, Yoshihiro; Inokuchi, Mizuho; Honsho, Kimiko; Lee, Ah-Reum; Nabekura, Hiroki; Ohta, Hiroshi; Tsukiyama, Tomoyuki; Ohinata, Yasuhide; Kuroiwa, Asato; Hishikawa, Yoshitaka; Saitou, Mitinori; Jogahara, Takamichi; Koshimoto, Chihiro

2017-05-01

In mammals, the Y chromosome strictly influences the maintenance of male germ cells. Almost all mammalian species require genetic contributors to generate testes. An endangered species, Tokudaia osimensis , has a unique sex chromosome composition XO/XO, and genetic differences between males and females have not been confirmed. Although a distinctive sex-determining mechanism may exist in T. osimensis , it has been difficult to examine thoroughly in this rare animal species. To elucidate the discriminative sex-determining mechanism in T. osimensis and to find a strategy to prevent its possible extinction, we have established induced pluripotent stem cells (iPSCs) and derived interspecific chimeras using mice as the hosts and recipients. Generated iPSCs are considered to be in the so-called "true naïve" state, and T. osimensis iPSCs may contribute as interspecific chimeras to several different tissues and cells in live animals. Surprisingly, female T. osimensis iPSCs not only contributed to the female germ line in the interspecific mouse ovary but also differentiated into spermatocytes and spermatids that survived in the adult interspecific mouse testes. Thus, T. osimensis cells have high sexual plasticity through which female somatic cells can be converted to male germline cells. These findings suggest flexibility in T. osimensis cells, which can adapt their germ cell sex to the gonadal niche. The probable reduction of the extinction risk of an endangered species through the use of iPSCs is indicated by this study.

Flexible adaptation of male germ cells from female iPSCs of endangered Tokudaia osimensis

PubMed Central

Honda, Arata; Choijookhuu, Narantsog; Izu, Haruna; Kawano, Yoshihiro; Inokuchi, Mizuho; Honsho, Kimiko; Lee, Ah-Reum; Nabekura, Hiroki; Ohta, Hiroshi; Tsukiyama, Tomoyuki; Ohinata, Yasuhide; Kuroiwa, Asato; Hishikawa, Yoshitaka; Saitou, Mitinori; Jogahara, Takamichi; Koshimoto, Chihiro

2017-01-01

In mammals, the Y chromosome strictly influences the maintenance of male germ cells. Almost all mammalian species require genetic contributors to generate testes. An endangered species, Tokudaia osimensis, has a unique sex chromosome composition XO/XO, and genetic differences between males and females have not been confirmed. Although a distinctive sex-determining mechanism may exist in T. osimensis, it has been difficult to examine thoroughly in this rare animal species. To elucidate the discriminative sex-determining mechanism in T. osimensis and to find a strategy to prevent its possible extinction, we have established induced pluripotent stem cells (iPSCs) and derived interspecific chimeras using mice as the hosts and recipients. Generated iPSCs are considered to be in the so-called “true naïve” state, and T. osimensis iPSCs may contribute as interspecific chimeras to several different tissues and cells in live animals. Surprisingly, female T. osimensis iPSCs not only contributed to the female germ line in the interspecific mouse ovary but also differentiated into spermatocytes and spermatids that survived in the adult interspecific mouse testes. Thus, T. osimensis cells have high sexual plasticity through which female somatic cells can be converted to male germline cells. These findings suggest flexibility in T. osimensis cells, which can adapt their germ cell sex to the gonadal niche. The probable reduction of the extinction risk of an endangered species through the use of iPSCs is indicated by this study. PMID:28508054

Repurposing tofacitinib as an anti-myeloma therapeutic to reverse growth-promoting effects of the bone marrow microenvironment.

PubMed

Lam, Christine; Ferguson, Ian D; Mariano, Margarette C; Lin, Yu-Hsiu T; Murnane, Megan; Liu, Hui; Smith, Geoffrey A; Wong, Sandy W; Taunton, Jack; Liu, Jun O; Mitsiades, Constantine S; Hann, Byron C; Aftab, Blake T; Wiita, Arun P

2018-04-05

The myeloma bone marrow microenvironment promotes proliferation of malignant plasma cells and resistance to therapy. Activation of JAK/STAT signaling is thought to be a central component of these microenvironment-induced phenotypes. In a prior drug repurposing screen, we identified tofacitinib, a pan-JAK inhibitor FDA-approved for rheumatoid arthritis, as an agent that may reverse the tumor-stimulating effects of bone marrow mesenchymal stromal cells. Here, we validated in vitro, in stromal-responsive human myeloma cell lines, and in vivo, in orthotopic disseminated xenograft models of myeloma, that tofacitinib showed efficacy in myeloma models. Furthermore, tofacitinib strongly synergized with venetoclax in co-culture with marrow stromal cells but not in monoculture. Surprisingly, we found that ruxolitinib, an FDA-approved agent targeting JAK1 and JAK2, did not lead to the same anti-myeloma effects. Combination with a novel irreversible JAK3-selective inhibitor also did not enhance ruxolitinib effects. RNA-seq and unbiased phosphoproteomics revealed that marrow stromal cells stimulate a JAK/STAT-mediated proliferative program in myeloma plasma cells, and tofacitinib reversed the large majority of these pro-growth signals. Taken together, our results suggest that tofacitinib reverses the growth-promoting effects of the tumor microenvironment. As tofacitinib is already FDA-approved, these results can be rapidly translated into potential clinical benefits for myeloma patients. Copyright © 2018, Ferrata Storti Foundation.

Hemi-fused structure mediates and controls fusion and fission in live cells

PubMed Central

Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J.; Krystofiak, Evan S.; Villarreal, Seth A.; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

2016-01-01

Membrane fusion and fission are vital to eukaryotes’ life1–5. For three decades, it has been proposed that fusion is mediated by fusion between proximal leaflets of two bilayers (hemi-fusion) that produces a hemi-fused structure, followed by fusion between distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission1, 4, 6–10. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion/hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed2, 11–15. Using confocal and super-resolution STED microscopy, we observed the hemi-fused Ω-shaped structure for the first time in live cells, neuroendocrine chromaffin cells and pancreatic β-cells. This structure was generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, its transition to full fusion or fission was determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and was surprisingly slow (seconds to tens of seconds) in a significant fraction of the events. These results provide key missing evidence over the past three decades proving the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion/fission, as fusion and fission mechanisms compete to determine its transition to fusion or fission. PMID:27309816

Presence of MUC4 in human milk and at the luminal surfaces of blood vessels.

PubMed

Zhang, Jin; Perez, Aymee; Yasin, Mohammad; Soto, Pedro; Rong, Min; Theodoropoulos, George; Carothers Carraway, Coralie A; Carraway, Kermit L

2005-07-01

MUC4 is a heterodimeric membrane mucin, composed of a mucin subunit ASGP-1 (MUC4alpha) and a transmembrane subunit ASGP-2 (MUC4beta), which has been implicated in the protection of epithelial cell surfaces. Surprisingly, development and characterization of a new monoclonal antibody (mAb), called 1G8, against ASGP-2 demonstrated by immunohistochemistry the presence of MUC4 at the luminal surfaces of blood vessels of both normal tissues and tumors. Muc4 was detected with 1G8 and other Muc4 antibodies in blood vessels from humans, rats and mice. This expression of MUC4 in endothelial cells was confirmed by immunoblotting with 1G8 in human umbilical vein endothelial cells (HUVECs), human iliac artery endothelial cells (HIAECs), and human microvascular endothelial cells (HMVECs). MUC4 could be observed on HUVECs grown on either plastic or Matrigel. Finally, MUC4 expression in the three types of endothelial cell lines was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). These results provide, to our knowledge, the first demonstration of a member of the MUC gene family and membrane mucin in blood vessels. As a luminal surface component, the MUC4 is situated to contribute to the non-adhesive luminal surface and to act as an intrinsic protection and survival factor. (c) 2004 Wiley-Liss, Inc.

Evaluation of copper-64-labeled somatostatin agonists and antagonist in sstr2-transfected cell lines that are positive and negative for p53: implications for cancer therapy

PubMed Central

Nguyen, Kim; Parry, Jesse J.; Rogers, Buck E.; Anderson, Carolyn J.

2011-01-01

Objectives Radiolabeled somatostatin analogs have become important agents for molecular imaging and targeted radiotherapy of somatostatin receptor-positive tumors. Here we determine the effect of the tumor suppressor protein, p53, on trafficking 64Cu to tumor cell nuclei from DOTA vs.CB-TE2A-conjugated agonist Y3-TATE and the antagonist 64Cu-CB-TE2A-sst2-ANT in cell lines that are positive or negative for p53. Methods Receptor binding, internalization, cAMP and nuclear localization studies were performed with the SSTr2 agonists, 64Cu-CB-TE2A-Y3-TATE and 64Cu-DOTA-Y3-TATE vs. antagonist, 64Cu-CB-TE2A-sst2-ANT, in SSTr2-transfected p53 +/+ and −/− HCT116 colorectal carcinoma cells. Results The antagonist, 64Cu-CB-TE2A-sst2-ANT, bound 8-9-fold more SSTr2 binding sites than did the 64Cu-labeled agonists. 64Cu-CB-TE2A-Y3-TATE was more efficiently internalized than 64Cu-DOTA-Y3-TATE, while 64Cu-CB-TE2A-sst2-ANT showed lower, yet significant levels of internalization. CB-TE2A-Y3-TATE acted as a full agonist, inhibiting cAMP production, whereas CB-TE2A-sst2-ANT showed no inhibition of cAMP production.The 64Cu from agonists 64Cu-DOTA-Y3-TATE and 64Cu-CB-TE2A-Y3-TATE showed greater nuclear localization at 24 h in p53 +/+ vs. −/− cells; however, there was no difference in the levels of 64Cu from the antagonist based on p53 status. Surprisingly, the DOTA and CB-TE2A-conjugated agonists showed similar nuclear localization in the p53 +/+ and −/− cells, suggesting no difference in 64Cu release from these chelators in the HCT116 cell lines. Conclusion Based on thesein vitro data, the agonist 64Cu-CB-TE2A-Y3-TATE demonstrated the most promise as an agent for targeted radiotherapy in p53 positive, SSTr2-positive tumors. PMID:22056254

Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Seula; Woo, Jong Kyu; Jung, Yuchae

In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulkmore » cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. - Highlights: • Evodiamine selectively kills breast cancer stem like cells at G1 phase. • Evodiamine utilizes different mechanism of cell cycle modulation in CSLC and in bulk cancer cells. • Evodiamine activate the p53, p21 and Rb pathway.« less

Allergen-specific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation.

PubMed

Hansen, G; Berry, G; DeKruyff, R H; Umetsu, D T

1999-01-01

Allergic asthma, which is present in as many as 10% of individuals in industrialized nations, is characterized by chronic airway inflammation and hyperreactivity induced by allergen-specific Th2 cells secreting interleukin-4 (IL-4) and IL-5. Because Th1 cells antagonize Th2 cell functions, it has been proposed that immune deviation toward Th1 can protect against asthma and allergies. Using an adoptive transfer system, we assessed the roles of Th1, Th2, and Th0 cells in a mouse model of asthma and examined the capacity of Th1 cells to counterbalance the proasthmatic effects of Th2 cells. Th1, Th2, and Th0 lines were generated from ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice and transferred into lymphocyte-deficient, OVA-treated severe combined immunodeficiency (SCID) mice. OVA-specific Th2 and Th0 cells induced significant airway hyperreactivity and inflammation. Surprisingly, Th1 cells did not attenuate Th2 cell-induced airway hyperreactivity and inflammation in either SCID mice or in OVA-immunized immunocompetent BALB/c mice, but rather caused severe airway inflammation. These results indicate that antigen-specific Th1 cells may not protect or prevent Th2-mediated allergic disease, but rather may cause acute lung pathology. These findings have significant implications with regard to current therapeutic goals in asthma and allergy and suggest that conversion of Th2-dominated allergic inflammatory responses into Th1-dominated responses may lead to further problems.

pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana.

PubMed

Tsutsui, Hiroki; Higashiyama, Tetsuya

2017-01-01

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is widely used as a tool for genome engineering in various organisms. A complex consisting of Cas9 and single guide RNA (sgRNA) induces a DNA double-strand break in a sequence-specific manner, resulting in knockout. Some binary vectors for CRISPR/Cas9 in plants have been reported, but there is a problem with low efficiency. Here, we present a newly developed, highly efficient CRISPR/Cas9 vector for Arabidopsis thaliana, pKAMA-ITACHI Red (pKIR), harboring the RIBOSOMAL PROTEIN S5 A (RPS5A) promoter to drive Cas9. The RPS5A promoter maintains high constitutive expression at all developmental stages starting from the egg cell and including meristematic cells. Even in the T1 generation, pKIR induced null phenotypes in some genes: PHYTOENE DESATURASE 3 (PDS3), AGAMOUS (AG) and DUO POLLEN 1 (DUO1). Mutations induced by pKIR were carried in the germ cell line of the T1 generation. Surprisingly, in some lines, 100% of the T2 plants had the adh1 (ALCOHOL DEHYDROGENASE 1) null phenotype, indicating that pKIR strongly induced heritable mutations. Cas9-free T2 mutant plants were obtained by removing T2 seeds expressing a fluorescent marker in pKIR. Our results suggest that the pKIR system is a powerful molecular tool for genome engineering in Arabidopsis. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Serendipitous discovery of potent human head and neck squamous cell carcinoma anti-cancer molecules: A fortunate failure of a rational molecular design.

PubMed

Zagni, Chiara; Pistarà, Venerando; Oliveira, Luciana A; Castilho, Rogerio M; Romeo, Giovanni; Chiacchio, Ugo; Rescifina, Antonio

2017-12-01

Histone deacetylase inhibitors (HDACis) play an important role as valuable drugs targeted to cancer therapy: several HDACis are currently being tested in clinical trials. Two new potential HDACis 1a and 1d, characterized by the presence of a biphenyl-4-sulfonamide group as a connection unit between the N-{4-[(E)-(2-formylhydrazinylidene)methyl]-3-hydroxyphenyl} and the 2-hydroxy-N-(trifluoroacetyl)benzamide moiety, respectively, as two zinc-binding group (ZBG), have been designed, synthesized and tested for their biological activity. Surprisingly, compounds 1a and 12, this last exclusively obtained in place of 1d, exhibited a very low HDAC inhibitory activity. A serendipitous assay of these two compounds, conducted on three chemoresistant cell lines of head and neck squamous cell carcinoma (HNSCC), showed their antiproliferative activity at low nanomolar concentrations, better than cisplatin. In vitro, biological assays indicated that compounds 1a and 12 are able to increase acetylation of histone H3 and to interfere with the PI3K/Akt/mTOR pathway by inducing the accumulation of PTEN protein. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

MAP/microtubule affinity-regulating kinases, microtubule dynamics, and spermatogenesis.

PubMed

Tang, Elizabeth I; Mruk, Dolores D; Cheng, C Yan

2013-05-01

During spermatogenesis, spermatids derived from meiosis simultaneously undergo extensive morphological transformation, to become highly specialized and metabolically quiescent cells, and transport across the seminiferous epithelium. Spermatids are also transported back-and-forth across the seminiferous epithelium during the epithelial cycle until they line up at the luminal edge of the tubule to prepare for spermiation at stage VIII of the cycle. Spermatid transport thus requires the intricate coordination of the cytoskeletons in Sertoli cells (SCs) as spermatids are nonmotile cells lacking the ultrastructures of lamellipodia and filopodia, as well as the organized components of the cytoskeletons. In the course of preparing this brief review, we were surprised to see that, except for some earlier eminent morphological studies, little is known about the regulation of the microtubule (MT) cytoskeleton and the coordination of MT with the actin-based cytoskeleton to regulate spermatid transport during the epithelia cycle, illustrating that this is a largely neglected area of research in the field. Herein, we summarize recent findings in the field regarding the significance of actin- and tubulin-based cytoskeletons in SCs that support spermatid transport; we also highlight specific areas of research that deserve attention in future studies.

Generation of transgenic Hydra by embryo microinjection.

PubMed

Juliano, Celina E; Lin, Haifan; Steele, Robert E

2014-09-11

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology(1). Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

The transcriptional landscape of αβ T cell differentiation

PubMed Central

Mingueneau, Michael; Kreslavsky, Taras; Gray, Daniel; Heng, Tracy; Cruse, Richard; Ericson, Jeffrey; Bendall, Sean; Spitzer, Matt; Nolan, Garry; Kobayashi, Koichi; von Boehmer, Harald; Mathis, Diane; Benoist, Christophe

2013-01-01

αβT cell differentiation from thymic precursors is a complex process, explored here with the breadth of ImmGen expression datasets, analyzing how differentiation of thymic precursors gives rise to transcriptomes. After surprisingly gradual changes though early T commitment, transit through the CD4+CD8+ stage involves a shutdown or rare breadth, and correlating tightly with MYC. MHC-driven selection promotes a large-scale transcriptional reactivation. We identify distinct signatures that mark cells destined for positive selection versus apoptotic deletion. Differential expression of surprisingly few genes accompany CD4 or CD8 commitment, a similarity that carries through to peripheral T cells and their activation, revealed by mass cytometry phosphoproteomics. The novel transcripts identified as candidate mediators of key transitions help define the “known unknown” of thymocyte differentiation. PMID:23644507

The Surprising Absence of Absorption in the Far-ultraviolet Spectrum of Mrk 231

NASA Technical Reports Server (NTRS)

Veilleux, S.; Trippe, M.; Hamann, F.; Rupke, D. S. N.; Tripp, T. M.; Netzer, H.; Lutz, D.; Sembach, K. R.; Krug, H.; Teng, Stacy H.;

Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

PubMed Central

Verdanova, Martina; Pytlik, Robert

2014-01-01

A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBS-free medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO. PMID:24749876

Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution.

PubMed

Johnson, Andrew D; Drum, Matthew; Bachvarova, Rosemary F; Masi, Thomas; White, Mary E; Crother, Brian I

2003-01-01

The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos.

Laser biomodulation on stem cells

NASA Astrophysics Data System (ADS)

Liu, Timon C.; Duan, Rui; Li, Yan; Li, Xue-Feng; Tan, Li-Ling; Liu, Songhao

2001-08-01

Stem cells are views from the perspectives of their function, evolution, development, and cause. Counterintuitively, most stem cells may arise late in development, to act principally in tissue renewal, thus ensuring an organisms long-term survival. Surprisingly, recent reports suggest that tissue-specific adult stem cells have the potential to contribute to replenishment of multiple adult tissues. Stem cells are currently in the news for two reasons: the successful cultivation of human embryonic stem cell lines and reports that adult stem cells can differentiate into developmentally unrelated cell types, such as nerve cells into blood cells. The spotlight on stem cells has revealed gaps in our knowledge that must be filled if we are to take advantage of their full potential for treating devastating degenerative diseases such as Parkinsons's disease and muscular dystrophy. We need to know more about the intrinsic controls that keep stem cells as stem cells or direct them along particular differentiation pathways. Such intrinsic regulators are, in turn, sensitive to the influences of the microenvironment, or niche, where stem cells normally reside. Both intrinsic and extrinsic signals regular stem cell fate and some of these signals have now been identified. Vacek et al and Wang et al have studied the effect of low intensity laser on the haemopoietic stem cells in vitro. There experiments show there is indeed the effect of low intensity laser on the haemopoietic stem cells in vitro, and the present effect is the promotion of haemopoietic stem cells proliferation. In other words, low intensity laser irradiation can act as an extrinsic signal regulating stem cell fate. In this paper, we study how low intensity laser can be used to regulate stem cell fate from the viewpoint of collective phototransduction.

Comparative analysis of the internalization of the macrophage receptor sialoadhesin in human and mouse primary macrophages and cell lines.

PubMed

De Schryver, Marjorie; Leemans, Annelies; Pintelon, Isabel; Cappoen, Davie; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

2017-06-01

Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab') 2 fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can impact the activity of antibody-based therapeutic interventions via Sn. Copyright © 2016 Elsevier GmbH. All rights reserved.

Construction of the physical map for three loci in chromosome band 13q14: comparison to the genetic map.

PubMed Central

Higgins, M J; Turmel, C; Noolandi, J; Neumann, P E; Lalande, M

1990-01-01

Pulsed-field gel electrophoresis (PFGE) and deletion mapping are being used to construct a physical map of the long arm of human chromosome 13. The present study reports a 2700-kilobase (kb) Not I long-range restriction map encompassing the 13q14-specific loci D13S10, D13S21, and D13S22, which are detected by the cloned DNA markers p7D2, pG24E2.4, and pG14E1.9, respectively. Analysis of a panel of seven cell lines that showed differential methylation at a Not I site between D13S10 and D13S21 proved physical linkage of the two loci to the same 875-kb Not I fragment. D13S22 mapped to a different Not I fragment, precluding the possibility that D13S22 is located between D13S10 and D13S21. PFGE analysis of Not I partial digests placed the 1850-kb Not I fragment containing D13S22 immediately adjacent to the 875-kb fragment containing the other two loci. The proximal rearrangement breakpoint in a cell line carrying a del13(q14.1q21.2) was detected by D13S21 but not by D13S10, demonstrating that D13S21 lies proximal to D13S10. Quantitative analysis of hybridization signals of the three DNA probes to DNA from the same cell line indicated that only D13S10 was deleted, establishing the order of these loci to be cen-D13S22-D13S21-D13S10-tel. Surprisingly, this order was estimated to be 35,000 times less likely than that favored by genetic linkage analysis. Images PMID:1970636

Salience and Attention in Surprisal-Based Accounts of Language Processing

PubMed Central

Zarcone, Alessandra; van Schijndel, Marten; Vogels, Jorrig; Demberg, Vera

2016-01-01

The notion of salience has been singled out as the explanatory factor for a diverse range of linguistic phenomena. In particular, perceptual salience (e.g., visual salience of objects in the world, acoustic prominence of linguistic sounds) and semantic-pragmatic salience (e.g., prominence of recently mentioned or topical referents) have been shown to influence language comprehension and production. A different line of research has sought to account for behavioral correlates of cognitive load during comprehension as well as for certain patterns in language usage using information-theoretic notions, such as surprisal. Surprisal and salience both affect language processing at different levels, but the relationship between the two has not been adequately elucidated, and the question of whether salience can be reduced to surprisal / predictability is still open. Our review identifies two main challenges in addressing this question: terminological inconsistency and lack of integration between high and low levels of representations in salience-based accounts and surprisal-based accounts. We capitalize upon work in visual cognition in order to orient ourselves in surveying the different facets of the notion of salience in linguistics and their relation with models of surprisal. We find that work on salience highlights aspects of linguistic communication that models of surprisal tend to overlook, namely the role of attention and relevance to current goals, and we argue that the Predictive Coding framework provides a unified view which can account for the role played by attention and predictability at different levels of processing and which can clarify the interplay between low and high levels of processes and between predictability-driven expectation and attention-driven focus. PMID:27375525

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

PubMed

Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

2014-10-13

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

PubMed

Marvel, Miranda; Spicer, Olivia Smith; Wong, Ten-Tsao; Zmora, Nilli; Zohar, Yonathan

2018-04-04

Gonadotropin-releasing hormone (GnRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a non-cell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

Acute virus control mediated by licensed NK cells sets primary CD8+ T cell dependence on CD27 costimulation1,2,3

PubMed Central

Teoh, Jeffrey J.; Gamache, Awndre E.; Gillespie, Alyssa L.; Stadnisky, Michael D.; Yagita, Hideo; Bullock, Timothy N.J.; Brown, Michael G.

2016-01-01

Natural killer (NK) cells represent a critical first-line of immune defense against a bevy of viral pathogens, and infection can provoke them to mediate both supportive and suppressive effects on virus-specific adaptive immunity. In mice expressing MHC I Dk, a major MCMV resistance factor and self-ligand of the inhibitory Ly49G2 (G2) receptor, licensed G2+ NK cells provide essential host resistance against murine (M)CMV infection. Additionally G2+ NK cell responses to MCMV increase the rate and extent of dendritic cell (DC) recovery, as well as early priming of CD8+ T-cell effectors in response to MCMV. However, relatively little is known about the NK-cell effect on co-stimulatory ligand patterns displayed by DCs, or ensuing effector and memory T-cell responses. Here we found that CD27-dependent CD8+ T-cell priming and differentiation is shaped by the efficiency of NK responses to virus infection. Surprisingly, differences in specific NK responses to MCMV in Dk-disparate mice failed to distinguish early DC co-stimulatory patterns. Nonetheless, while CD27 deficiency did not impede licensed NK-mediated resistance, both CD70 and CD27 were required to efficiently prime and regulate effector CD8+ T-cell differentiation in response to MCMV, which eventually resulted in biased memory T-cell precursor formation in Dk mice. In contrast, CD8+ T-cells accrued more slowly in non-Dk mice, and eventually differentiated into terminal effector cells regardless of CD27 stimulation. Disparity in this requirement for CD27 signaling indicates that specific virus control mediated by NK cells can shape DC co-stimulatory signals needed to prime CD8+ T cells and eventual T-cell fate decisions. PMID:27798162

Coherent optical transients observed in rubidium atomic line filtered Doppler velocimetry experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fajardo, Mario E., E-mail: mario.fajardo@eglin.af.mil; Molek, Christopher D.; Vesely, Annamaria L.

2015-10-14

We report the first successful results from our novel Rubidium Atomic Line Filtered (RALF) Doppler velocimetry apparatus, along with unanticipated oscillatory signals due to coherent optical transients generated within pure Rb vapor cells. RALF is a high-velocity and high-acceleration extension of the well-known Doppler Global Velocimetry (DGV) technique for constructing multi-dimensional flow velocity vector maps in aerodynamics experiments [H. Komine, U.S. Patent No. 4,919,536 (24 April 1990)]. RALF exploits the frequency dependence of pressure-broadened Rb atom optical absorptions in a heated Rb/N{sub 2} gas cell to encode the Doppler shift of reflected near-resonant (λ{sub 0} ≈ 780.24 nm) laser light onto the intensitymore » transmitted by the cell. The present RALF apparatus combines fiber optic and free-space components and was built to determine suitable operating conditions and performance parameters for the Rb/N{sub 2} gas cells. It yields single-spot velocities of thin laser-driven-flyer test surfaces and incorporates a simultaneous Photonic Doppler Velocimetry (PDV) channel [Strand et al., Rev. Sci. Instrum. 77, 083108 (2006)] for validation of the RALF results, which we demonstrate here over the v = 0 to 1 km/s range. Both RALF and DGV presume the vapor cells to be simple Beer's Law optical absorbers, so we were quite surprised to observe oscillatory signals in experiments employing low pressure pure Rb vapor cells. We interpret these oscillations as interference between the Doppler shifted reflected light and the Free Induction Decay (FID) coherent optical transient produced within the pure Rb cells at the original laser frequency; this is confirmed by direct comparison of the PDV and FID signals. We attribute the different behaviors of the Rb/N{sub 2} vs. Rb gas cells to efficient dephasing of the atomic/optical coherences by Rb-N{sub 2} collisions. The minimum necessary N{sub 2} buffer gas density ≈0.3 amagat translates into a smallest useful velocity range of 0 to 2 km/s, which can readily be extended to cover the 0 to 10 km/s range, and beyond. The recognition that coherent optical transients can be produced within low pressure vapor cells during velocimetry experiments may offer new insights into some quantitative discrepancies reported in earlier DGV studies. Future plans include “line-RALF” experiments with streak camera detection, and two-dimensional surface velocity mapping using pulsed laser illumination and/or gated intensified CCD camera detection.« less

NPM and BRG1 Mediate Transcriptional Resistance to Retinoic Acid in Acute Promyelocytic Leukemia.

PubMed

Nichol, Jessica N; Galbraith, Matthew D; Kleinman, Claudia L; Espinosa, Joaquín M; Miller, Wilson H

2016-03-29

Perturbation in the transcriptional control of genes driving differentiation is an established paradigm whereby oncogenic fusion proteins promote leukemia. From a retinoic acid (RA)-sensitive acute promyelocytic leukemia (APL) cell line, we derived an RA-resistant clone characterized by a block in transcription initiation, despite maintaining wild-type PML/RARA expression. We uncovered an aberrant interaction among PML/RARA, nucleophosmin (NPM), and topoisomerase II beta (TOP2B). Surprisingly, RA stimulation in these cells results in enhanced chromatin association of the nucleosome remodeler BRG1. Inhibition of NPM or TOP2B abrogated BRG1 recruitment. Furthermore, NPM inhibition and targeting BRG1 restored differentiation when combined with RA. Here, we demonstrate a role for NPM and BRG1 in obstructing RA differentiation and implicate chromatin remodeling in mediating therapeutic resistance in malignancies. NPM mutations are the most common genetic change in patients with acute leukemia (AML); therefore, our model may be applicable to other more common leukemias driven by NPM. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

An extremely rare case of small-cell lung cancer harboring variant 2 of the EML4-ALK fusion gene.

PubMed

Toyokawa, Gouji; Takenoyama, Mitsuhiro; Taguchi, Kenichi; Toyozawa, Ryo; Inamasu, Eiko; Kojo, Miyako; Shiraishi, Yoshimasa; Morodomi, Yosuke; Takenaka, Tomoyoshi; Hirai, Fumihiko; Yamaguchi, Masafumi; Seto, Takashi; Shimokawa, Mototsugu; Ichinose, Yukito

2013-09-01

Anaplastic lymphoma kinase (ALK) fuses echinoderm microtubule-associated protein-like 4 (EML4) to acquire a transforming activity in lung adenocarcinomas. However, the presence of an EML4-ALK fusion gene in other lung cancer histologies is an extremely rare phenomenon. A 43-year-old female was referred to our department due to dyspnea on effort and left back pain. Computed tomography (CT) showed a large mass in the upper lobe of the left lung and a massive left pleural effusion, while a CT-guided needle biopsy confirmed a diagnosis of small-cell lung cancer (SCLC). Surprisingly, the tumor was genetically considered to harbor the EML4-ALK fusion gene (variant 2). Although the patient underwent two regimens of cytotoxic chemotherapy for SCLC, she died approximately seven months after the administration of first-line chemotherapy. Our analysis of 30 consecutive patients with SCLC for EML4-ALK revealed that two patients, including the current patient and a patient we previously reported, harbored the EML4-ALK fusion gene. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Lysine acetyltransferase NuA4 and acetyl-CoA regulate glucose-deprived stress granule formation in Saccharomyces cerevisiae

PubMed Central

Huard, Sylvain; Morettin, Alan; Fullerton, Morgan D.; Côté, Jocelyn

2017-01-01

Eukaryotic cells form stress granules under a variety of stresses, however the signaling pathways regulating their formation remain largely unknown. We have determined that the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 is required for stress granule formation upon glucose deprivation but not heat stress. Further, the Tip60 complex, the human homolog of the NuA4 complex, is required for stress granule formation in cancer cell lines. Surprisingly, the impact of NuA4 on glucose-deprived stress granule formation is partially mediated through regulation of acetyl-CoA levels, which are elevated in NuA4 mutants. While elevated acetyl-CoA levels suppress the formation of glucose-deprived stress granules, decreased acetyl-CoA levels enhance stress granule formation upon glucose deprivation. Further our work suggests that NuA4 regulates acetyl-CoA levels through the Acetyl-CoA carboxylase Acc1. Altogether this work establishes both NuA4 and the metabolite acetyl-CoA as critical signaling pathways regulating the formation of glucose-deprived stress granules. PMID:28231279

Specification of embryonic stem cell-derived tissues into eye fields by Wnt signaling using rostral diencephalic tissue-inducing culture.

PubMed

Sakakura, Eriko; Eiraku, Mototsugu; Takata, Nozomu

2016-08-01

The eyes are subdivided from the rostral diencephalon in early development. How the neuroectoderm regulates this subdivision, however, is largely unknown. Taking advantage of embryonic stem cell (ESC) culture using a Rax reporter line to monitor rostral diencephalon formation, we found that ESC-derived tissues at day 7 grown in Glasgow Minimum Expression Media (GMEM) containing knockout serum replacement (KSR) exhibited higher levels of expression of axin2, a Wnt target gene, than those grown in chemically defined medium (CDM). Surprisingly, Wnt agonist facilitated eye field-like tissue specification in CDM. In contrast, the addition of Wnt antagonist diminished eye field tissue formation in GMEM+KSR. Furthermore, the morphological formation of the eye tissue anlage, including the optic vesicle, was accompanied by Wnt signaling activation. Additionally, using CDM culture, we developed an efficient method for generating Rax+/Chx10+ retinal progenitors, which could become fully stratified retina. Here we provide a new avenue for exploring the mechanisms of eye field specification in vitro. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Extracellular calcium controls the expression of two different forms of ripple-like hippocampal oscillations.

PubMed

Aivar, Paloma; Valero, Manuel; Bellistri, Elisa; Menendez de la Prida, Liset

2014-02-19

Hippocampal high-frequency oscillations (HFOs) are prominent in physiological and pathological conditions. During physiological ripples (100-200 Hz), few pyramidal cells fire together coordinated by rhythmic inhibitory potentials. In the epileptic hippocampus, fast ripples (>200 Hz) reflect population spikes (PSs) from clusters of bursting cells, but HFOs in the ripple and the fast ripple range are vastly intermixed. What is the meaning of this frequency range? What determines the expression of different HFOs? Here, we used different concentrations of Ca(2+) in a physiological range (1-3 mM) to record local field potentials and single cells in hippocampal slices from normal rats. Surprisingly, we found that this sole manipulation results in the emergence of two forms of HFOs reminiscent of ripples and fast ripples recorded in vivo from normal and epileptic rats, respectively. We scrutinized the cellular correlates and mechanisms underlying the emergence of these two forms of HFOs by combining multisite, single-cell and paired-cell recordings in slices prepared from a rat reporter line that facilitates identification of GABAergic cells. We found a major effect of extracellular Ca(2+) in modulating intrinsic excitability and disynaptic inhibition, two critical factors shaping network dynamics. Moreover, locally modulating the extracellular Ca(2+) concentration in an in vivo environment had a similar effect on disynaptic inhibition, pyramidal cell excitability, and ripple dynamics. Therefore, the HFO frequency band reflects a range of firing dynamics of hippocampal networks.

Curcumin and Turmeric Modulate the Tumor-Promoting Effects of Iron In Vitro.

PubMed

Messner, Donald J; Robinson, Todd; Kowdley, Kris V

2017-04-01

Free or loosely chelated iron has tumor-promoting properties in vitro. Curcumin, a polyphenol derived from the food spice turmeric (Curcuma longa), is a potent antioxidant that binds iron. The primary aim of this study was to investigate whether curcuminoids prevent tumor-promoting effects of iron in T51B cells, a non-neoplastic rat liver epithelial cell line. Purified curcuminoids (curcumin) or a standardized turmeric extract similarly reduced oxidative stress and cytotoxicity associated with iron overload (IC 50 values near 10 μM, P < 0.05). Inhibition of iron-induced tumor promotion (seen upon treatment with 200 μM ferric ammonium citrate ± curcumin/turmeric for 16 wk in culture; subsequently assayed by soft agar colony formation) was nearly complete at 20 μM of total curcuminoids (P < 0.05), a concentration predicted to only partially chelate the added iron. Surprisingly, lower curcumin concentrations (10 μM) increased tumor promotion (P < 0.01). Curcuminoids delivered as a standardized turmeric extract were taken up better by cells, had a longer half-life, and appeared more effective in blocking tumor promotion (P < 0.01), suggesting enhanced curcuminoid delivery to cells in culture. The primary finding that curcuminoids can inhibit tumor promotion caused by iron in T51B cells is tempered by evidence for an underlying increase in neoplastic transformation at lower concentrations.

Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum

PubMed Central

Mitsuda, Satoshi; Yokomichi, Tomonobu; Yokoigawa, Junpei; Kataoka, Takao

2014-01-01

Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule-1 (ICAM-1) in response to interleukin-1α (IL-1α). We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL-1α-induced cell-surface ICAM-1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1. Ursolic acid induced the accumulation of ICAM-1 in the endoplasmic reticulum, which was linked mainly to high-mannose-type glycans. Moreover, in ursolic-acid-treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum. PMID:24649404

Curcumin and Turmeric Modulate the Tumor-Promoting Effects of Iron In Vitro

PubMed Central

Messner, Donald J.; Robinson, Todd; Kowdley, Kris V.

2018-01-01

Free or loosely chelated iron has tumor-promoting properties in vitro. Curcumin, a polyphenol derived from the food spice turmeric (Curcuma longa), is a potent antioxidant that binds iron. The primary aim of this study was to investigate whether curcuminoids prevent tumor-promoting effects of iron in T51B cells, a non-neoplastic rat liver epithelial cell line. Purified curcuminoids (curcumin) or a standardized turmeric extract similarly reduced oxidative stress and cytotoxicity associated with iron overload (IC50 values near 10 μM, P < 0.05). Inhibition of iron-induced tumor promotion (seen upon treatment with 200 μM ferric ammonium citrate ± curcumin/turmeric for 16 wk in culture; subsequently assayed by soft agar colony formation) was nearly complete at 20 μM of total curcuminoids (P < 0.05), a concentration predicted to only partially chelate the added iron. Surprisingly, lower curcumin concentrations (10 μM) increased tumor promotion (P < 0.01). Curcuminoids delivered as a standardized turmeric extract were taken up better by cells, had a longer half-life, and appeared more effective in blocking tumor promotion (P < 0.01), suggesting enhanced curcuminoid delivery to cells in culture. The primary finding that curcuminoids can inhibit tumor promotion caused by iron in T51B cells is tempered by evidence for an underlying increase in neoplastic transformation at lower concentrations. PMID:28129008

The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

PubMed

Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

2014-04-01

Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation. Copyright © 2014 Elsevier B.V. All rights reserved.

Hexon modification to improve the activity of oncolytic adenovirus vectors against neoplastic and stromal cells in pancreatic cancer.

PubMed

Lucas, Tanja; Benihoud, Karim; Vigant, Frédéric; Schmidt, Christoph Q; Schmidt, Christoph Q Andreas; Wortmann, Andreas; Bachem, Max G; Simmet, Thomas; Kochanek, Stefan

2015-01-01

Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFβ receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety.

Innate immunity and the regulation and mobilization of keratinocyte stem cells: are the old players playing a new game?

PubMed

Singh, Ashok; Morris, Rebecca J

2012-09-01

The skin provides an anatomical barrier to physical, chemical and biological agents. Hence, it is not surprising that it has well-developed innate immunity. What we find surprising is that the CD49f(+) /CD34(+) hair follicle stem cells should have an enriched expression profile of so many genes involved in innate immunity. Do these stem cells require extra protection from environmental insults? Or, could there be a new role for these genes? To probe these questions, we first summarize the roles of some key players in epidermal innate immunity. We next focus on their expression in CD49f(+) /CD34(+) hair follicle stem cells. Then, we consider recent data suggesting a new role for these 'old players' in the regulation and mobilization of haematopoietic and mesenchymal stem cells. Finally, we hypothesize that the 'old players' in these hair follicle stem cells may be playing a 'new game'. © 2012 John Wiley & Sons A/S.

Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics

PubMed Central

Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl MJ; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie

2015-01-01

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems. PMID:26214591

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes – A method for quantitative transcriptional reprogramming

PubMed Central

Halley-Stott, R.P.; Pasque, V.; Astrand, C.; Miyamoto, K.; Simeoni, I.; Jullien, J.; Gurdon, J.B.

2010-01-01

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein. PMID:20123126

Cell culture's spider silk road.

PubMed

Perkel, Jeffrey

2014-06-01

A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk.

The relationship of aplastic anemia and PNH.

PubMed

Young, Neal S; Maciejewski, Jaroslaw P; Sloand, Elaine; Chen, Guiben; Zeng, Weihua; Risitano, Antonio; Miyazato, Akira

2002-08-01

Bone marrow failure has been regarded as one of the triad of clinical manifestations of paroxysmal noctumal hemoglobinuria (PNH), and PNH in turn has been described as a late clonal disease evolving in patients recovering from aplastic anemia. Better understanding of the pathophysiology of both diseases and improved tests for cell surface glycosylphosphatidylinositol (GPI)-linked proteins has radically altered this view. Flow cytometry of granulocytes shows evidence of an expanded PNH clone in a large proportion of marrow failure patients at the time of presentation: in our large NIH series, about 1/3 of over 200 aplastic anemia cases and almost 20% of more than 100 myelodysplasia cases. Clonal PNH expansion (rather than bone marrow failure) is strongly linked to the histocompatability antigen HLA.-DR2 in all clinical varieties of the disease, suggesting an immune component to its pathophysiology. An extrinsic mechanism of clonal expansion is also more consistent with knock-out mouse models and culture experiments with primary cells and cell lines, which have failed to demonstrate an intrinsic proliferative advantage for PNH cells. DNA chip analysis of multiple paired normal and PIG-A mutant cell lines and lymphoblastoid cells do not show any consistent differences in levels of gene expression. In aplastic anemia/PNH there is surprisingly limited utilization of the V-beta chain of the T cell receptor, and patients' dominant T cell clones, which are functionally inhibitory of autologous hematopoiesis, use identical CDR3 regions for antigen binding. Phenotypically normal cells from PNH patients proliferate more poorly in culture than do the same patient's PNH cells, and the normal cells are damaged as a result of apoptosis and overexpress Fas. Differences in protein degradation might play a dual role in pathophysiology, as GPI-linked proteins lacking an anchor would be predicted to be processed by the proteasome machinery and displayed in a class I H.A. context, in contrast to the normal pathway of cell surface membrane recycling, lysosomal degradation, and presentation by class II HLA. The strong relationship between a chronic, organ-specific immune destructive process and the expansion of a single mutant stem cell clone remains frustratingly enigmatic but likely to be the result of interesting biologic processes, with mechanisms that potentially can be extended to the role of inflammation in producing premalignant syndromes.

An Acidic Microenvironment Increases NK Cell Killing of Cryptococcus neoformans and Cryptococcus gattii by Enhancing Perforin Degranulation

PubMed Central

Islam, Anowara; Li, Shu Shun; Oykhman, Paul; Timm-McCann, Martina; Huston, Shaunna M.; Stack, Danuta; Xiang, Richard F.; Kelly, Margaret M.; Mody, Christopher H.

2013-01-01

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment. PMID:23853583

Low redshift Lyman alpha absorption lines and the dark matter halos of disk galaxies

NASA Technical Reports Server (NTRS)

Maloney, Philip

1993-01-01

Recent observations using the Hubble Space Telescope of the z = 0.156 QSO 3C 273 have discovered a surprisingly large number of Ly-alpha absorption lines. In particular, Morris et al. found 9 certain and 7 possible Ly-alpha lines with equivalent widths above 25 mA. This is much larger (by a factor of 5-10) than the number expected from extrapolation of the high-redshift behavior of the Ly-alpha forest. Within the context of pressure-confined models for the Ly-alpha clouds, this behavior can be understood if the ionizing background declines sharply between z is approximately 2 and z is approximately 0. However, this requires that the ionizing photon flux drop as rapidly as the QSO volume emissivity; moreover, the absorbers must have a space density n(sub O) is approximately 2.6(N/10)h/((D/100 kpc)(sup 2)) Mpc(sup -3) where D is the present-day diameter of the absorbers. It is somewhat surprising that such necessarily fragile objects could have survived in such numbers to the present day. It is shown that it is plausible that the atomic hydrogen extents of spiral and irregular galaxies are large enough to produce the observed number of Ly-alpha absorption lines toward 3C 273, and that the neutral column densities and doppler b-values expected under these conditions fall in the range found by Morris et al. (1991).

Linking transcriptional and genetic tumor heterogeneity through allele analysis of single-cell RNA-seq data.

PubMed

Fan, Jean; Lee, Hae-Ock; Lee, Soohyun; Ryu, Da-Eun; Lee, Semin; Xue, Catherine; Kim, Seok Jin; Kim, Kihyun; Barkas, Nikolas; Park, Peter J; Park, Woong-Yang; Kharchenko, Peter V

2018-06-13

Characterization of intratumoral heterogeneity is critical to cancer therapy, as presence of phenotypically diverse cell populations commonly fuels relapse and resistance to treatment. Although genetic variation is a well-studied source of intratumoral heterogeneity, the functional impact of most genetic alterations remains unclear. Even less understood is the relative importance of other factors influencing heterogeneity, such as epigenetic state or tumor microenvironment. To investigate the relationship between genetic and transcriptional heterogeneity in a context of cancer progression, we devised a computational approach called HoneyBADGER to identify copy number variation and loss-of-heterozygosity in individual cells from single-cell RNA-sequencing data. By integrating allele and normalized expression information, HoneyBADGER is able to identify and infer the presence of subclone-specific alterations in individual cells and reconstruct underlying subclonal architecture. Examining several tumor types, we show that HoneyBADGER is effective at identifying deletion, amplifications, and copy-neutral loss-of-heterozygosity events, and is capable of robustly identifying subclonal focal alterations as small as 10 megabases. We further apply HoneyBADGER to analyze single cells from a progressive multiple myeloma patient to identify major genetic subclones that exhibit distinct transcriptional signatures relevant to cancer progression. Surprisingly, other prominent transcriptional subpopulations within these tumors did not line up with the genetic subclonal structure, and were likely driven by alternative, non-clonal mechanisms. These results highlight the need for integrative analysis to understand the molecular and phenotypic heterogeneity in cancer. Published by Cold Spring Harbor Laboratory Press.

Glycosylation-related genes in NS0 cells are insensitive to moderately elevated ammonium concentrations

PubMed Central

Brodsky, Arthur Nathan; Caldwell, Mary; Bae, Sooneon; Harcum, Sarah W.

2014-01-01

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, the of these genes was analysed in response to various culture conditions – elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analysed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium. PMID:25062658

Evolution of robustness in the signaling network of Pristionchus vulva development

PubMed Central

Zauner, Hans; Sommer, Ralf J.

2007-01-01

Robustness to environmental or genetic perturbation, like any other trait, is affected by evolutionary change. However, direct studies on the interplay of robustness and evolvability are limited and require experimental microevolutionary studies of developmental processes. One system in which such microevolutionary studies can be performed is vulva development in the nematode Pristionchus pacificus. Three vulval precursor cells respond to redundant cell–cell interactions, including signals from the gonad and the epidermal cell P8.p. Interestingly, P. pacificus P8.p is involved in cell fate specification of the future vulva cells by lateral inhibition but is incompetent to respond to the inductive signal from the gonad itself. These functional properties of P8.p are unknown from other nematodes, such as Caenorhabditis elegans. We began an experimental and genetic analysis of the microevolution of P8.p function. We show that vulva misspecification events differ between Pristionchus strains and species. Similarly, lateral inhibition and developmental competence of P8.p evolved within the genus Pristionchus and between natural isolates of P. pacificus. Surprisingly, in some recombinant inbred lines of two distinct P. pacificus isolates, P8.p gained competence to form vulva tissue, a trait that was never observed in P. pacificus isolates. Our results suggest differences in developmental stability between natural isolates, and we hypothesize that the remarkable evolvability of redundant cell–cell interactions allows for adaptive evolution of robustness to developmental noise. PMID:17551021

Regulation and Function of TMEM16F in Renal Podocytes.

PubMed

Schenk, Laura K; Ousingsawat, Jiraporn; Skryabin, Boris V; Schreiber, Rainer; Pavenstädt, Hermann; Kunzelmann, Karl

2018-06-18

The Ca 2+ -activated phospholipid scramblase and ion channel TMEM16F is expressed in podocytes of renal glomeruli. Podocytes are specialized cells that form interdigitating foot processes as an essential component of the glomerular filter. These cells, which participate in generation of the primary urine, are often affected during primary glomerular diseases, such as glomerulonephritis and secondary hypertensive or diabetic nephropathy, which always leads to proteinuria. Because the function of podocytes is known to be controlled by intracellular Ca 2+ signaling, it is important to know about the role of Ca 2+ -activated TMEM16F in these cells. To that end, we generated an inducible TMEM16F knockdown in the podocyte cell line AB8, and produced a conditional mouse model with knockout of TMEM16F in podocytes and renal epithelial cells of the nephron. We found that knockdown of TMEM16F did not produce proteinuria or any obvious phenotypic changes. Knockdown of TMEM16F affected cell death of tubular epithelial cells but not of glomerular podocytes when analyzed in TUNEL assays. Surprisingly, and in contrast to other cell types, TMEM16F did not control intracellular Ca 2+ signaling and was not responsible for Ca 2+ -activated whole cell currents in podocytes. TMEM16F levels in podocytes were enhanced after inhibition of the endolysosomal pathway and after treatment with angiotensin II. Renal knockout of TMEM16F did not compromise renal morphology and serum electrolytes. Taken together, in contrast to other cell types, such as platelets, bone cells, and immune cells, TMEM16F shows little effect on basal properties of podocytes and does not appear to be essential for renal function.

Heterotic line bundle models on elliptically fibered Calabi-Yau three-folds

NASA Astrophysics Data System (ADS)

Braun, Andreas P.; Brodie, Callum R.; Lukas, Andre

2018-04-01

We analyze heterotic line bundle models on elliptically fibered Calabi-Yau three-folds over weak Fano bases. In order to facilitate Wilson line breaking to the standard model group, we focus on elliptically fibered three-folds with a second section and a freely-acting involution. Specifically, we consider toric weak Fano surfaces as base manifolds and identify six such manifolds with the required properties. The requisite mathematical tools for the construction of line bundle models on these spaces, including the calculation of line bundle cohomology, are developed. A computer scan leads to more than 400 line bundle models with the right number of families and an SU(5) GUT group which could descend to standard-like models after taking the ℤ2 quotient. A common and surprising feature of these models is the presence of a large number of vector-like states.

Paradoxical Roles of Elongation Factor-2 Kinase in Stem Cell Survival *

PubMed Central

Liao, Yi; Chu, Hsueh-Ping; Hu, Zhixian; Merkin, Jason J.; Chen, Jianmin; Liu, Zuguo; Degenhardt, Kurt; White, Eileen; Ryazanov, Alexey G.

2016-01-01

Protein synthesis inhibition is an immediate response during stress to switch the composition of protein pool in order to adapt to the new environment. It was reported that this response could be either protective or deleterious. However, how cells choose to live or die upon protein synthesis inhibition is largely unknown. Previously, we have shown that elongation factor-2 kinase (eEF2K), a protein kinase that suppresses protein synthesis during elongation phase, is a positive regulator of apoptosis both in vivo and in vitro. Consistently, here we report that knock-out of eEF2K protects mice from a lethal dose of whole-body ionizing radiation at 8 Gy by reducing apoptosis levels in both bone marrow and gastrointestinal tracts. Surprisingly, similar to the loss of p53, eEF2K deficiency results in more severe damage to the gastrointestinal tract at 20 Gy with the increased mitotic cell death in small intestinal stem cells. Furthermore, using epithelial cell lines, we showed that eEF2K is required for G2/M arrest induced by radiation to prevent mitotic catastrophe in a p53-independent manner. Specifically, we observed the elevation of Akt/ERK activity as well as the reduction of p21 expression in Eef2k−/− cells. Therefore, eEF2K also provides a protective strategy to maintain genomic integrity by arresting cell cycle in response to stress. Our results suggest that protective versus pro-apoptotic roles of eEF2K depend on the type of cells: eEF2K is protective in highly proliferative cells, such as small intestinal stem cells and cancer cells, which are more susceptible to mitotic catastrophe. PMID:27466362

Elementary Visual Hallucinations and Their Relationships to Neural Pattern-Forming Mechanisms

ERIC Educational Resources Information Center

Billock, Vincent A.; Tsou, Brian H.

2012-01-01

An extraordinary variety of experimental (e.g., flicker, magnetic fields) and clinical (epilepsy, migraine) conditions give rise to a surprisingly common set of elementary hallucinations, including spots, geometric patterns, and jagged lines, some of which also have color, depth, motion, and texture. Many of these simple hallucinations fall into a…

Breakdown of Spatial Parallel Coding in Children's Drawing

ERIC Educational Resources Information Center

De Bruyn, Bart; Davis, Alyson

2005-01-01

When drawing real scenes or copying simple geometric figures young children are highly sensitive to parallel cues and use them effectively. However, this sensitivity can break down in surprisingly simple tasks such as copying a single line where robust directional errors occur despite the presence of parallel cues. Before we can conclude that this…

Teaching Literature in Virtual Worlds: Immersive Learning in English Studies

ERIC Educational Resources Information Center

Webb, Allen, Ed.

2011-01-01

What are the realities and possibilities of utilizing on-line virtual worlds as teaching tools for specific literary works? Through engaging and surprising stories from classrooms where virtual worlds are in use, this book invites readers to understand and participate in this emerging and valuable pedagogy. It examines the experience of high…

Cytoplasmic long noncoding RNAs are frequently bound to and degraded at ribosomes in human cells

PubMed Central

Carlevaro-Fita, Joana; Rahim, Anisa; Guigó, Roderic; Vardy, Leah A.; Johnson, Rory

2016-01-01

Recent footprinting studies have made the surprising observation that long noncoding RNAs (lncRNAs) physically interact with ribosomes. However, these findings remain controversial, and the overall proportion of cytoplasmic lncRNAs involved is unknown. Here we make a global, absolute estimate of the cytoplasmic and ribosome-associated population of stringently filtered lncRNAs in a human cell line using polysome profiling coupled to spike-in normalized microarray analysis. Fifty-four percent of expressed lncRNAs are detected in the cytoplasm. The majority of these (70%) have >50% of their cytoplasmic copies associated with polysomal fractions. These interactions are lost upon disruption of ribosomes by puromycin. Polysomal lncRNAs are distinguished by a number of 5′ mRNA-like features, including capping and 5′UTR length. On the other hand, nonpolysomal “free cytoplasmic” lncRNAs have more conserved promoters and a wider range of expression across cell types. Exons of polysomal lncRNAs are depleted of endogenous retroviral insertions, suggesting a role for repetitive elements in lncRNA localization. Finally, we show that blocking of ribosomal elongation results in stabilization of many associated lncRNAs. Together these findings suggest that the ribosome is the default destination for the majority of cytoplasmic long noncoding RNAs and may play a role in their degradation. PMID:27090285

HIV turns plasmacytoid dendritic cells (pDC) into TRAIL-expressing killer pDC and down-regulates HIV coreceptors by Toll-like receptor 7-induced IFN-alpha.

PubMed

Hardy, Andrew W; Graham, David R; Shearer, Gene M; Herbeuval, Jean-Philippe

2007-10-30

Plasmacytoid dendritic cells (pDC) are key players in viral immunity and produce IFN-alpha after HIV-1 exposure, which in turn regulates TNF-related apoptosis-inducing ligand (TRAIL) expression by CD4(+) T cells. We show here that infectious and noninfectious HIV-1 virions induce activation of pDC into TRAIL-expressing IFN-producing killer pDC (IKpDC). IKpDC expressed high levels of activation markers (HLA-DR, CD80, CD83, and CD86) and the migration marker CCR7. Surprisingly, CXCR4 and CCR5 were down-regulated on IKpDC. We also show that HIV-1-induced IKpDC depended on Toll-like receptor 7 (TLR7) activation. HIV-1 or TLR7 agonistexposed IKpDC induced apoptosis of the CD4(+) T cell line SupT1 via the TRAIL pathway. Furthermore, IFN-alpha produced after HIV-induced TLR7 stimulation was responsible for TRAIL expression and the down-regulation of both CXCR4 and CCR5 by IKpDC. In contrast, activation and migration markers were not regulated by IFN-alpha. Finally, IFN-alpha increased the survival of IKpDC. We characterized a subset of pDC with a killer activity that is activated by endosomal-associated viral RNA and not by infection.

Regulatory T cells may participate in Helicobacter pylori persistence in gastric MALT lymphoma: lessons from an animal model.

PubMed

Laur, Amandine Marine; Floch, Pauline; Chambonnier, Lucie; Benejat, Lucie; Korolik, Victoria; Giese, Alban; Dubus, Pierre; Mégraud, Francis; Bandeira, Antonio; Lehours, Philippe

2016-01-19

It has been postulated that the emergence of autoimmune gastritis in neonatal thymectomised (d3Tx) BALB/c mice may be a consequence of post-surgery deficit in Tregs. In this study, previously obtained samples from d3Tx mice were used in order to determine whether thymectomy creates a deficit in this T cell subset thereby allowing the emergence of autoimmune phenomena as a prerequisite for GML. The splenic Treg reserve and the local recruitment of these cells in the gastric mucosa were investigated using complementary molecular and immunohistochemistry approaches. Higher Foxp3/CD3 ratios were found in the spleen of non-infected d3Tx mice compared to non-thymectomised (NTx) controls. These results indicate a relative enrichment of Tregs following thymectomy in adult mice. The absence of Treg depletion in d3Tx mice is in line with the absence of auto-immune gastritis in non-infected d3Tx mice. Higher levels of T cell and Treg infiltration were also found in the stomach of GML-developing d3Tx mice versus NTx mice. Surprisingly, inflammatory scores inversely correlated with the bacterial inoculum. The presence of a small Treg containing compartment among gastric biopsies of GML developing d3Tx mice may play a role in perseverance of a minimal bacterial numbers thereby maintaining an antigen-dependent stimulation and proliferation.

Azadirachtin interacts with the tumor necrosis factor (TNF) binding domain of its receptors and inhibits TNF-induced biological responses.

PubMed

Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A; Manna, Sunil K

2010-02-19

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses*

PubMed Central

Thoh, Maikho; Kumar, Pankaj; Nagarajaram, Hampathalu A.; Manna, Sunil K.

2010-01-01

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy. PMID:20018848

Targeting the receptor tyrosine kinase RET in combination with aromatase inhibitors in ER positive breast cancer xenografts.

PubMed

Andreucci, Elena; Francica, Paola; Fearns, Antony; Martin, Lesley-Ann; Chiarugi, Paola; Isacke, Clare M; Morandi, Andrea

2016-12-06

The majority of breast cancers are estrogen receptor positive (ER+). Blockade of estrogen biosynthesis by aromatase inhibitors (AIs) is the first-line endocrine therapy for post-menopausal women with ER+ breast cancers. However, AI resistance remains a major challenge. We have demonstrated previously that increased GDNF/RET signaling in ER+ breast cancers promotes AI resistance. Here we investigated the efficacy of different small molecule RET kinase inhibitors, sunitinib, cabozantinib, NVP-BBT594 and NVP-AST487, and the potential of combining a RET inhibitor with the AI letrozole in ER+ breast cancers. The most effective inhibitor identified, NVP-AST487, suppressed GDNF-stimulated RET downstream signaling and 3D tumor spheroid growth. Ovariectomized mice were inoculated with ER+ aromatase-overexpressing MCF7-AROM1 cells and treated with letrozole, NVP-AST487 or the two drugs in combination. Surprisingly, the three treatment regimens showed similar efficacy in impairing MCF7-AROM1 tumor growth in vivo. However in vitro, NVP-AST487 was superior to letrozole in inhibiting the GDNF-induced motility and tumor spheroid growth of MCF7-AROM1 cells and required in combination with letrozole to inhibit GDNF-induced motility in BT474-AROM3 aromatase expressing cells. These data indicate that inhibiting RET is as effective as the current therapeutic regimen of AI therapy but that a combination treatment may delay cancer cell dissemination and metastasis.

Surprisingly high-pressure shocks in the supernova remnant IC 443

NASA Technical Reports Server (NTRS)

Moorhouse, A.; Brand, P. W. J. L.; Geballe, T. R.; Burton, M. G.

1991-01-01

The intensities of several lines of molecular hydrogen have been measured from two regions of the supernova-remnant/molecular-cloud shock in IC 443. The lines measured have upper-state energies ranging from 7000 K to 23,000 K. Their relative intensities differ in the two regions, but are consistent with those predicted from the post-shock regions of simple jump-type shocks of different pressure. The pressures so derived are far higher than the pressure in the supernova remnant itself, and a possible reason for this discrepancy is discussed.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor.

PubMed

Stahl, Sebastian; da Silva Mateus Seidl, Ana Rita; Ducret, Axel; Kux van Geijtenbeek, Sabine; Michel, Sven; Racek, Tomas; Birzele, Fabian; Haas, Alexander K; Rueger, Ruediger; Gerg, Michael; Niederfellner, Gerhard; Pastan, Ira; Brinkmann, Ulrich

2015-08-25

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

mTORC1 Inhibition Induces Resistance to Methotrexate and 6-Mercaptopurine in Ph+ and Ph-like B-ALL.

PubMed

Vo, Thanh-Trang T; Lee, J Scott; Nguyen, Duc; Lui, Brandon; Pandori, William; Khaw, Andrew; Mallya, Sharmila; Lu, Mengrou; Müschen, Markus; Konopleva, Marina; Fruman, David A

2017-09-01

Elevated activity of mTOR is associated with poor prognosis and higher incidence of relapse in B-cell acute lymphoblastic leukemia (B-ALL). Thus, ongoing clinical trials are testing mTOR inhibitors in combination with chemotherapy in B-ALL. However, the combination of mTOR inhibitors with standard of care chemotherapy drugs has not been studied extensively in high-risk B-ALL subtypes. Therefore, we tested whether mTOR inhibition can augment the efficacy of current chemotherapy agents in Ph + and Ph-like B-ALL models. Surprisingly, inhibiting mTOR complex 1 (mTORC1) protected B-ALL cells from killing by methotrexate and 6-mercaptopurine, two antimetabolite drugs used in maintenance chemotherapy. The cytoprotective effects correlated with decreased cell-cycle progression and were recapitulated using cell-cycle inhibitors, palbociclib or aphidicolin. Dasatinib, a tyrosine kinase inhibitor currently used in Ph + patients, inhibits ABL kinase upstream of mTOR. Dasatinib resistance is mainly caused by ABL kinase mutations, but is also observed in a subset of ABL unmutated cases. We identified dasatinib-resistant Ph+ cell lines and patient samples in which dasatinib can effectively reduce ABL kinase activity and mTORC1 signaling without causing cell death. In these cases, dasatinib protected leukemia cells from killing by 6-mercaptopurine. Using xenograft models, we observed that mTOR inhibition or dasatinib increased the numbers of leukemia cells that emerge after cessation of chemotherapy treatment. These results demonstrate that inhibitors targeting mTOR or upstream signaling nodes should be used with caution when combined with chemotherapeutic agents that rely on cell-cycle progression to kill B-ALL cells. Mol Cancer Ther; 16(9); 1942-53. ©2017 AACR . ©2017 American Association for Cancer Research.

Physical Conditions in Low Ionization Regions of the Orion Nebula

NASA Technical Reports Server (NTRS)

Baldwin, J. A.; Crotts, A.; DuFour, R. J.; Ferland, G. J.; Heathcote, S.; Hester, J. J.; Korista, K. T.; Martin, P. J.; ODell, C. R.

1996-01-01

We reexamine the spectroscopic underpinnings of recent claims that low ionization (O(I)) and (Fe(II)) lines from the Orion H(II) region are produced in a region where the iron-carrying grains have been destroyed and the electron density is surprisingly high. Our new HST and CTIO observations show that previous reported detections of(O(I)) lambda 5577 were strongly affected by telluric emission. Our line limits consistent with a moderate density (approx. 10(exp 4)/cu. cm photoionized gas. We show that a previously proposed model of the Orion H(II) region reproduces the observed (O(I)) and (Fe(II)) spectrum. These lines are fully consistent with formation in a moderate density dusty region.

Force Spectrum Microscopy Using Mitochondrial Fluctuations of Control and ATP-Depleted Cells.

PubMed

Xu, Wenlong; Alizadeh, Elaheh; Prasad, Ashok

2018-06-19

A single-cell assay of active and passive intracellular mechanical properties of mammalian cells could give significant insight into cellular processes. Force spectrum microscopy (FSM) is one such technique, which combines the spontaneous motion of probe particles and the mechanical properties of the cytoskeleton measured by active microrheology using optical tweezers to determine the force spectrum of the cytoskeleton. A simpler and noninvasive method to perform FSM would be very useful, enabling its widespread adoption. Here, we develop an alternative method of FSM using measurement of the fluctuating motion of mitochondria. Mitochondria of the C3H-10T1/2 cell line were labeled and tracked using confocal microscopy. Mitochondrial probes were selected based on morphological characteristics, and their mean-square displacement, creep compliance, and distributions of directional change were measured. We found that the creep compliance of mitochondria resembles that of particles in viscoelastic media. However, comparisons of creep compliance between controls and cells treated with pharmacological agents showed that perturbations to the actomysoin network had surprisingly small effects on mitochondrial fluctuations, whereas microtubule disruption and ATP depletion led to a significantly decreased creep compliance. We used properties of the distribution of directional change to identify a regime of thermally dominated fluctuations in ATP-depleted cells, allowing us to estimate the viscoelastic parameters for a range of timescales. We then determined the force spectrum by combining these viscoelastic properties with measurements of spontaneous fluctuations tracked in control cells. Comparisons with previous measurements made using FSM revealed an excellent match. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis

PubMed Central

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O.; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-01-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim–Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. PMID:28982759

Chemopreventive Effect of PSP Through Targeting of Prostate Cancer Stem Cell-Like Population

PubMed Central

Liu, Ji; Lee, Davy Tak-Wing; Chiu, Yung-Tuen; Ma, Stephanie; Ng, Irene Oi-Lin; Wong, Yong-Chuan; Chan, Franky Leung; Ling, Ming-Tat

2011-01-01

Recent evidence suggested that prostate cancer stem/progenitor cells (CSC) are responsible for cancer initiation as well as disease progression. Unfortunately, conventional therapies are only effective in targeting the more differentiated cancer cells and spare the CSCs. Here, we report that PSP, an active component extracted from the mushroom Turkey tail (also known as Coriolus versicolor), is effective in targeting prostate CSCs. We found that treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, PSP treatment not only suppressed the ability of PC-3 cells to form prostaspheres under non-adherent culture conditions, but also inhibited their tumorigenicity in vivo, further proving that PSP can suppress prostate CSC properties. To investigate if the anti-CSC effect of PSP may lead to prostate cancer chemoprevention, transgenic mice (TgMAP) that spontaneously develop prostate tumors were orally fed with PSP for 20 weeks. Whereas 100% of the mice that fed with water only developed prostate tumors at the end of experiment, no tumors could be found in any of the mice fed with PSP, suggesting that PSP treatment can completely inhibit prostate tumor formation. Our results not only demonstrated the intriguing anti-CSC effect of PSP, but also revealed, for the first time, the surprising chemopreventive property of oral PSP consumption against prostate cancer. PMID:21603625

C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

NASA Astrophysics Data System (ADS)

Russ, K. A.; Elvati, P.; Parsonage, T. L.; Dews, A.; Jarvis, J. A.; Ray, M.; Schneider, B.; Smith, P. J. S.; Williamson, P. T. F.; Violi, A.; Philbert, M. A.

2016-02-01

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07003a

VOLATILE-RICH CIRCUMSTELLAR GAS IN THE UNUSUAL 49 CETI DEBRIS DISK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roberge, Aki; Grady, Carol A.; Welsh, Barry Y.

2014-11-20

We present Hubble Space Telescope Space Telescope Imaging Spectrograph far-UV spectra of the edge-on disk around 49 Ceti, one of the very few debris disks showing submillimeter CO emission. Many atomic absorption lines are present in the spectra, most of which arise from circumstellar gas lying along the line-of-sight to the central star. We determined the line-of-sight C I column density, estimated the total carbon column density, and set limits on the O I column density. Surprisingly, no line-of-sight CO absorption was seen. We discuss possible explanations for this non-detection, and present preliminary estimates of the carbon abundances in themore » line-of-sight gas. The C/Fe ratio is much greater than the solar value, suggesting that 49 Cet harbors a volatile-rich gas disk similar to that of β Pictoris.« less

Competitive learning with pairwise constraints.

PubMed

Covões, Thiago F; Hruschka, Eduardo R; Ghosh, Joydeep

2013-01-01

Constrained clustering has been an active research topic since the last decade. Most studies focus on batch-mode algorithms. This brief introduces two algorithms for on-line constrained learning, named on-line linear constrained vector quantization error (O-LCVQE) and constrained rival penalized competitive learning (C-RPCL). The former is a variant of the LCVQE algorithm for on-line settings, whereas the latter is an adaptation of the (on-line) RPCL algorithm to deal with constrained clustering. The accuracy results--in terms of the normalized mutual information (NMI)--from experiments with nine datasets show that the partitions induced by O-LCVQE are competitive with those found by the (batch-mode) LCVQE. Compared with this formidable baseline algorithm, it is surprising that C-RPCL can provide better partitions (in terms of the NMI) for most of the datasets. Also, experiments on a large dataset show that on-line algorithms for constrained clustering can significantly reduce the computational time.

Human osteocalcin and bone sialoprotein mediating osteomimicry of prostate cancer cells: role of cAMP-dependent protein kinase A signaling pathway.

PubMed

Huang, Wen-Chin; Xie, Zhihui; Konaka, Hiroyuki; Sodek, Jaro; Zhau, Haiyen E; Chung, Leland W K

2005-03-15

Osteocalcin and bone sialoprotein are the most abundant noncollagenous bone matrix proteins expressed by osteoblasts. Surprisingly, osteocalcin and bone sialoprotein are also expressed by malignant but not normal prostate epithelial cells. The purpose of this study is to investigate how osteocalcin and bone sialoprotein expression is regulated in prostate cancer cells. Our investigation revealed that (a) human osteocalcin and bone sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b) Deletion analysis of human osteocalcin and bone sialoprotein promoter regions identified cyclic AMP (cAMP)-responsive elements (CRE) as the critical determinants for conditioned medium-mediated osteocalcin and bone sialoprotein gene expression in prostate cancer cells. Consistent with these results, the protein kinase A (PKA) pathway activators forskolin and dibutyryl cAMP and the PKA pathway inhibitor H-89, respectively, increased or repressed human osteocalcin and bone sialoprotein promoter activities. (c) Electrophoretic mobility shift assay showed that conditioned medium-mediated stimulation of human osteocalcin and bone sialoprotein promoter activities occurs through increased interaction between CRE and CRE-binding protein. (d) Conditioned medium was found to induce human osteocalcin and bone sialoprotein promoter activities via increased CRE/CRE-binding protein interaction in a cell background-dependent manner, with marked stimulation in selected prostate cancer but not bone stromal cells. Collectively, these results suggest that osteocalcin and bone sialoprotein expression is coordinated and regulated through cAMP-dependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.

Endogenous channels in HEK cells and potential roles in HCN ionic current measurements.

PubMed

Varghese, Anthony; Tenbroek, Erica M; Coles, James; Sigg, Daniel C

2006-01-01

A transformed line of human embryonic kidney epithelial cells (HEK 293) is commonly used as an expression system for exogenous ion channel genes. Previously, it has been shown that these cells contain mRNAs for a variety of ion channels. Expression of some of these genes has been confirmed at the protein level. Patch-clamp electrophysiology experiments confirm the presence of multiple ion channels and molecular data agree with pharmacological profiles of identified channels. In this work, we show that endogenous voltage-gated potassium channels in HEK cells are a significant source of outward current at positive potentials. We show that both non-transfected HEK cells and HEK cells transfected with hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have a significant amount of voltage-gated potassium (K(V)) current when certain tail current voltage-clamp protocols are used to assay HCN current activation. Specifically, tail current protocols that use a depolarized holding potential of -40 mV followed by hyperpolarizing pulses (-80 to -140 mV) and then a tail pulse potential of +20 mV indicate K(V) channels undergo closed-state inactivation at the more depolarized holding potential of -40 mV, followed by recovery from inactivation (but no activation) at hyperpolarizing potentials and high amount of activation at the positive tail potential. Our results indicate that pulse protocols with positive tail pulses are inaccurate assays for HCN current in certain HEK cells. Surprisingly, HEK-293 cells were found to contain mRNA for HCN2 and HCN3 although we have not detected a significant and consistent endogenous I(f)-like current in these cells.

Combining metabolic and process engineering strategies to improve recombinant glycoprotein production and quality.

PubMed

Karengera, Eric; Durocher, Yves; De Crescenzo, Gregory; Henry, Olivier

2017-11-01

Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon α2b (IFNα2b), were cultured in media deprived of glutamine but containing chosen substitutes. Among the tested substitutes, pyruvate led to the best improvement in growth (integral of viable cell density) for both cell lines in batch cultures, whereas the culture of PYC2 cells without neither glutamine nor any substitute displayed surprisingly enhanced IFNα2b production. The drastic reduction in both lactate and ammonia in the cultures translated into extended high viability conditions and an increase in recombinant protein titer by up to 47% for the parental cells and the PYC2 cells. Product characterization performed by surface plasmon resonance biosensing using Sambucus nigra (SNA) lectin revealed that the increase in yield was however accompanied by a reduction in the degree of sialylation of the product. Supplementing cultures with glycosylation precursors and a cofactor were effective at counterbalancing the lack of glutamine and allowed improvement in IFNα2b quality as evaluated by lectin affinity. Our study provides a strategy to reconcile protein productivity and quality and highlights the advantages of PYC2-overexpressing cells in glutamine-free conditions.

Morphometry, ultrastructure, myosin isoforms, and metabolic capacities of the "mini muscles" favoured by selection for high activity in house mice.

PubMed

Guderley, Helga; Houle-Leroy, Philippe; Diffee, Gary M; Camp, Dana M; Garland, Theodore

2006-07-01

Prolonged selective breeding of mice (Mus musculus) for high levels of voluntary wheel running has favoured an unusual phenotype ("mini muscles"), apparently caused by a single Mendelian recessive allele, in which most hind-limb muscles are markedly reduced in mass, but have increased mass-specific activities of mitochondrial enzymes. We examined whether these changes reflect changes in fibre size, number or ultrastructure in normal and "mini-muscle" mice within the two (of four) selectively bred lines (lab designations L3 and L6) that exhibit the phenotype at generations 26 and 27. In both lines, the gastrocnemius and plantaris muscles are smaller in mass (by >50% and 20%, respectively) in affected individuals. The mass-specific activities of mitochondrial enzymes in the gastrocnemius and plantaris muscles were increased in the mini phenotype in both lines, with stronger effects in the gastrocnemius muscle. In the gastrocnemius, the % myosin heavy chain (MHC) IIb was reduced by 50% in L3 and by 30% in L6, whereas the % MHC IIa and I were higher, particularly in L3. Fibre number in the plantaris muscle did not significantly differ between mini and normal muscles, although muscle mass was a significant positive correlate of fibre number. Small fibres were more abundant in mini than normal muscles in L3. Mitochondrial volume density was significantly higher in mini than normal muscle fibres in L3, but not in L6. Microscopy revealed a surprising attribute of the mini muscles: an abundance of small, minimally differentiated, myofibril-containing cells positioned in a disorderly fashion, particularly in the surface layer. We hypothesise that these unusual cells may be satellite cells or type IIb fibres that did not complete their differentiation. Together, these observations suggest that mice with the mini phenotype have reduced numbers of type IIb fibres in many of their hind-limb muscles, leading to a decrease in mass and an increase in mass-specific aerobic capacity in muscles that typically have a high proportion of type IIb fibres. Moreover, the several statistically significant interactions between muscle phenotype and line indicate that the effect of the underlying allele is altered by genetic background.

Safety, efficacy and efficiency of laser-assisted IVF in subfertile mutant mouse strains

PubMed Central

Li, Ming-Wen; Kinchen, Kristy L; Vallelunga, Jadine M; Young, Diana L; Wright, Kaleb D K; Gorano, Lisa N; Wasson, Katherine; Lloyd, K C Kent

2013-01-01

In the present report we studied the safety, efficacy and efficiency of using an infrared laser to facilitate IVF by assessing fertilization, development and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate four to ten times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved two-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen–thawed embryos derived by regular IVF. Surprisingly after surgical transfer to pseudopregnant recipients the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space. By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious and efficient assisted reproductive technology for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm–zona penetration defects. PMID:23315689

Plant Breeding: Surprisingly, Less Sex Is Better.

PubMed

van Dijk, Peter J; Rigola, Diana; Schauer, Stephen E

2016-02-08

Introduction of apomixis, asexual reproduction through seeds, into crop species has the potential to dramatically transform plant breeding. A new study demonstrates that traits can be stably transferred between generations in newly produced apomictic lines, and heralds a breeding revolution needed to increase food production for the growing planet. Copyright © 2016 Elsevier Ltd. All rights reserved.

The observed human sperm mutation frequency cannot explain the achondroplasia paternal age effect

PubMed Central

Tiemann-Boege, Irene; Navidi, William; Grewal, Raji; Cohn, Dan; Eskenazi, Brenda; Wyrobek, Andrew J.; Arnheim, Norman

2002-01-01

The lifelong spermatogonial stem cell divisions unique to male germ cell production are thought to contribute to a higher mutation frequency in males. The fact that certain de novo human genetic conditions (e.g., achondroplasia) increase in incidence with the age of the father is consistent with this idea. Although it is assumed that the paternal age effect is the result of an increasing frequency of mutant sperm as a man grows older, no direct molecular measurement of the germ-line mutation frequency has been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the nucleotide substitution in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. Surprisingly, the magnitude of the increase in mutation frequency with age appears insufficient to explain why older fathers have a greater chance of having a child with this condition. A number of alternatives may explain this discrepancy, including selection for sperm that carry the mutation or an age-dependent increase in premutagenic lesions that remain unrepaired in sperm and are inefficiently detected by the PCR assay. PMID:12397172

Wnt signaling potentiates nevogenesis

PubMed Central

Pawlikowski, Jeff S.; McBryan, Tony; van Tuyn, John; Drotar, Mark E.; Hewitt, Rachael N.; Maier, Andrea B.; King, Ayala; Blyth, Karen; Wu, Hong; Adams, Peter D.

2013-01-01

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway (senescence-associated secretory phenotype). Cellular senescence is also a tumor suppressor mechanism, to which both proliferation arrest and senescence-associated secretory phenotype are thought to contribute. The melanocytes within benign human nevi are a paradigm for tumor-suppressive senescent cells in a premalignant neoplasm. Here a comparison of proliferating and senescent melanocytes and melanoma cell lines by RNA sequencing emphasizes the importance of senescence-associated proliferation arrest in suppression of transformation. Previous studies showed that activation of the Wnt signaling pathway can delay or bypass senescence. Consistent with this, we present evidence that repression of Wnt signaling contributes to melanocyte senescence in vitro. Surprisingly, Wnt signaling is active in many senescent human melanocytes in nevi, and this is linked to histological indicators of higher proliferative and malignant potential. In a mouse, activated Wnt signaling delays senescence-associated proliferation arrest to expand the population of senescent oncogene-expressing melanocytes. These results suggest that Wnt signaling can potentiate nevogenesis in vivo by delaying senescence. Further, we suggest that activated Wnt signaling in human nevi undermines senescence-mediated tumor suppression and enhances the probability of malignancy. PMID:24043806

Wnt signaling potentiates nevogenesis.

PubMed

Pawlikowski, Jeff S; McBryan, Tony; van Tuyn, John; Drotar, Mark E; Hewitt, Rachael N; Maier, Andrea B; King, Ayala; Blyth, Karen; Wu, Hong; Adams, Peter D

2013-10-01

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway (senescence-associated secretory phenotype). Cellular senescence is also a tumor suppressor mechanism, to which both proliferation arrest and senescence-associated secretory phenotype are thought to contribute. The melanocytes within benign human nevi are a paradigm for tumor-suppressive senescent cells in a premalignant neoplasm. Here a comparison of proliferating and senescent melanocytes and melanoma cell lines by RNA sequencing emphasizes the importance of senescence-associated proliferation arrest in suppression of transformation. Previous studies showed that activation of the Wnt signaling pathway can delay or bypass senescence. Consistent with this, we present evidence that repression of Wnt signaling contributes to melanocyte senescence in vitro. Surprisingly, Wnt signaling is active in many senescent human melanocytes in nevi, and this is linked to histological indicators of higher proliferative and malignant potential. In a mouse, activated Wnt signaling delays senescence-associated proliferation arrest to expand the population of senescent oncogene-expressing melanocytes. These results suggest that Wnt signaling can potentiate nevogenesis in vivo by delaying senescence. Further, we suggest that activated Wnt signaling in human nevi undermines senescence-mediated tumor suppression and enhances the probability of malignancy.

Secretory pathway Ca2+ -ATPases promote in vitro microcalcifications in breast cancer cells.

PubMed

Dang, Donna; Prasad, Hari; Rao, Rajini

2017-11-01

Calcification of the breast is often an outward manifestation of underlying molecular changes that drive carcinogenesis. Up to 50% of all non-palpable breast tumors and 90% of ductal carcinoma in situ present with radiographically dense mineralization in mammographic scans. However, surprisingly little is known about the molecular pathways that lead to microcalcifications in the breast. Here, we report on a rapid and quantitative in vitro assay to monitor microcalcifications in breast cancer cell lines, including MCF7, MDA-MB-231, and Hs578T. We show that the Secretory Pathway Ca 2+ -ATPases SPCA1 and SPCA2 are strongly induced under osteogenic conditions that elicit microcalcifications. SPCA gene expression is significantly elevated in breast cancer subtypes that are associated with microcalcifications. Ectopic expression of SPCA genes drives microcalcifications and is dependent on pumping activity. Conversely, knockdown of SPCA expression significantly attenuates formation of microcalcifications. We propose that high levels of SPCA pumps may initiate mineralization in the secretory pathway by elevating luminal Ca 2+ . Our new findings offer mechanistic insight and functional implications on a widely observed, yet poorly understood radiographic signature of breast cancer. © 2017 Wiley Periodicals, Inc.

Msi2 Regulates the Aggressiveness of Non-Small Cell Lung Cancer (NSCLC)

DTIC Science & Technology

2016-12-01

Non-small cell lung cancer, invasion, metastasis, pro-invasive signaling, RNA binding proteins, Musashi, TGF-beta, epithelial mesenchymal transition...Non-small cell lung cancer, invasion, metastasis, pro-invasive signaling, RNA binding proteins, Musashi, TGF- beta, epithelial mesenchymal...NOTCH-1 RNA and protein expression in 344SQ and 531LN2 cells (NICD protein level was tested in 344SQ cells as well), Fig. 2 D-F. Surprisingly

Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang Shurong; Risques, Rosa Ana; Martin, George M.

2008-01-01

LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. Tomore » our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.« less

Plant cell wall: Never too much acetate

DOE PAGES

Scheller, Henrik V.

2017-03-03

Here, plant cell walls incorporate a variety of acetylated polysaccharides. In addition to enzymes catalysing acetylation (acetyltransferases), plants could produce enzymes to remove acetyl groups (acetylesterases). Previously, pectin acetylesterases were known and now a xylan acetylesterase has been identified — and it has many surprises.

Entrapment of curcumin into monoolein-based liquid crystalline nanoparticle dispersion for enhancement of stability and anticancer activity

PubMed Central

Baskaran, Rengarajan; Madheswaran, Thiagarajan; Sundaramoorthy, Pasupathi; Kim, Hwan Mook; Yoo, Bong Kyu

2014-01-01

Despite the promising anticancer potential of curcumin, its therapeutic application has been limited, owing to its poor solubility, bioavailability, and chemical fragility. Therefore, various formulation approaches have been attempted to address these problems. In this study, we entrapped curcumin into monoolein (MO)-based liquid crystalline nanoparticles (LCNs) and evaluated the physicochemical properties and anticancer activity of the LCN dispersion. The results revealed that particles in the curcumin-loaded LCN dispersion were discrete and monodispersed, and that the entrapment efficiency was almost 100%. The stability of curcumin in the dispersion was surprisingly enhanced (about 75% of the curcumin survived after 45 days of storage at 40°C), and the in vitro release of curcumin was sustained (10% or less over 15 days). Fluorescence-activated cell sorting (FACS) analysis using a human colon cancer cell line (HCT116) exhibited 99.1% fluorescence gating for 5 μM curcumin-loaded LCN dispersion compared to 1.36% for the same concentration of the drug in dimethyl sulfoxide (DMSO), indicating markedly enhanced cellular uptake. Consistent with the enhanced cellular uptake of curcumin-loaded LCNs, anticancer activity and cell cycle studies demonstrated apoptosis induction when the cells were treated with the LCN dispersion; however, there was neither noticeable cell death nor significant changes in the cell cycle for the same concentration of the drug in DMSO. In conclusion, entrapping curcumin into MO-based LCNs may provide, in the future, a strategy for overcoming the hurdles associated with both the stability and cellular uptake issues of the drug in the treatment of various cancers. PMID:25061290

Insulin-independent role of adiponectin receptor signaling in Drosophila germline stem cell maintenance.

PubMed

Laws, Kaitlin M; Sampson, Leesa L; Drummond-Barbosa, Daniela

2015-03-15

Adipocytes have key endocrine roles, mediated in large part by secreted protein hormones termed adipokines. The adipokine adiponectin is well known for its role in sensitizing peripheral tissues to insulin, and several lines of evidence suggest that adiponectin might also modulate stem cells/precursors. It remains unclear, however, how adiponectin signaling controls stem cells and whether this role is secondary to its insulin-sensitizing effects or distinct. Drosophila adipocytes also function as an endocrine organ and, although no obvious adiponectin homolog has been identified, Drosophila AdipoR encodes a well-conserved homolog of mammalian adiponectin receptors. Here, we generate a null AdipoR allele and use clonal analysis to demonstrate an intrinsic requirement for AdipoR in germline stem cell (GSC) maintenance in the Drosophila ovary. AdipoR null GSCs are not fully responsive to bone morphogenetic protein ligands from the niche and have a slight reduction in E-cadherin levels at the GSC-niche junction. Conversely, germline-specific overexpression of AdipoR inhibits natural GSC loss, suggesting that reduction in adiponectin signaling might contribute to the normal decline in GSC numbers observed over time in wild-type females. Surprisingly, AdipoR is not required for insulin sensitization of the germline, leading us to speculate that insulin sensitization is a more recently acquired function than stem cell regulation in the evolutionary history of adiponectin signaling. Our findings establish Drosophila female GSCs as a new system for future studies addressing the molecular mechanisms whereby adiponectin receptor signaling modulates stem cell fate. Copyright © 2015 Elsevier Inc. All rights reserved.

Entrapment of curcumin into monoolein-based liquid crystalline nanoparticle dispersion for enhancement of stability and anticancer activity.

PubMed

Baskaran, Rengarajan; Madheswaran, Thiagarajan; Sundaramoorthy, Pasupathi; Kim, Hwan Mook; Yoo, Bong Kyu

2014-01-01

Despite the promising anticancer potential of curcumin, its therapeutic application has been limited, owing to its poor solubility, bioavailability, and chemical fragility. Therefore, various formulation approaches have been attempted to address these problems. In this study, we entrapped curcumin into monoolein (MO)-based liquid crystalline nanoparticles (LCNs) and evaluated the physicochemical properties and anticancer activity of the LCN dispersion. The results revealed that particles in the curcumin-loaded LCN dispersion were discrete and monodispersed, and that the entrapment efficiency was almost 100%. The stability of curcumin in the dispersion was surprisingly enhanced (about 75% of the curcumin survived after 45 days of storage at 40°C), and the in vitro release of curcumin was sustained (10% or less over 15 days). Fluorescence-activated cell sorting (FACS) analysis using a human colon cancer cell line (HCT116) exhibited 99.1% fluorescence gating for 5 μM curcumin-loaded LCN dispersion compared to 1.36% for the same concentration of the drug in dimethyl sulfoxide (DMSO), indicating markedly enhanced cellular uptake. Consistent with the enhanced cellular uptake of curcumin-loaded LCNs, anticancer activity and cell cycle studies demonstrated apoptosis induction when the cells were treated with the LCN dispersion; however, there was neither noticeable cell death nor significant changes in the cell cycle for the same concentration of the drug in DMSO. In conclusion, entrapping curcumin into MO-based LCNs may provide, in the future, a strategy for overcoming the hurdles associated with both the stability and cellular uptake issues of the drug in the treatment of various cancers.

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-{kappa}B ligand (RANKL) expression in rheumatoid arthritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takeshita, Harunori; Kitano, Masayasu, E-mail: mkitano6@hyo-med.ac.jp; Iwasaki, Tsuyoshi

Highlights: Black-Right-Pointing-Pointer MH7A cells and CD4{sup +} T cells expressed S1P1 and RANKL. Black-Right-Pointing-Pointer S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells. Black-Right-Pointing-Pointer The effect of S1P in MH7A cells was inhibited by specific Gi/Go inhibitors. -- Abstract: Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-{kappa}B ligand (RANKL) in RA synoviocytes and CD4{sup +} T cells. We demonstrated MH7Amore » cells, a human RA synovial cell line, and CD4{sup +} T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4{sup +} T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-{alpha} in MH7A cells and CD4{sup +} T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4{sup +} T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.« less

hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3.

PubMed

Søndergaard, Helle Bach; Brodin, Birger; Nielsen, Carsten Uhd

2008-06-01

The purpose of this work was to investigate the apical uptake and transepithelial transport of Gly-Sar along with the expression of the di-/tripeptide transporters hPEPT1 and hPEPT2 in human Calu-3 bronchial epithelial cells. The apical Gly-Sar uptake rate in Calu-3 cells followed Michaelis-Menten kinetics with a Km value of 1.3 +/- 0.3 mM and a Vmax value of 0.60 +/- 0.06 nmol cm(-2) min(-1). Transepithelial apical to basolateral transport of 50 microM [3H]-labelled Gly-Sar across the Calu-3 cell monolayer was pH-dependent. The Gly-Sar flux was significantly reduced in the presence of delta-aminolevulinic acid (2.5 mM), cephalexin (25 mM), and captopril (25 mM; p < 0.05, n = 3). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of both hPEPT1 and hPEPT2 mRNA in the Calu-3 cells. These findings were confirmed in healthy human bronchial cDNA. Restriction-endonuclease analysis identified hPEPT2 in Calu-3 cells to be the hPEPT2*1 haplotype. Western blotting demonstrated expression of the hPEPT1 protein (approximately 80 kDa), and the immunolabel was mainly localized in the apical membrane as judged by immunolocalization studies using confocal laser scanning microscopy (CLSM). This work presents for the first time hPEPT1 and hPEPT2*1 expression in human Calu-3 cells. Surprisingly, the results indicate that Gly-Sar uptake and transport in Calu-3 cells are hPEPT1-mediated rather than hPEPT2-mediated.

A Distinctive Cytoplasmic Tail Contributes to Low Surface Expression and Intracellular Retention of the Patr-AL MHC class I molecule1

PubMed Central

Goyos, Ana; Guethlein, Lisbeth A.; Horowitz, Amir; Hilton, Hugo G.; Gleimer, Michael; Brodsky, Frances M.; Parham, Peter

2015-01-01

Chimpanzees have orthologs of the six, fixed, functional human MHC class I genes. But in addition, the chimpanzee has a seventh functional gene, Patr-AL, which is not polymorphic but contributes substantially to population diversity by its presence on only 50% of MHC haplotypes. The ancestral AL gene emerged long before the separation of human and chimpanzee ancestors and then subsequently and specifically lost function during human evolution, but was maintained in chimpanzees. Patr-AL is an alloantigen that participates in negative and positive selection of the T-cell repertoire. The three-dimensional structure and the peptide-binding repertoire of Patr-AL and HLA-A*02 are surprisingly similar. In contrast, the expression of these two molecules is very different as shown using specific monoclonal and polyclonal antibodies made against Patr-AL. Peripheral blood cells and B cell lines express low levels of Patr-AL at the cell surface. Higher levels are seen for 221-cell transfectants expressing Patr-AL, but in these cells a large majority of Patr-AL molecules are retained in the early compartments of the secretory pathway: mainly the endoplasmic reticulum but also cis-Golgi. Replacing the cytoplasmic tail of Patr-AL with that of HLA-A*02 increased the cell-surface expression of Patr-AL substantially. Four substitutions distinguish the Patr-AL and HLA-A*02 cytoplasmic tails. Systematic mutagenesis showed that each substitution contributes changes in cell-surface expression. The combination of residues present in Patr-AL appears unique, but each individual residue is present in other primate MHC class I molecules, notably MHC-E, the most ancient of the functional human MHC class I molecules. PMID:26371256

CLO: The cell line ontology

PubMed Central

2014-01-01

Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

Translational Genomics for the Improvement of Switchgrass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carpita, Nicholas; McCann, Maureen

2014-05-07

Our objectives were to apply bioinformatics and high throughput sequencing technologies to identify and classify the genes involved in cell wall formation in maize and switchgrass. Targets for genetic modification were to be identified and cell wall materials isolated and assayed for enhanced performance in bioprocessing. We annotated and assembled over 750 maize genes into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice, and Arabidopsis sequences revealed differences in gene family structure. In addition, differences in expression between gene family members of Arabidopsis, maize and rice underscored the need for a grass-specific genetic modelmore » for functional analyses. A forward screen of mature leaves of field-grown maize lines by near-infrared spectroscopy yielded several dozen lines with heritable spectroscopic phenotypes, several of which near-infrared (nir) mutants had altered carbohydrate-lignin compositions. Our contributions to the maize genome sequencing effort built on knowledge of copy number variation showing that uneven gene losses between duplicated regions were involved in returning an ancient allotetraploid to a genetically diploid state. For example, although about 25% of all duplicated genes remain genome-wide, all of the cellulose synthase (CesA) homologs were retained. We showed that guaiacyl and syringyl lignin in lignocellulosic cell-wall materials from stems demonstrate a two-fold natural variation in content across a population of maize Intermated B73 x Mo7 (IBM) recombinant inbred lines, a maize Association Panel of 282 inbreds and landraces, and three populations of the maize Nested Association Mapping (NAM) recombinant inbred lines grown in three years. We then defined quantitative trait loci (QTL) for stem lignin content measured using pyrolysis molecular-beam mass spectrometry, and glucose and xylose yield measured using an enzymatic hydrolysis assay. Among five multi-year QTL for lignin abundance, two for 4-vinylphenol abundance, and four for glucose and/or xylose yield, not a single QTL for aromatic abundance and sugar yield was shared. A genome-wide association study (GWAS) for lignin abundance and sugar yield of the 282-member maize Association Panel provided candidate genes in the eleven QTL and showed that many other alleles impacting these traits exist in the broader pool of maize genetic diversity. The maize B73 and Mo17 genotypes exhibited surprisingly large differences in gene expression in developing stem tissues, suggesting certain regulatory elements can significantly enhance activity of biomass synthesis pathways. Candidate genes, identified by GWAS or by differential expression, include genes of cell-wall metabolism, transcription factors associated with vascularization and fiber formation, and components of cellular signaling pathways. Our work provides new insights and strategies beyond modification of lignin to enhance yields of biofuels from genetically tailored biomass.« less

Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells

PubMed Central

Raghupathi, Ravinarayan; Duffield, Michael D; Zelkas, Leah; Meedeniya, Adrian; Brookes, Simon J H; Sia, Tiong Cheng; Wattchow, David A; Spencer, Nick J; Keating, Damien J

2013-01-01

The major source of serotonin (5-HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ∼95% of total body 5-HT, and that this 5-HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5-HT release from single primary EC cells. We have developed a rapid primary culture of guinea-pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca2+ imaging, immunocytochemistry and 3D modelling. Ca2+ enters EC cells upon stimulation and triggers quantal 5-HT release via L-type Ca2+ channels. Real time amperometric techniques reveal that EC cells release 5-HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5-HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5-HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three-dimensional (3D) simulations of 5-HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5-HT release in the whole-tissue milieu. These models indicate that local 5-HT levels are likely to be maintained around the activation threshold for mucosal 5-HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5-HT release in primary EC cells. The mode of 5-HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function. PMID:24099799

Ultra-relativistic Au+Au and d+Au collisions:

NASA Astrophysics Data System (ADS)

Back, B. B.; Baker, M. D.; Ballintijn, M.; Barton, D. S.; Betts, R. R.; Bickley, A. A.; Bindel, R.; Budzanowski, A.; Busza, W.; Carroll, A.; Chai, Z.; Decowski, M. P.; García, E.; Gburek, T.; George, N.; Gulbrandsen, K.; Gushue, S.; Halliwell, C.; Hamblen, J.; Hauer, M.; Heintzelman, G. A.; Henderson, C.; Hofman, D. J.; Hollis, R. S.; Hołyński, R.; Holzman, B.; Iordanova, A.; Johnson, E.; Kane, J. L.; Katzy, J.; Khan, N.; Kucewicz, W.; Kulinich, P.; Kuo, C. M.; Lin, W. T.; Manly, S.; McLeod, D.; Mignerey, A. C.; Nouicer, R.; Olszewski, A.; Pak, R.; Park, I. C.; Pernegger, H.; Reed, C.; Remsberg, L. P.; Reuter, M.; Roland, C.; Roland, G.; Rosenberg, L.; Sagerer, J.; Sarin, P.; Sawicki, P.; Seals, H.; Sedykh, I.; Skulski, W.; Smith, C. E.; Stankiewicz, M. A.; Steinberg, P.; Stephans, G. S. F.; Sukhanov, A.; Tang, J.-L.; Tonjes, M. B.; Trzupek, A.; Vale, C.; van Nieuwenhuizen, G. J.; Vaurynovich, S. S.; Verdier, R.; Veres, G. I.; Wenger, E.; Wolfs, F. L. H.; Wosiek, B.; Woźniak, K.; Wuosmaa, A. H.; Wysłouch, B.

In this talk I will review PHOBOS data on charged particle multiplicities, obtained in Au+Au and d+Au collisions at RHIC. The general features of the Au+Au pseudorapidity distributions results will be discussed and compared to those of /line{p}p collisions. The total charged particle multiplicity, scaled by the number of participant pairs, is observed to be about 40% higher in Au+Au collisions than in /line{p}p and d+Au systems, but, surprisingly at the same level of e+e- collisions. Limiting fragmentation scaling is seen to be obeyed in Au+Au collisions.

mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic TILLING approach.

PubMed

Acevedo-Garcia, Johanna; Spencer, David; Thieron, Hannah; Reinstädler, Anja; Hammond-Kosack, Kim; Phillips, Andrew L; Panstruga, Ralph

2017-03-01

Wheat is one of the most widely grown cereal crops in the world and is an important food grain source for humans. However, wheat yields can be reduced by many abiotic and biotic stress factors, including powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt). Generating resistant varieties is thus a major effort in plant breeding. Here, we took advantage of the non-transgenic Targeting Induced Lesions IN Genomes (TILLING) technology to select partial loss-of-function alleles of TaMlo, the orthologue of the barley Mlo (Mildew resistance locus o) gene. Natural and induced loss-of-function alleles (mlo) of barley Mlo are known to confer durable broad-spectrum powdery mildew resistance, typically at the expense of pleiotropic phenotypes such as premature leaf senescence. We identified 16 missense mutations in the three wheat TaMlo homoeologues, TaMlo-A1, TaMlo-B1 and TaMlo-D1 that each lead to single amino acid exchanges. Using transient gene expression assays in barley single cells, we functionally analysed the different missense mutants and identified the most promising candidates affecting powdery mildew susceptibility. By stacking of selected mutant alleles we generated four independent lines with non-conservative mutations in each of the three TaMlo homoeologues. Homozygous triple mutant lines and surprisingly also some of the homozygous double mutant lines showed enhanced, yet incomplete, Bgt resistance without the occurrence of discernible pleiotropic phenotypes. These lines thus represent an important step towards the production of commercial non-transgenic, powdery mildew-resistant bread wheat varieties. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Cyclotron resonance of interacting quantum Hall droplets

NASA Astrophysics Data System (ADS)

Widmann, M.; Merkt, U.; Cortés, M.; Häusler, W.; Eberl, K.

1998-06-01

The line shape and position of cyclotron resonance in gated GaAs/GaAlAs heterojunctions with δ-doped layers of negatively charged beryllium acceptors, that provide strong potential fluctuations in the channels of the quasi-two-dimensional electron systems, are examined. Specifically, the magnetic quantum limit is considered when the electrons are localized in separate quantum Hall droplets in the valleys of the disorder potential. A model treating disorder and electron-electron interaction on an equal footing accounts for all of the principal experimental findings: blue shifts from the unperturbed cyclotron frequency that decrease when the electron density is reduced, surprisingly narrow lines in the magnetic quantum limit, and asymmetric lines due to additional oscillator strength on their high-frequency sides.

Puzzling Out the Cell's Power Plant.

ERIC Educational Resources Information Center

Miller, Julie Ann

1979-01-01

The biological research, of Gottfried Schatz at the University of Basel and Gunter Blobel at Rockefeller University, which explains a mechanism by which mitochondrial proteins are transported across membranes is described. Results indicate that the construction and heredity of mitochondria have surprising differences from other cell processes. (BT)

Thyroid cell lines in research on goitrogenesis.

PubMed

Gerber, H; Peter, H J; Asmis, L; Studer, H

1991-12-01

Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

NASA Astrophysics Data System (ADS)

Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari

2017-05-01

The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

Who Gives and Who Gets: Why, when, and with Whom Young People Engage in Oral Sex

ERIC Educational Resources Information Center

Vannier, Sarah A.; O'Sullivan, Lucia F.

2012-01-01

Surprisingly little is known about oral sex experiences among emerging adults, including the motives behind their participation in this sexual activity. The current study examined the characteristics of emerging adults' most recent oral sex experience. A total of 431 young people (M age = 21.7 years; 71.7% female) completed an on-line survey…

Evaluating cell lines as tumour models by comparison of genomic profiles

PubMed Central

Domcke, Silvia; Sinha, Rileen; Levine, Douglas A.; Sander, Chris; Schultz, Nikolaus

2013-01-01

Cancer cell lines are frequently used as in vitro tumour models. Recent molecular profiles of hundreds of cell lines from The Cancer Cell Line Encyclopedia and thousands of tumour samples from the Cancer Genome Atlas now allow a systematic genomic comparison of cell lines and tumours. Here we analyse a panel of 47 ovarian cancer cell lines and identify those that have the highest genetic similarity to ovarian tumours. Our comparison of copy-number changes, mutations and mRNA expression profiles reveals pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and high-grade serous ovarian cancer tumour samples. We identify several rarely used cell lines that more closely resemble cognate tumour profiles than commonly used cell lines, and we propose these lines as the most suitable models of ovarian cancer. Our results indicate that the gap between cell lines and tumours can be bridged by genomically informed choices of cell line models for all tumour types. PMID:23839242

Estimating human cochlear tuning behaviorally via forward masking

NASA Astrophysics Data System (ADS)

Oxenham, Andrew J.; Kreft, Heather A.

2018-05-01

The cochlea is where sound vibrations are transduced into the initial neural code for hearing. Despite the intervening stages of auditory processing, a surprising number of auditory perceptual phenomena can be explained in terms of the cochlea's biomechanical transformations. The quest to relate perception to these transformations has a long and distinguished history. Given its long history, it is perhaps surprising that something as fundamental as the link between frequency tuning in the cochlea and perception remains a controversial and active topic of investigation. Here we review some recent developments in our understanding of the relationship between cochlear frequency tuning and behavioral measures of frequency selectivity in humans. We show that forward masking using the notched-noise technique can produce reliable estimates of tuning that are in line with predictions from stimulus frequency otoacoustic emissions.

Continuous hematopoietic cell lines as model systems for leukemia-lymphoma research.

PubMed

Drexler, H G; Matsuo, A Y; MacLeod, R A

2000-11-01

Along with other improvements, the advent of continuous human leukemia-lymphoma (LL) cell lines as a rich resource of abundant, accessible and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first LL cell lines, Burkitt's lymphoma-derived lines, were established in 1963. Since then, more than 1000 cell lines have been described, although not all of them in full detail. The major advantages of continuous cell lines is the unlimited supply and worldwide availability of identical cell material, and the infinite viable storability in liquid nitrogen. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and specific genetic alterations. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines must be discerned from Epstein-Barr virus (EBV)-immortalized normal cells, using various distinguishing parameters. However, the picture is not quite so straightforward, as some types of LL cell lines are indeed EBV+, and some EBV+ normal cell lines carry also genetic aberrations and may mimic malignancy-associated features. Apart from EBV and human T-cell leukemia virus in some lines, the majority of wild-type LL cell lines are virus-negative. The efficiency of cell line establishment is rather low and the deliberate establishment of new LL cell lines remains by and large an unpredictable random process. Difficulties in establishing continuous cell lines may be caused by the inappropriate selection of nutrients and growth factors for these cells. Clearly, a generally suitable microenvironment for hematopoietic cells, either malignant or normal, cannot yet be created in vitro. The characterization and publication of new LL cell lines should provide important and informative core data, attesting to their scientific significance. Large percentages of LL cell lines are contaminated with mycoplasma (about 30%) or are cross-contaminated with other cell lines (about 15-20%). Solutions to these problems are sensitive detection, effective elimination and rigorous prevention of mycoplasma infection, and proper, regular authentication of cell lines. The underlying cause, however, appears to be negligent cell culture practice. The willingness of investigators to make their LL cell lines available to others is all too often limited. There is a need in the scientific community for clean and authenticated high-quality LL cell lines to which every scientist has access. These are offered by various institutionalized public cell line banks. It has been argued that LL cell lines are genetically unstable (both cytogenetically and molecular genetically). For instance, cell lines are supposed to acquire numerical and structural chromosomal alterations and various types of mutations (e.g. point mutations) in vitro. We present evidence that while nearly 100% of all LL cell lines indeed carry genetic alterations, these alterations appear to be stable rather than unstable. As an example of the practical utility of LL cell lines, the recent advances in studies of classical and molecular cytogenetics, which in large part were made possible by cell lines, are highlighted. A list of the most useful, robust and publicly available reference cell lines that may be used for a variety of experimental purposes is proposed. Clearly, by opening new avenues for investigation, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia. Over a period of nearly four decades, these initially rather exotic cell cultures, known only to a few specialists, have become ubiquitous powerful research tools that are available to every investigator.

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

PubMed

de Angelis, Martin Hrabě; Nicholson, George; Selloum, Mohammed; White, Jacqui; Morgan, Hugh; Ramirez-Solis, Ramiro; Sorg, Tania; Wells, Sara; Fuchs, Helmut; Fray, Martin; Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl Mj; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie; Holmes, Chris; Steel, Karen P; Herault, Yann; Gailus-Durner, Valérie; Mallon, Ann-Marie; Brown, Steve Dm

2015-09-01

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.

Gene Expression in Bone

NASA Astrophysics Data System (ADS)

D'Ambrogio, A.

Skeletal system has two main functions, to provide mechanical integrity for both locomotion and protection and to play an important role in mineral homeostasis. There is extensive evidence showing loss of bone mass during long-term Space-Flights. The loss is due to a break in the equilibrium between the activity of osteoblasts (the cells that forms bone) and the activity of osteoclasts (the cells that resorbs bone). Surprisingly, there is scanty information about the possible altered gene expression occurring in cells that form bone in microgravity.(Just 69 articles result from a "gene expression in microgravity" MedLine query.) Gene-chip or microarray technology allows to screen thousands of genes at the same time: the use of this technology on samples coming from cells exposed to microgravity could provide us with many important informations. For example, the identification of the molecules or structures which are the first sensors of the mechanical stress derived from lack of gravity, could help in understanding which is the first event leading to bone loss due to long-term exposure to microgravity. Consequently, this structure could become a target for a custom-designed drug. It is evident that bone mass loss, observed during long-time stay in Space, represents an accelerated model of what happens in aging osteoporosis. Therefore, the discovery and design of drugs able to interfere with the bone-loss process, could help also in preventing negative physiological processes normally observed on Earth. Considering the aims stated above, my research is designed to:

Developmental Patterning: Putting the Squeeze on Mis-specified Cells.

PubMed

Nakajima, Yu-Ichiro; Gibson, Matthew C

2016-03-07

Widely implicated in human disease, abnormal cellular cysts reflect dramatic defects in the maintenance of epithelial integrity. A new study reports that epithelial cysts may arise as a surprisingly general consequence of clonal defects in the specification of cell identity. Copyright © 2016 Elsevier Ltd. All rights reserved.

TGF-β-Induced Transcription Sustains Amoeboid Melanoma Migration and Dissemination

PubMed Central

Cantelli, Gaia; Orgaz, Jose L.; Rodriguez-Hernandez, Irene; Karagiannis, Panagiotis; Maiques, Oscar; Matias-Guiu, Xavier; Nestle, Frank O.; Marti, Rosa M.; Karagiannis, Sophia N.; Sanz-Moreno, Victoria

2015-01-01

Summary Cell migration underlies metastatic dissemination of cancer cells, and fast “amoeboid” migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility. How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood. Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development. Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion. Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature. We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces. Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis. CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients. Its expression is also enriched in the invasive fronts of lesions. Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth. We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT. PMID:26526369

Radiosensitization of head/neck sqaumous cell carcinoma by adenovirus-mediated expression of the Nbs1 protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rhee, Juong G.; Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD; Li, Daqing

2007-01-01

Purpose: Local failure and toxicity to adjacent critical structures is a significant problem in radiation therapy of cancers of the head and neck. We are developing a gene therapy based method of sensitizing head/neck squamous cell carcinoma (HNSCC) to radiation treatment. As patients with the rare hereditary disorder, Nijmegen breakage syndrome, show radiation sensitivity we hypothesized that tumor-specific disruption of the function of the Nbs1 protein would lead to enhanced cellular sensitivity to ionizing radiation. Experimental Procedures: We constructed two recombinant adenoviruses by cloning the full-length Nbs1 cDNA as well as the C-terminal 300 amino acids of Nbs1 into anmore » adenovirus backbone under the control of a CMV promoter. The resulting adenoviruses were used to infect HNSCC cell line JHU011. These cells were evaluated for expression of the viral based constructs and assayed for clonogenic survival following radiation exposure. Results: Exposure of cells expressing Nbs1-300 to ionizing radiation resulted in a small reduction in survival relative to cells infected with control virus. Surprisingly, expression of full-length Nbs1 protein resulted in markedly enhanced sensitivity to ionizing radiation. Furthermore, the use of a fractionated radiation scheme following virus infection demonstrates that expression of full-length Nbs1 protein results in significant reduction in cell survival. Conclusions: These results provide a proof of principle that disruption of Nbs1 function may provide a means of enhancing the radiosensitivity of head and neck tumors. Additionally, this work highlights the Mre11 complex as an attractive target for development of radiation sensitizers.« less

Independent replication of mitochondrial genes supports the transcriptional program in developing fiber cells of cotton (Gossypium hirsutum L.).

PubMed

Thyssen, Gregory N; Song, Xianliang; Naoumkina, Marina; Kim, Hee-Jin; Fang, David D

2014-07-01

The mitochondrial genomes of flowering plants exist both as a "master circle" chromosome and as numerous subgenomic sublimons that are generated by intramolecular recombination. Differential stability or replication of these sublimons allows individual mitochondrial gene copy numbers to vary independently between different cell types and developmental stages. Our objective was to determine the relationship between mitochondrial gene copy number and transcript abundance in the elongating fiber cells of Upland cotton (Gossypium hirsutum L.). We compared RNA and DNA from cotton fiber cells at five developmental time points from early elongation through secondary cell wall thickening from the Ligon-lintless 2 (Li2) short fiber mutant and its wild type near isogenic line (NIL) DP5690. Mitochondrial gene copy number decreased from 3 to 8-DPA in the developing cotton fiber cells while transcript levels remained low. As secondary cell wall biosynthesis began in developing fibers, the expression levels and copy numbers of mitochondrial genes involved in energy production and respiration were up-regulated in wild type cotton DP5690. However, the short fiber mutant Li2, failed to increase expression of these genes, which include three subunits of ATP synthase, atp1, atp8 and atp9 and two cytochrome genes cox1 and cob. At the same time, Li2 failed to increase the copy numbers of these highly expressed genes. Surprisingly, we found that when mitochondrial genes were highly transcribed, they also had very high copy numbers. This observation suggests that in developing cotton fibers, increased mitochondrial sublimon replication may support increases in gene transcription. Published by Elsevier B.V.

An in vitro correlation of mechanical forces and metastatic capacity

NASA Astrophysics Data System (ADS)

Indra, Indrajyoti; Undyala, Vishnu; Kandow, Casey; Thirumurthi, Umadevi; Dembo, Micah; Beningo, Karen A.

2011-02-01

Mechanical forces have a major influence on cell migration and are predicted to significantly impact cancer metastasis, yet this idea is currently poorly defined. In this study we have asked if changes in traction stress and migratory properties correlate with the metastatic progression of tumor cells. For this purpose, four murine breast cancer cell lines derived from the same primary tumor, but possessing increasing metastatic capacity, were tested for adhesion strength, traction stress, focal adhesion organization and for differential migration rates in two-dimensional and three-dimensional environments. Using traction force microscopy (TFM), we were surprised to find an inverse relationship between traction stress and metastatic capacity, such that force production decreased as the metastatic capacity increased. Consistent with this observation, adhesion strength exhibited an identical profile to the traction data. A count of adhesions indicated a general reduction in the number as metastatic capacity increased but no difference in the maturation as determined by the ratio of nascent to mature adhesions. These changes correlated well with a reduction in active beta-1 integrin with increasing metastatic ability. Finally, in two dimensions, wound healing, migration and persistence were relatively low in the entire panel, maintaining a downward trend with increasing metastatic capacity. Why metastatic cells would migrate so poorly prompted us to ask if the loss of adhesive parameters in the most metastatic cells indicated a switch to a less adhesive mode of migration that would only be detected in a three-dimensional environment. Indeed, in three-dimensional migration assays, the most metastatic cells now showed the greatest linear speed. We conclude that traction stress, adhesion strength and rate of migration do indeed change as tumor cells progress in metastatic capacity and do so in a dimension-sensitive manner.

Characterization of prmt7alpha and beta isozymes from Chinese hamster cells sensitive and resistant to topoisomerase II inhibitors.

PubMed

Gros, Laurent; Renodon-Cornière, Axelle; de Saint Vincent, Bruno Robert; Feder, Marcin; Bujnicki, Janusz M; Jacquemin-Sablon, Alain

2006-11-01

By selection of genetic suppressor elements (GSEs) conferring resistance to topoisomerase II inhibitors in Chinese hamster cells (DC-3F), we identified a gene encoding two proteins of 78 and 82 kDa which belong to the protein arginine methyltransferase (PRMT) family. Down-regulation of these enzymes (named PRMT7alpha and beta), either induced by an antisense GSE or as observed in the 9-OH-ellipticine (9-OH-E) resistant mutant DC-3F/9-OH-E, was responsible for cell resistance to various DNA damaging agents. Alternative splicing alterations in the 5'-terminal region and changes of the polyadenylation site of PRMT7 mRNAs were observed in these resistant mutant cells. PRMT7alpha and beta are isoforms of a highly conserved protein containing two copies of a module common to all PRMTs, comprising a Rossmann-fold domain and a beta-barrel domain. The C-terminal repeat appears to be degenerate and catalytically inactive. PRMT7alpha and beta form homo- and hetero-dimers but differ by their sub-cellular localization and in vitro recognize different substrates. PRMT7beta was only observed in Chinese hamster cells while mouse 10T1/2 fibroblasts only contain PRMT7alpha. Surprisingly, in human cells the anti-PRMT7 antibody essentially recognized an approximately 37 kDa peptide, which is not formed during extraction, and a faint band at 78 kDa. Analysis of in vitro and in vivo methylation patterns in cell lines under- or over-expressing PRMT7alpha and beta detected a discrete number of proteins which methylation and/or expression are under the control of these enzymes.

Ontological representation, integration, and analysis of LINCS cell line cells and their cellular responses.

PubMed

Ong, Edison; Xie, Jiangan; Ni, Zhaohui; Liu, Qingping; Sarntivijai, Sirarat; Lin, Yu; Cooper, Daniel; Terryn, Raymond; Stathias, Vasileios; Chung, Caty; Schürer, Stephan; He, Yongqun

2017-12-21

Aiming to understand cellular responses to different perturbations, the NIH Common Fund Library of Integrated Network-based Cellular Signatures (LINCS) program involves many institutes and laboratories working on over a thousand cell lines. The community-based Cell Line Ontology (CLO) is selected as the default ontology for LINCS cell line representation and integration. CLO has consistently represented all 1097 LINCS cell lines and included information extracted from the LINCS Data Portal and ChEMBL. Using MCF 10A cell line cells as an example, we demonstrated how to ontologically model LINCS cellular signatures such as their non-tumorigenic epithelial cell type, three-dimensional growth, latrunculin-A-induced actin depolymerization and apoptosis, and cell line transfection. A CLO subset view of LINCS cell lines, named LINCS-CLOview, was generated to support systematic LINCS cell line analysis and queries. In summary, LINCS cell lines are currently associated with 43 cell types, 131 tissues and organs, and 121 cancer types. The LINCS-CLO view information can be queried using SPARQL scripts. CLO was used to support ontological representation, integration, and analysis of over a thousand LINCS cell line cells and their cellular responses.

CCMC Modeling of Magnetic Reconnection in Electron Diffusion Region Events

NASA Astrophysics Data System (ADS)

Marshall, A.; Reiff, P. H.; Daou, A.; Webster, J.; Sazykin, S. Y.; Kuznetsova, M.; Grocer, A.; Rastaetter, L.; Welling, D. T.; DeZeeuw, D.; Russell, C. T.

2017-12-01

We use the unprecedented spatial and temporal cadence of the Magnetospheric Multiscale Mission to study four electron diffusion events, and infer important physical properties of their respective magnetic reconnection processes. We couple these observations with numerical simulations using tools such as SWMF with RCM, and RECON-X, from the Coordinated Community Modeling Center, to provide, for a first time, a coherent temporal description of the magnetic reconnection process through tracing the coupling of IMF and closed Earth magnetic field lines, leading to the corresponding polar cap open field lines. We note that the reconnection geometry is far from slab-like: the IMF field lines drape over the magnetopause, lending to a stretching of the field lines. The stretched field lines become parallel to, and merge with the dayside separator. Surprisingly, the inner closed field lines also distort to become parallel to the separator. This parallel geometry allows a very sharp boundary between open and closed field lines. In three of the events, the MMS location was near the predicted separator location; in the fourth it was near the outflow region.

Nerve growth factor in the adult brain of a teleostean model for aging research: Nothobranchius furzeri.

PubMed

D'Angelo, L; Castaldo, L; Cellerino, A; de Girolamo, P; Lucini, C

2014-07-01

Nerve growth factor (NGF) acts on central nervous system neurons, regulating naturally occurring cell death, synaptic connectivity, fiber guidance and dendritic morphology. The dynamically regulated production of NGF beginning in development, extends throughout adult life and aging, exerting numerous roles through a surprising variety of neurons and glial cells. This study analyzes the localization of NGF in the brain of the teleost fish Nothobranchius furzeri, an emerging model for aging research due to its short lifespan. Immunochemical and immunohistochemical experiments were performed by employing an antibody mapping at the N-terminus of the mature chain human origin NGF. Western blot analysis revealed an intense and well defined band of 20 kDa, which corresponds to proNGF of N. furzeri. Immunohistochemistry revealed NGF immunoreactivity (IR) diffused throughout all regions of telencephalon, diencephalon, mesencephalon and rhomboencephalon. It was detected in neurons and in glial cells, the latter mostly lining the mesencephalic and rhomboencephalic ventricles. Particularly in neurons, NGF IR was localized in perikarya and, to a less extent, in fibers. The widespread distribution of proNGF suggests that it might modulate numerous physiological functions in the adult brain of N. furzeri. The present survey constitutes a baseline study to enhance the understanding of the mechanisms underlying the role of NGF during aging processes. Copyright © 2014 Elsevier GmbH. All rights reserved.

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

PubMed

Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

2017-03-01

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

The chemokine receptor CXCR4 is required for outgrowth of colon carcinoma micrometastases.

PubMed

Zeelenberg, Ingrid S; Ruuls-Van Stalle, Lisette; Roos, Ed

2003-07-01

CXCR4, the receptor for the chemokine stromal cell-derived factor (SDF)-1 (CXCL12), is involved in lymphocyte trafficking. We have demonstrated previously that it is required for invasion of lymphoma cells into tissues and therefore essential for lymphoma metastasis. CXCR4 is also expressed by carcinoma cells, and CXCR4 antibodies were recently shown to reduce metastasis of a mammary carcinoma cell line. This was also ascribed to impaired invasion. We have blocked CXCR4 function in CT-26 colon carcinoma cells by transfection of SDF-1, extended with a KDEL sequence. The SDF-KDEL protein is retained in the endoplasmic reticulum by the KDEL-receptor and binds CXCR4, which is thus prevented from reaching the cell surface. We found that metastasis of these cells to liver and lungs was greatly reduced and often completely blocked. Surprisingly, however, our observations indicate that this was not attributable to inhibition of invasion but rather to impairment of outgrowth of micrometastases: (a) in contrast to the lymphoma cells, metastasis was not affected by the transfected S1 subunit of pertussis toxin. S1 completely inhibited Gi protein signaling, which is required for SDF-1-induced invasion; (b) CXCR4 levels were very low in CT-26 cells grown in vitro but strongly up-regulated in vivo. Strong up-regulation was not seen in the lungs until 7 days after tail vein injection. CXCR4 can thus have no role in initial invasion in the lungs; and (c) CXCR4-deficient cells did colonize the lungs to the same extent as control cells and survived. However, they did not expand, whereas control cells proliferated rapidly after a lag period of > or = 7 days. We conclude that CXCR4 is up-regulated by the microenvironment and that isolated metastatic cells are likely to require CXCR4 signals to initiate proliferation. Our results suggest that CXCR4 inhibitors have potential as anticancer agents to suppress outgrowth of micrometastases.

Establishment and characterization of three immortal bovine muscular epithelial cell lines.

PubMed

Jin, Xun; Lee, Joong-Seob; Kwak, Sungwook; Lee, Soo-Yeon; Jung, Ji-Eun; Kim, Tae-Kyung; Xu, Chenxiong; Hong, Zhongshan; Li, Zhehu; Kim, Sun-Myung; Pian, Xumin; Lee, Dong-Hee; Yoon, Jong-Taek; You, Seungkwon; Choi, Yun-Jaie; Kim, Huunggee

2006-02-28

We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.

Investigation of the expression of RIF1 gene on head and neck, pancreatic and brain cancer and cancer stem cells.

PubMed

GursesCila, Hacer E; Acar, Muradiye; Barut, Furkan B; Gunduz, Mehmet; Grenman, Reidar; Gunduz, Esra

2016-12-01

Recent studies have shown that cancer stem cells are resistant to chemotherapy. The aim of this study was to compare RIF1 gene expression in head and neck, pancreatic cancer and glioma cell lines and the cancer stem cells isolated from these cell lines. UT-SCC-74 from Turku University and UT-SCC-74B primary tumor metastasis and neck cancer cell lines, YKG-1 glioma cancer cell line from RIKEN, pancreatic cancer cell lines and ASPC-1 cells from ATCC were grown in cell culture. To isolate cancer stem cells, ALDH-1 for UT-SCC-74 and UT-SCC-74B cell line, CD-133 for YKG-1 cell line and CD-24 for ASPC-1 cell line, were used as markers of cancer stem cells. RNA isolation was performed for both cancer lines and cancer stem cells. RNAs were converted to cDNA. RIF1 gene expression was performed by qRT-PCR analysis. RIF1 gene expression was compared with cancer cell lines and cancer stem cells isolated from these cell lines. The possible effect of RIF1 gene was evaluated. In the pancreatic cells, RIF1 gene expression in the stem cell-positive cell line was 256 time that seen in the stem cell-negative cell line. Considering the importance of RIF1 in NHEJ and of NHEJ in pancreatic cancer, RIF1 may be one of the genes that plays an important role in the diagnoses and therapeutic treatment of pancreatic cancer. The results of head and neck and brain cancers are inconclusive and further studies are required to elucidate the connection between RIF1 gene and these other types of cancers.

Mix-ups and mycoplasma: the enemies within.

PubMed

Drexler, Hans G; Uphoff, Cord C; Dirks, Willy G; MacLeod, Roderick A F

2002-04-01

Human leukemia-lymphoma (LL) cell lines represent important tools for experimental research. Among the various problems associated with cell lines, the two most common concern contaminations: (1) cross-contamination with unrelated cells and (2) contamination with microorganisms, in particular mycoplasma. The bad news is that about one-third of the cell lines are either cross-contaminated or mycoplasma-infected or both. The good news is that there are means to recognize and overcome these problems. In cases where, during attempts to establish new LL cell lines, primary LL cultures are cross-contaminated with continuous cell lines, intended new cell lines simply cannot be established ("early" cross-contamination). In cases of "late" cross-contamination of existing LL cell lines where the intrusive cells have a growth advantage, the original ("uncontaminated") cell lines may still be available elsewhere. DNA fingerprinting and cytogenetic analysis appear to be the most suitable approaches to detect cross-contaminations and to authenticate LL cell lines. A different but related aspect of "false" LL cell lines is the frequent misclassification of cell lines whereby the actual cell type of the cell line does not correspond to the purported model character of the cell line. Mycoplasma infection can have a multitude of effects on the eukaryotic cells which, due to the variety of infecting mycoplasma species and many other contributing parameters, cannot be predicted, rendering resulting data questionable at best. Practical procedures for the detection and elimination of mycoplasma contamination have been developed. Diagnostic and preventive strategies in order to hem the alarming increase in "false" and mycoplasma-positive LL cell lines are recommended.

Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

USDA-ARS?s Scientific Manuscript database

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

Preclinical investigation of ibrutinib, a Bruton's kinase tyrosine (Btk) inhibitor, in suppressing glioma tumorigenesis and stem cell phenotypes

PubMed Central

Wei, Li; Su, Yu-Kai; Lin, Chien-Min; Chao, Tsu-Yi; Huang, Shang-Pen; Huynh, Thanh-Tuan; Jan, Hsun-Jin; Whang-Peng, Jacqueline; Chiou, Jeng-Fong; Wu, Alexander T.H.; Hsiao, Michael

2016-01-01

Standard interventions for glioma include surgery, radiation and chemotherapies but the prognosis for malignant cases such as glioblastoma multiforme remain grim. Even with targeted therapeutic agent, bevacitumab, malignant glioma often develops resistance and recurrence. Thus, developing alternative interventions (therapeutic targets, biomarkers) is urgently required. Bruton's tyrosine kinase (Btk) has been long implicated in B cell malignancies but surprisingly it has recently been shown to also play a tumorigenic role in solid tumors such as ovarian and prostate cancer. Bioinformatics data indicates that Btk is significantly higher in clinical glioma samples as compared to normal brain cells and Btk expression level is associated with stage progression. This prompts us to investigate the potential role of Btk as a therapeutic target for glioma. Here, we demonstrate Btk expression is associated with GBM tumorigenesis. Down-regulation of Btk in GBM cell lines showed a significantly reduced abilities in colony formation, migration and GBM sphere-forming potential. Mechanistically, Btk-silenced cells showed a concomitant reduction in the expression of CD133 and Akt/mTOR signaling. In parallel, Ibrutinib (a Btk inhibitor) treatment led to a similar anti-tumorigenic response. Using xenograft mouse model, tumorigenesis was significantly reduced in Btk-silenced or ibrutinib-treated mice as compared to control counterparts. Finally, our glioma tissue microarray analysis indicated a higher Btk staining in the malignant tumors than less malignant and normal brain tissues. Collectively, Btk may represent a novel therapeutic target for glioma and ibrunitib may be used as an adjuvant treatment for malignant GBM. PMID:27564106

Preclinical investigation of ibrutinib, a Bruton's kinase tyrosine (Btk) inhibitor, in suppressing glioma tumorigenesis and stem cell phenotypes.

PubMed

Wei, Li; Su, Yu-Kai; Lin, Chien-Min; Chao, Tsu-Yi; Huang, Shang-Pen; Huynh, Thanh-Tuan; Jan, Hsun-Jin; Whang-Peng, Jacqueline; Chiou, Jeng-Fong; Wu, Alexander T H; Hsiao, Michael

2016-10-25

Standard interventions for glioma include surgery, radiation and chemotherapies but the prognosis for malignant cases such as glioblastoma multiforme remain grim. Even with targeted therapeutic agent, bevacitumab, malignant glioma often develops resistance and recurrence. Thus, developing alternative interventions (therapeutic targets, biomarkers) is urgently required. Bruton's tyrosine kinase (Btk) has been long implicated in B cell malignancies but surprisingly it has recently been shown to also play a tumorigenic role in solid tumors such as ovarian and prostate cancer. Bioinformatics data indicates that Btk is significantly higher in clinical glioma samples as compared to normal brain cells and Btk expression level is associated with stage progression. This prompts us to investigate the potential role of Btk as a therapeutic target for glioma. Here, we demonstrate Btk expression is associated with GBM tumorigenesis. Down-regulation of Btk in GBM cell lines showed a significantly reduced abilities in colony formation, migration and GBM sphere-forming potential. Mechanistically, Btk-silenced cells showed a concomitant reduction in the expression of CD133 and Akt/mTOR signaling. In parallel, Ibrutinib (a Btk inhibitor) treatment led to a similar anti-tumorigenic response. Using xenograft mouse model, tumorigenesis was significantly reduced in Btk-silenced or ibrutinib-treated mice as compared to control counterparts. Finally, our glioma tissue microarray analysis indicated a higher Btk staining in the malignant tumors than less malignant and normal brain tissues. Collectively, Btk may represent a novel therapeutic target for glioma and ibrunitib may be used as an adjuvant treatment for malignant GBM.

The zipper mechanism in phagocytosis: energetic requirements and variability in phagocytic cup shape

PubMed Central

2010-01-01

Background Phagocytosis is the fundamental cellular process by which eukaryotic cells bind and engulf particles by their cell membrane. Particle engulfment involves particle recognition by cell-surface receptors, signaling and remodeling of the actin cytoskeleton to guide the membrane around the particle in a zipper-like fashion. Despite the signaling complexity, phagocytosis also depends strongly on biophysical parameters, such as particle shape, and the need for actin-driven force generation remains poorly understood. Results Here, we propose a novel, three-dimensional and stochastic biophysical model of phagocytosis, and study the engulfment of particles of various sizes and shapes, including spiral and rod-shaped particles reminiscent of bacteria. Highly curved shapes are not taken up, in line with recent experimental results. Furthermore, we surprisingly find that even without actin-driven force generation, engulfment proceeds in a large regime of parameter values, albeit more slowly and with highly variable phagocytic cups. We experimentally confirm these predictions using fibroblasts, transfected with immunoreceptor FcγRIIa for engulfment of immunoglobulin G-opsonized particles. Specifically, we compare the wild-type receptor with a mutant receptor, unable to signal to the actin cytoskeleton. Based on the reconstruction of phagocytic cups from imaging data, we indeed show that cells are able to engulf small particles even without support from biological actin-driven processes. Conclusions This suggests that biochemical pathways render the evolutionary ancient process of phagocytic highly robust, allowing cells to engulf even very large particles. The particle-shape dependence of phagocytosis makes a systematic investigation of host-pathogen interactions and an efficient design of a vehicle for drug delivery possible. PMID:21059234

Identification of sites of STAT3 action in the female reproductive tract through conditional gene deletion.

PubMed

Robker, Rebecca L; Watson, Laura N; Robertson, Sarah A; Dunning, Kylie R; McLaughlin, Eileen A; Russell, Darryl L

2014-01-01

The STAT3 transcription factor is a pleiotropic transducer of signalling by hormones, growth factors and cytokines that has been identified in the female reproductive tract from oocytes and granulosa cells of the ovary to uterine epithelial and stromal cells. In the present study we used transgenic models to investigate the importance of STAT3 for reproductive performance in these different tissues. The Cre-LoxP system was used to delete STAT3 in oocytes by crossing Stat3fl/fl with Zp3-cre+ mice, or in ovarian granulosa cells and uterine stroma by crossing with Amhr2-Cre+ mice. Surprisingly, deletion of STAT3 in oocytes had no effect on fertility indicating that the abundance of STAT3 protein in maturing oocytes and fertilized zygotes is not essential to these developmental stages. In Stat3fl/fl;Amhr2-cre+ females impaired fertility was observed through significantly fewer litters and smaller litter size. Ovulation rate, oocyte fertilization and development to blastocyst were unaffected in this line; however, poor recombination efficiency in granulosa cells had yielded no net change in STAT3 protein abundance. In contrast, uteri from these mice showed STAT3 protein depletion selectively from the stomal compartment. A significant reduction in number of viable fetuses on gestational day 18, increased fetal resorptions and disrupted placental morphology were evident causes of the reduced fertility. In conclusion, this study defines an important role for STAT3 in uterine stromal cells during embryo implantation and the development of a functional placenta.

Hepatocyte growth factor sensitizes brain tumors to c-MET kinase inhibition

PubMed Central

Zhang, Ying; Farenholtz, Kaitlyn E.; Yang, Yanzhi; Guessous, Fadila; diPierro, Charles G.; Calvert, Valerie S.; Deng, Jianghong; Schiff, David; Xin, Wenjun; Lee, Jae K.; Purow, Benjamin; Christensen, James; Petricoin, Emanuel; Abounader, Roger

2013-01-01

Purpose The receptor tyrosine kinase (RTK) c-MET and its ligand hepatocyte growth factor (HGF) are deregulated and promote malignancy in cancer and brain tumors. Consequently, clinically applicable c-MET inhibitors have been developed. The purpose of this study was to investigate the not well known molecular determinants that predict responsiveness to c-MET inhibitors, and to explore new strategies for improving inhibitor efficacy in brain tumors. Experimental design We investigated the molecular factors and pathway activation signatures that determine sensitivity to c-MET inhibitors in a panel of glioblastoma and medulloblastoma cells, glioblastoma stem cells (GSCs), and established cell line-derived xenografts using functional assays, reverse protein microarrays, and in vivo tumor volume measurements, but validation with animal survival analyses remains to be done. We also explored new approaches for improving the efficacy of the inhibitors in vitro and in vivo. Results We found that HGF co-expression is a key predictor of response to c-MET inhibition among the examined factors, and identified an ERK/JAK/p53 pathway activation signature that differentiates c-MET inhibition in responsive and non-responsive cells. Surprisingly, we also found that short pre-treatment of cells and tumors with exogenous HGF moderately but statistically significantly enhanced the anti-tumor effects of c-MET inhibition. We observed a similar ligand-induced sensitization effect to an EGFR small molecule kinase inhibitor. Conclusions These findings allow the identification of a subset of patients that will be responsive to c-MET inhibition, and propose ligand pre-treatment as a potential new strategy for improving the anti-cancer efficacy of RTK inhibitors. PMID:23386689

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

PubMed

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-09-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-X L , recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-X L inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

New dualities and misleading anomaly matchings from outer-automorphism twists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pal, Sridip; Song, Jaewon

We study four-dimensional N=1, 2 superconformal theories in class S obtained by compactifying the 6d N=(2, 0) theory on a Riemann surface C with outer-automorphism twist lines. From the pair-of-pants decompositions of C, we find various dual descriptions for the same theory having distinct gauge groups. We show that the various configurations of the twist line give rise to dual descriptions for the identical theory. We compute the ’t Hooft anomaly coefficients and the superconformal indices to test dualities. Surprisingly, we find that the class S theories with twist lines wrapping 1-cycles of C have the identical ’t Hooft anomaliesmore » as the ones without the twist line, whereas the superconformal indices differ. As a result, this provides a large set of examples where the anomaly matching is insufficient to test dualities.« less

New dualities and misleading anomaly matchings from outer-automorphism twists

DOE PAGES

Pal, Sridip; Song, Jaewon

2017-03-29

We study four-dimensional N=1, 2 superconformal theories in class S obtained by compactifying the 6d N=(2, 0) theory on a Riemann surface C with outer-automorphism twist lines. From the pair-of-pants decompositions of C, we find various dual descriptions for the same theory having distinct gauge groups. We show that the various configurations of the twist line give rise to dual descriptions for the identical theory. We compute the ’t Hooft anomaly coefficients and the superconformal indices to test dualities. Surprisingly, we find that the class S theories with twist lines wrapping 1-cycles of C have the identical ’t Hooft anomaliesmore » as the ones without the twist line, whereas the superconformal indices differ. As a result, this provides a large set of examples where the anomaly matching is insufficient to test dualities.« less

Molecularly Targeted Dose-Enhancement Radiotherapy Using Gold and Luminescent Nanoparticles in an Orthotopic Human Prostate Cancer Rat Model

DTIC Science & Technology

2013-10-01

cell lines, such as cervix cancer cell line (HeLa) and breast cancer cell line (MDA-MB-231), were also employed. The experiments with other cell lines...breast cancer cell line (MDA-MB- 231), and cervix cancer cell line (HeLa). Different from our hypothesis, prostate cancer cell lines did not present...Radiotherapy Using Gold and Luminescent Nanoparticles in an Orthotopic Human Prostate Cancer Rat Model PRINCIPAL INVESTIGATOR: Kwang Song

[The factors involved in invasive ability of endometrial carcinoma cells].

PubMed

Mori, Y; Mizuuchi, H; Sato, K; Okamura, N; Kudo, R

1994-06-01

The in vitro invasive ability, the expression of cell adhesion molecule E-cadherin, activity of matrix metalloproteinase (MMP) and K-ras point mutation were investigated in eight human endometrial carcinoma cell lines. 1) In vitro invasive abilities of endometrial carcinoma cell lines depend on the degree of cell differentiation and the origin of cell lines. A poorly-differentiated carcinoma cell line (NUE-1) and a cell line derived from metastatic lymph node (SNG-M) were more invasive than moderately-(HEC-1A, HEC-1BE) and well-differentiated (HEC-6, Ishikawa) cell lines. 2) Immunohistochemically, less or non-invasive cell lines expressed E-cadherin strongly, whereas a highly invasive cell line (NUE-1) expressed E-cadherin weakly. 3) When cultured on Matrigel-coated dishes, the tumor cells derived from moderately- and well-differentiated carcinoma aggregated with each other and did not invade Matrigel in the invasion assay. The aggregated cells expressed E-cadherin more strongly when cultured on Matrigel. 4) 72-kD gelatinase (MMP-2) was secreted in serum-free conditioned medium of all cell lines. In an invasive cell line (NUE-1,SNG-M), the activity of MMP-2 was stronger than in other cell lines. And the activity of 92-kDa gelatinase (MMP-9) was detected in most invasive cell line (NUE-1). 5) Point mutation of K-ras codon 12 was detected in four of eight (50%) cell lines by the PCR-RFLP method. The changes in the DNA sequence were identified, but K-ras point mutation was not correlated with in vitro invasiveness of the tumor cells.

PEG-PE/clay composite carriers for doxorubicin: Effect of composite structure on release, cell interaction and cytotoxicity.

PubMed

Kohay, Hagay; Sarisozen, Can; Sawant, Rupa; Jhaveri, Aditi; Torchilin, Vladimir P; Mishael, Yael G

2017-06-01

A novel drug delivery system for doxorubicin (DOX), based on organic-inorganic composites was developed. DOX was incorporated in micelles (M-DOX) of polyethylene glycol-phosphatidylethanolamine (PEG-PE) which in turn were adsorbed by the clay, montmorillonite (MMT). The nano-structures of the PEG-PE/MMT composites of LOW and HIGH polymer loadings were characterized by XRD, TGA, FTIR, size (DLS) and zeta measurements. These measurements suggest that for the LOW composite a single layer of polymer intercalates in the clay platelets and the polymer only partially covers the external surface, while for the HIGH composite two layers of polymer intercalate and a bilayer may form on the external surface. These nanostructures have a direct effect on formulation stability and on the rate of DOX release. The release rate was reversely correlated with the degree of DOX interaction with the clay and followed the sequence: M-DOX>HIGH formulation>LOW formulation>DOX/MMT. Despite the slower release from the HIGH formulation, its cytotoxicity effect on sensitive cells was as high as the "free" DOX. Surprisingly, the LOW formulation, with the slowest release, demonstrated the highest cytotoxicity in the case of Adriamycin (ADR) resistant cells. Confocal microscopy images and association tests provided an insight into the contribution of formulation-cell interactions vs. the contribution of DOX release rate. Internalization of the formulations was suggested as a mechanism that increases DOX efficiency, particularly in the ADR resistant cell line. The employment of organic-inorganic hybrid materials as drug delivery systems, has not reached its full potential, however, its functionality as an efficient tunable release system was demonstrated. DOX PEG-PE/clay formulations were design as an efficient drug delivery system. The main aim was to develop PEG-PE/clay formulations of different structures based on various PEG-PE/clay ratios in order to achieve tunable release rates, to control the external surface characteristics and formulation stability. The formulations showed significantly higher toxicity in comparison to "free" DOX, explained by formulation internalization. For each cell line tested, sensitive and ADR resistant, a different formulation structure was found most efficient. The potential of PEG-PE/clay-DOX formulations to improve DOX administration efficacy was demonstrated and should be further explored and implemented for other cancer drugs and cells. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The Stem Cell Marker Lgr5 Defines a Subset of Postmitotic Neurons in the Olfactory Bulb.

PubMed

Yu, Yiqun; Moberly, Andrew H; Bhattarai, Janardhan P; Duan, Chen; Zheng, Qian; Li, Fangqi; Huang, Hugh; Olson, William; Luo, Wenqin; Wen, Tieqiao; Yu, Hongmeng; Ma, Minghong

2017-09-27

Lgr5, leucine-rich repeat-containing G-protein coupled receptor 5, is a bona fide biomarker for stem cells in multiple tissues. Lgr5 is also expressed in the brain, but the identities and properties of these Lgr5 + cells are still elusive. Using an Lgr5-EGFP reporter mouse line, we found that, from early development to adulthood, Lgr5 is highly expressed in the olfactory bulb (OB), an area with ongoing neurogenesis. Immunostaining with stem cell, glial, and neuronal markers reveals that Lgr5 does not label stem cells in the OB but instead labels a heterogeneous population of neurons with preference in certain subtypes. Patch-clamp recordings in OB slices reveal that Lgr5-EGFP + cells fire action potentials and display spontaneous excitatory postsynaptic events, indicating that these neurons are integrated into OB circuits. Interestingly, R-spondin 3, a potential ligand of Lgr5, is also expressed in the adult OB. Collectively, our data indicate that Lgr5-expressing cells in the OB are fully differentiated neurons and imply distinct roles of Lgr5 and its ligand in postmitotic cells. SIGNIFICANCE STATEMENT Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) is a bona fide stem cell marker in many body organs. Here we report that Lgr5 is also highly expressed in the olfactory bulb (OB), the first relay station in the brain for processing odor information and one of the few neural structures that undergo continuous neurogenesis. Surprisingly, Lgr5 is not expressed in the OB stem cells, but instead in a few subtypes of terminally differentiated neurons, which are incorporated into the OB circuit. This study reveals that Lgr5 + cells in the brain represent a nonstem cell lineage, implying distinct roles of Lgr5 in postmitotic neurons. Copyright © 2017 the authors 0270-6474/17/379403-12$15.00/0.

Very-Low-Density Lipoprotein (VLDL)-Producing and Hepatitis C Virus-Replicating HepG2 Cells Secrete No More Lipoviroparticles than VLDL-Deficient Huh7.5 Cells

PubMed Central

Jammart, Baptiste; Michelet, Maud; Pécheur, Eve-Isabelle; Parent, Romain; Bartosch, Birke; Zoulim, Fabien

2013-01-01

In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs. PMID:23427158

Very-low-density lipoprotein (VLDL)-producing and hepatitis C virus-replicating HepG2 cells secrete no more lipoviroparticles than VLDL-deficient Huh7.5 cells.

PubMed

Jammart, Baptiste; Michelet, Maud; Pécheur, Eve-Isabelle; Parent, Romain; Bartosch, Birke; Zoulim, Fabien; Durantel, David

2013-05-01

In the plasma samples of hepatitis C virus (HCV)-infected patients, lipoviroparticles (LVPs), defined as (very-) low-density viral particles immunoprecipitated with anti-β-lipoproteins antibodies are observed. This HCV-lipoprotein association has major implications with respect to our understanding of HCV assembly, secretion, and entry. However, cell culture-grown HCV (HCVcc) virions produced in Huh7 cells, which are deficient for very-low-density lipoprotein (VLDL) secretion, are only associated with and dependent on apolipoprotein E (apoE), not apolipoprotein B (apoB), for assembly and infectivity. In contrast to Huh7, HepG2 cells can be stimulated to produce VLDL by both oleic acid treatment and inhibition of the MEK/extracellular signal-regulated kinase (ERK) pathway but are not permissive for persistent HCV replication. Here, we developed a new HCV cell culture model to study the interaction between HCV and lipoproteins, based on engineered HepG2 cells stably replicating a blasticidin-tagged HCV JFH1 strain (JB). Control Huh7.5-JB as well as HepG2-JB cell lines persistently replicated viral RNA and expressed viral proteins with a subcellular colocalization of double-stranded RNA (dsRNA), core, gpE2, and NS5A compatible with virion assembly. The intracellular RNA replication level was increased in HepG2-JB cells upon dimethyl sulfoxide (DMSO) treatment, MEK/ERK inhibition, and NS5A overexpression to a level similar to that observed in Huh7.5-JB cells. Both cell culture systems produced infectious virions, which were surprisingly biophysically and biochemically similar. They floated at similar densities on gradients, contained mainly apoE but not apoB, and were not neutralized by anti-apoB antibodies. This suggests that there is no correlation between the ability of cells to simultaneously replicate HCV as well as secrete VLDL and their capacity to produce LVPs.

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

PubMed Central

McDermott, Martina; Eustace, Alex J.; Busschots, Steven; Breen, Laura; Crown, John; Clynes, Martin; O’Donovan, Norma; Stordal, Britta

2014-01-01

The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance. PMID:24639951

Immortal, telomerase-negative cell lines derived from a Li-Fraumeni syndrome patient exhibit telomere length variability and chromosomal and minisatellite instabilities.

PubMed

Tsutsui, Takeki; Kumakura, Shin-Ichi; Tamura, Yukiko; Tsutsui, Takeo W; Sekiguchi, Mizuki; Higuchi, Tokihiro; Barrett, J Carl

2003-05-01

Five immortal cell lines derived from a Li-Fraumeni syndrome patient (MDAH 087) with a germline mutant p53 allele were characterized with respect to telomere length and genomic instability. The remaining wild-type p53 allele is lost in the cell lines. Telomerase activity was undetectable in all immortal cell lines. Five subclones of each cell line and five re-subclones of each of the subclones also showed undetectable telomerase activity. All five immortal cell lines exhibited variability in the mean length of terminal restriction fragments (TRFs). Subclones of each cell line, and re-subclones of the subclones also showed TRF variability, indicating that the variability is owing to clonal heterogeneity. Chromosome aberrations were observed at high frequencies in these cell lines including the subclones and re-subclones, and the principal types of aberrations were breaks, double minute chromosomes and dicentric chromosomes. In addition, minisatellite instability detected by DNA fingerprints was observed in the immortal cell lines. However, all of the cell lines were negative for microsatellite instability. As minisatellite sequences are considered recombinogenic in mammalian cells, these results suggest that recombination rates can be increased in these cell lines. Tumor-derived human cell lines, HT1080 cells and HeLa cells that also lack p53 function, exhibited little genomic instability involving chromosomal and minisatellite instabilities, indicating that chromosomal and minisatellite instabilities observed in the immortal cell lines lacking telomerase activity could not result from loss of p53 function.

B7-H4 as a Target for Breast Cancer Immunotherapy

DTIC Science & Technology

2013-06-01

T cell proliferation isolated from peripheral blood of health donors. Surprisingly, however, our data could not reproduce the in vitro suppression...peripheral blood using magnetic cell sorting and incubated with anti-CD3/CD28 Dynabeads (Invitrogen) for 3 days. Cells were stained with B7-H4-Fc-APC... blood using magnetic isolation kits from Miltenyi Biotec and stained with CFSE. Cells are treated accordingly and measured for proliferation by

Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line.

PubMed

Lee, Suk Kyoo; Lee, Gyun Min

2003-06-30

Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 872-876, 2003.

The Human Microbiome and Skin and Soft-Tissue Infections

DTIC Science & Technology

2015-09-23

254   Cell harvesting, RNA extraction, and RT-PCR ...................................................... 257   Primer extension and 5...contribution of the human microbiota to human health is not surprising given that the number of human-associated bacterial cells far exceeds the 37.5 trillion... cells of the human body by a factor of ~10 (27; 266). Additionally, the human 13 Figure 1. Microbiome and disease-related PubMed

Leukemia-lymphoma cell lines as model systems for hematopoietic research.

PubMed

Drexler, Hans G; MacLeod, Roderick A F

2003-01-01

Continuous human leukemia-lymphoma (LL) cell lines comprise a rich self-renewing resource of accessible and manipulable living cells which has illuminated the pathophysiology of hematopoietic tumors as well as basic cell biology. The major key advantages of continuous cell lines are the unlimited supply and worldwide availability of identical cell material and their cryopreservation. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro with preservation of most cellular features, and specific genetic alterations. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines may be distinguished from Epstein-Barr virus (EBV)-immortalized normal cells, using various operational and conceptual parameters. The characterization and publication of new LL cell lines provides important and informative core data which, by opening new avenues for investigation, have become ubiquitous powerful research tools that are available to every investigator by reference cell repositories. There is a need in the scientific community for clean and authenticated LL cell lines to which every scientist has access as offered by these institutionalized public cell line banks. A list of the most useful, robust and freely available reference cell lines is proposed in this review. Clearly, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia.

Antigen-Specific CD8+ T Cells Fail To Respond to Shigella flexneri ▿

PubMed Central

Jehl, Stephanie P.; Doling, Amy M.; Giddings, Kara S.; Phalipon, Armelle; Sansonetti, Philippe J.; Goldberg, Marcia B.; Starnbach, Michael N.

2011-01-01

CD8+ T lymphocytes often play a primary role in adaptive immunity to cytosolic microbial pathogens. Surprisingly, CD8+ T cells are not required for protective immunity to the enteric pathogen Shigella flexneri, despite the ability of Shigella to actively secrete proteins into the host cytoplasm, a location from which antigenic peptides are processed for presentation to CD8+ T cells. To determine why CD8+ T cells fail to play a role in adaptive immunity to S. flexneri, we investigated whether antigen-specific CD8+ T cells are primed during infection but are unable to confer protection or, alternatively, whether T cells fail to be primed. To test whether Shigella is capable of stimulating an antigen-specific CD8+ T-cell response, we created an S. flexneri strain that constitutively secretes a viral CD8+ T-cell epitope via the Shigella type III secretion system and characterized the CD8+ T-cell response to this strain both in mice and in cultured cells. Surprisingly, no T cells specific for the viral epitope were stimulated in mice infected with this strain, and cells infected with the recombinant strain were not targeted by epitope-specific T cells. Additionally, we found that the usually robust T-cell response to antigens artificially introduced into the cytoplasm of cultured cells was significantly reduced when the antigen-presenting cell was infected with Shigella. Collectively, these results suggest that antigen-specific CD8+ T cells are not primed during S. flexneri infection and, as a result, afford little protection to the host during primary or subsequent infection. PMID:21357720

Lateral offsets on surveyed cultural features resulting from the 1999 İzmit and Düzce earthquakes, Turkey

USGS Publications Warehouse

Rockwell, Thomas K.; Lindvall, Scott; Dawson, Tim; Langridge, Rob; Lettis, William; Klinger, Yann

2002-01-01

Surveys of multiple tree lines within groves of poplar trees, planted in straight lines across the fault prior to the earthquake, show surprisingly large lateral variations. In one grove, slip increases by nearly 1.8 m, or 35% of the maximum measured value, over a lateral distance of nearly 100 m. This and other observations along the 1999 ruptures suggest that the lateral variability of slip observed from displaced geomorphic features in many earthquakes of the past may represent a combination of (1) actual differences in slip at the surface and (2) the difficulty in recognizing distributed nonbrittle deformation.

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation

PubMed Central

Naegeli, Kaleb M.; Chi, Qiuyi; Ziel, Joshua W.; Hagedorn, Elliott J.; Park, Jieun E.; Jayadev, Ranjay; Sherwood, David R.

2016-01-01

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue. PMID:26765257

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

PubMed

Lohmer, Lauren L; Clay, Matthew R; Naegeli, Kaleb M; Chi, Qiuyi; Ziel, Joshua W; Hagedorn, Elliott J; Park, Jieun E; Jayadev, Ranjay; Sherwood, David R

2016-01-01

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.

[Establishment of human embryonic stem cell lines and their therapeutic application].

PubMed

Suemori, Hirofumi

2004-03-01

Embryonic stem (ES) cell lines are pluripotent stem cell lines that can be propagated indefinitely in culture, retaining their potency to differentiate into every type of cell and tissue in the body. ES cell lines were first established from mouse blastocysts, and have been used for research in developmental biology. ES cells have been proven to be very valuable in the genetic modification of the mouse, especially in producing knockout mice. Since establishment of human ES cell lines was reported, their use in cell replacement therapies has been enthusiastically expected. There have been reports of the differentiation of several useful cell types from human ES cell lines, and clinical use of functional tissues and cells from human ES cells is anticipated. In Japan, there have also been many demands for the use of human ES cells in basic and pre-clinical research. We obtained governmental permission to establish human ES cell lines in April 2002 and started research using donated frozen embryos in January 2003. We successfully established three ES cell line from three blastocysts. These cell lines will be distributed at cost to researchers who have governmental permission to use human ES cells.

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

PubMed

Jadi, R S; Sudeep, A B; Kumar, Satyendra; Arankalle, V A; Mishra, A C

2010-08-01

Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID(50)) and indirect immunofluorescence assay (IFA). All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

Evaluation of the anti-adhesive effect of milk fat globule membrane glycoproteins on Helicobacter pylori in the human NCI-N87 cell line and C57BL/6 mouse model.

PubMed

Horemans, Tessa; Kerstens, Monique; Clais, Sofie; Struijs, Karin; van den Abbeele, Pieter; Van Assche, Tim; Maes, Louis; Cos, Paul

2012-08-01

 The interest in non-antibiotic therapies for Helicobacter pylori infections in man has considerably grown because increasing numbers of antibiotic-resistant strains are being reported. Intervention at the stage of bacterial attachment to the gastric mucosa could be an approach to improve the control/eradication rate of this infection.  Fractions of purified milk fat globule membrane glycoproteins were tested in vitro for their cytotoxic and direct antibacterial effect. The anti-adhesive effect on H. pylori was determined first in a cell model using the mucus-producing gastric epithelial cell line NCI-N87 and next in the C57BL/6 mouse model after dosing at 400 mg/kg protein once or twice daily from day -2 to day 4 post-infection. Bacterial loads were determined by using quantitative real-time PCR and the standard plate count method.  The milk fat globule membrane fractions did not show in vitro cytotoxicity, and a marginal antibacterial effect was demonstrated for defatted milk fat globule membrane at 256 μg/mL. In the anti-adhesion assay, the results varied from 56.0 ± 5.3% inhibition for 0.3% crude milk fat globule membrane to 79.3 ± 3.5% for defatted milk fat globule membrane. Quite surprisingly, in vivo administration of the same milk fat globule membrane fractions did not confirm the anti-adhesive effects and even caused an increase in bacterial load in the stomach.  The promising anti-adhesion in vitro results could not be confirmed in the mouse model, even after the highest attainable exposure. It is concluded that raw or defatted milk fat globule membrane fractions do not have any prophylactic or therapeutic potential against Helicobacter infection. © 2012 Blackwell Publishing Ltd.

The Wnt signaling antagonist Kremen1 is required for development of thymic architecture.

PubMed

Osada, Masako; Ito, Emi; Fermin, Hector A; Vazquez-Cintron, Edwin; Venkatesh, Tadmiri; Friedel, Roland H; Pezzano, Mark

2006-01-01

Wnt signaling has been reported to regulate thymocyte proliferation and selection at several stages during T cell ontogeny, as well as the expression of FoxN1 in thymic epithelial cells (TECs). Kremen1 (Krm1) is a negative regulator of the canonical Wnt signaling pathway, and functions together with the secreted Wnt inhibitor Dickkopf (Dkk) by competing for the lipoprotein receptor-related protein (LRP)-6 co-receptor for Wnts. Here krm1 knockout mice were used to examine krm1 expression in the thymus and its function in thymocyte and TEC development. Krm1 expression was detected in both cortical and medullary TEC subsets, as well as in immature thymocyte subsets, beginning at the CD25+CD44+ (DN2) stage and continuing until the CD4+CD8+(DP) stage. Neonatal mice show elevated expression of krm1 in all TEC subsets. krm1(-/-) mice exhibit a severe defect in thymic cortical architecture, including large epithelial free regions. Much of the epithelial component remains at an immature Keratin 5+ (K5) Keratin 8(+)(K8) stage, with a loss of defined cortical and medullary regions. A TOPFlash assay revealed a 2-fold increase in canonical Wnt signaling in TEC lines derived from krm1(-/-) mice, when compared with krm1(+/+) derived TEC lines. Fluorescence activated cell sorting (FACS) analysis of dissociated thymus revealed a reduced frequency of both cortical (BP1(+)EpCAM(+)) and medullary (UEA-1(+) EpCAM(hi)) epithelial subsets, within the krm1(-/-) thymus. Surprisingly, no change in thymus size, total thymocyte number or the frequency of thymocyte subsets was detected in krm1(-/-) mice. However, our data suggest that a loss of Krm1 leads to a severe defect in thymic architecture. Taken together, this study revealed a new role for Krm1 in proper development of thymic epithelium.

Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma.

PubMed

Drexler, H G; Matsuo, Y

2000-05-01

Malignancies involving natural killer (NK) cells are rare disorders. The complexity of NK cell-involving disorders has only recently been appreciated. Modern classifications discern immature (precursor) from mature NK cell leukemias-lymphomas. Continuous NK leukemia-lymphoma cell lines represent important model systems to study these neoplasms. While there are a number of putative NK cell lines which are, however, either not characterized, not immortalized, non-malignant, non-NK, or plain false cell lines, six bona fide malignant NK cell lines have been established and are sufficiently well characterized: HANK1, KHYG-1, NK-92, NKL, NK-YS and YT. Except for YT which was derived from a not further defined acute lymphoblastic lymphoma, these cell lines were established from patients with various NK cell malignancies. Five of the six cell lines are constitutively interleukin-2-dependent. Their immunoprofile is remarkably similar: CD1-, CD2+, surface CD3 (but cytoplasmic CD3epsilon+), CD4-, CD5-, CD7+, CD8-, CD16-, CD56+, CD57-, TCRalphabeta-, TCRgammadelta-, negative for B cell and myelomonocytic markers. The immunoglobulin heavy chain and T cell receptor genes are all in germline configuration. All six lines show complex chromosomal alterations, with both numerical and structural aberrations, attesting to their malignant and monoclonal nature. Functionally, these cells which contain azurophilic granules in their cytoplasm are nearly universally positive in NK activity assays. Three of five cell lines are Epstein-Barr virus-positive (type II latency). The composite data on these six cell lines allow for the operational definition of a typical malignant NK cell line profile. NK leukemia-lymphoma cell lines will prove invaluable for studies of normal and malignant NK cell biology.

Simultaneous sequencing of coding and noncoding RNA reveals a human transcriptome dominated by a small number of highly expressed noncoding genes.

PubMed

Boivin, Vincent; Deschamps-Francoeur, Gabrielle; Couture, Sonia; Nottingham, Ryan M; Bouchard-Bourelle, Philia; Lambowitz, Alan M; Scott, Michelle S; Abou-Elela, Sherif

2018-07-01

Comparing the abundance of one RNA molecule to another is crucial for understanding cellular functions but most sequencing techniques can target only specific subsets of RNA. In this study, we used a new fragmented ribodepleted TGIRT sequencing method that uses a thermostable group II intron reverse transcriptase (TGIRT) to generate a portrait of the human transcriptome depicting the quantitative relationship of all classes of nonribosomal RNA longer than 60 nt. Comparison between different sequencing methods indicated that FRT is more accurate in ranking both mRNA and noncoding RNA than viral reverse transcriptase-based sequencing methods, even those that specifically target these species. Measurements of RNA abundance in different cell lines using this method correlate with biochemical estimates, confirming tRNA as the most abundant nonribosomal RNA biotype. However, the single most abundant transcript is 7SL RNA, a component of the signal recognition particle. S tructured n on c oding RNAs (sncRNAs) associated with the same biological process are expressed at similar levels, with the exception of RNAs with multiple functions like U1 snRNA. In general, sncRNAs forming RNPs are hundreds to thousands of times more abundant than their mRNA counterparts. Surprisingly, only 50 sncRNA genes produce half of the non-rRNA transcripts detected in two different cell lines. Together the results indicate that the human transcriptome is dominated by a small number of highly expressed sncRNAs specializing in functions related to translation and splicing. © 2018 Boivin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Specific c-Jun target genes in malignant melanoma.

PubMed

Schummer, Patrick; Kuphal, Silke; Vardimon, Lily; Bosserhoff, Anja K; Kappelmann, Melanie

2016-05-03

A fundamental event in the development and progression of malignant melanoma is the de-regulation of cancer-relevant transcription factors. We recently showed that c-Jun is a main regulator of melanoma progression and, thus, is the most important member of the AP-1 transcription factor family in this disease. Surprisingly, no cancer-related specific c-Jun target genes in melanoma were described in the literature, so far. Therefore, we focused on pre-existing ChIP-Seq data (Encyclopedia of DNA Elements) of 3 different non-melanoma cell lines to screen direct c-Jun target genes. Here, a specific c-Jun antibody to immunoprecipitate the associated promoter DNA was used. Consequently, we identified 44 direct c-Jun targets and a detailed analysis of 6 selected genes confirmed their deregulation in malignant melanoma. The identified genes were differentially regulated comparing 4 melanoma cell lines and normal human melanocytes and we confirmed their c-Jun dependency. Direct interaction between c-Jun and the promoter/enhancer regions of the identified genes was confirmed by us via ChIP experiments. Interestingly, we revealed that the direct regulation of target gene expression via c-Jun can be independent of the existence of the classical AP-1 (5´-TGA(C/G)TCA-3´) consensus sequence allowing for the subsequent down- or up-regulation of the expression of these cancer-relevant genes. In summary, the results of this study indicate that c-Jun plays a crucial role in the development and progression of malignant melanoma via direct regulation of cancer-relevant target genes and that inhibition of direct c-Jun targets through inhibition of c-Jun is a potential novel therapeutic option for treatment of malignant melanoma.

Output-driven feedback system control platform optimizes combinatorial therapy of tuberculosis using a macrophage cell culture model.

PubMed

Silva, Aleidy; Lee, Bai-Yu; Clemens, Daniel L; Kee, Theodore; Ding, Xianting; Ho, Chih-Ming; Horwitz, Marcus A

2016-04-12

Tuberculosis (TB) remains a major global public health problem, and improved treatments are needed to shorten duration of therapy, decrease disease burden, improve compliance, and combat emergence of drug resistance. Ideally, the most effective regimen would be identified by a systematic and comprehensive combinatorial search of large numbers of TB drugs. However, optimization of regimens by standard methods is challenging, especially as the number of drugs increases, because of the extremely large number of drug-dose combinations requiring testing. Herein, we used an optimization platform, feedback system control (FSC) methodology, to identify improved drug-dose combinations for TB treatment using a fluorescence-based human macrophage cell culture model of TB, in which macrophages are infected with isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible green fluorescent protein (GFP)-expressing Mycobacterium tuberculosis (Mtb). On the basis of only a single screening test and three iterations, we identified highly efficacious three- and four-drug combinations. To verify the efficacy of these combinations, we further evaluated them using a methodologically independent assay for intramacrophage killing of Mtb; the optimized combinations showed greater efficacy than the current standard TB drug regimen. Surprisingly, all top three- and four-drug optimized regimens included the third-line drug clofazimine, and none included the first-line drugs isoniazid and rifampin, which had insignificant or antagonistic impacts on efficacy. Because top regimens also did not include a fluoroquinolone or aminoglycoside, they are potentially of use for treating many cases of multidrug- and extensively drug-resistant TB. Our study shows the power of an FSC platform to identify promising previously unidentified drug-dose combinations for treatment of TB.

High-Resolution Array CGH Profiling Identifies Na/K Transporting ATPase Interacting 2 (NKAIN2) as a Predisposing Candidate Gene in Neuroblastoma

PubMed Central

Romania, Paolo; Castellano, Aurora; Surace, Cecilia; Citti, Arianna; De Ioris, Maria Antonietta; Sirleto, Pietro; De Mariano, Marilena; Longo, Luca; Boldrini, Renata; Angioni, Adriano; Locatelli, Franco; Fruci, Doriana

2013-01-01

Neuroblastoma (NB), the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the ALK and PHOX2B genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an ALK germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the PHOX2B gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH) analysis revealed a germline gain at NKAIN2 (Na/K transporting ATPase interacting 2) locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation. PMID:24205241

High-resolution array CGH profiling identifies Na/K transporting ATPase interacting 2 (NKAIN2) as a predisposing candidate gene in neuroblastoma.

PubMed

Romania, Paolo; Castellano, Aurora; Surace, Cecilia; Citti, Arianna; De Ioris, Maria Antonietta; Sirleto, Pietro; De Mariano, Marilena; Longo, Luca; Boldrini, Renata; Angioni, Adriano; Locatelli, Franco; Fruci, Doriana

2013-01-01

Neuroblastoma (NB), the most common solid cancer in early childhood, usually occurs sporadically but also its familial occurance is known in 1-2% of NB patients. Germline mutations in the ALK and PHOX2B genes have been found in a subset of familial NBs. However, because some individuals harbouring mutations in these genes do not develop this tumor, additional genetic alterations appear to be required for NB pathogenesis. Herein, we studied an Italian family with three NB patients, two siblings and a first cousin, carrying an ALK germline-activating mutation R1192P, that was inherited from their unaffected mothers and with no mutations in the PHOX2B gene. A comparison between somatic and germline DNA copy number changes in the two affected siblings by a high resolution array-based Comparative Genomic Hybridization (CGH) analysis revealed a germline gain at NKAIN2 (Na/K transporting ATPase interacting 2) locus in one of the sibling, that was inherited from the parent who does not carry the ALK mutation. Surprisingly, NKAIN2 was expressed at high levels also in the affected sibling that lacks the genomic gain at this locus, clearly suggesting the existance of other regulatory mechanisms. High levels of NKAIN2 were detected in the MYCN-amplified NB cell lines and in the most aggressive NB lesions as well as in the peripheral blood of a large cohort of NB patients. Consistent with a role of NKAIN2 in NB development, NKAIN2 was down-regulated during all-trans retinoic acid differentiation in two NB cell lines. Taken together, these data indicate a potential role of NKAIN2 gene in NB growth and differentiation.

Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells.

PubMed

Shi, Cheng; Shen, Huan; Jiang, Wei; Song, Zhi-Hua; Wang, Cheng-Yan; Wei, Li-Hui

2011-04-01

Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability. Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propagate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells. We generated a new Chinese human embryonic stem cells line, CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines: normal morphology, karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line. This newly established Chinese cell line, CH1, which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells, will provide a useful tool for embryo development research, along with clinical treatments for diabetes and some hepatic diseases.

77 FR 5489 - Identification of Human Cell Lines Project

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-03

...-01] Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology... cell line samples as part of the Identification of Human Cell Lines Project. All data and corresponding... cell lines accepted on the NIST Applied Genetics Group Web site at http://www.nist.gov/mml/biochemical...

Analysis of rubella virus capsid protein-mediated enhancement of replicon replication and mutant rescue.

PubMed

Tzeng, Wen-Pin; Matthews, Jason D; Frey, Teryl K

2006-04-01

The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed deltaNotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (Cdelta8-Vero cells), a construct previously shown to be unable to complement DeltaNotI. In C-Vero cells but not Vero or Cdelta8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with deltaNotI (RUBrep/GFP-deltaNotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5' and 3' cis-acting elements were also rescued in C-Vero cells. Interestingly, the Cdelta8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in Cdelta8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.

Development and characterization of two new cell lines from milkfish (Chanos chanos) and grouper (Epinephelus coioides) for virus isolation.

PubMed

Parameswaran, V; Ishaq Ahmed, V P; Shukla, Ravi; Bhonde, R R; Sahul Hameed, A S

2007-01-01

Two new cell lines, SIMH and SIGE, were derived from the heart of milkfish (Chanos chanos), a euryhaline teleost, and from the eye of grouper (Epinephelus coioides), respectively. These cell lines were maintained in Leibovitz's L-15 supplemented with 20% fetal bovine serum (FBS). The SIMH cell line was subcultured more than 50 times over a period of 210 days and SIGE cell line has been subcultured 100 times over a period of 1 1/2 years. The SIMH cell line consists predominantly of fibroblastic-like cells. The SIGE cell line consists predominantly of epithelial cells. Both the cell lines were able to grow at temperatures between 25 and 32 degrees C with an optimum temperature of 28 degrees C. The growth rate of these cells increased as the proportion of FBS increased from 2% to 20% at 28 degrees C with optimum growth at the concentrations of 15% or 20% FBS. Seven marine fish viruses were tested to determine the susceptibility of these cell lines. The SIGE cell line was found to be susceptible to nodavirus, MABV NC-1 and Y6, and the infection was confirmed by cytopathic effect (CPE) and reverse transcriptase-polymerase chain reaction. When these cells were transfected with pEGFP-N1 vector DNA, significant fluorescent signals were observed, suggesting that these cell lines can be a useful tool for transgenic and genetic manipulation studies. Further, these cell lines are characterized by immunocytochemistry using confocal laser scanning microscopy (CFLSM).

Authentication of M14 melanoma cell line proves misidentification of MDA‐MB‐435 breast cancer cell line

PubMed Central

Korch, Christopher; Hall, Erin M.; Dirks, Wilhelm G.; Ewing, Margaret; Faries, Mark; Varella‐Garcia, Marileila; Robinson, Steven; Storts, Douglas; Turner, Jacqueline A.; Wang, Ying; Burnett, Edward C.; Healy, Lyn; Kniss, Douglas; Neve, Richard M.; Nims, Raymond W.; Reid, Yvonne A.; Robinson, William A.

2017-01-01

A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research. PMID:28940260

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling.

PubMed

Povey, Jane F; O'Malley, Christopher J; Root, Tracy; Martin, Elaine B; Montague, Gary A; Feary, Marc; Trim, Carol; Lang, Dietmar A; Alldread, Richard; Racher, Andrew J; Smales, C Mark

2014-08-20

Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10L bioreactor model of Lonza's large-scale (up to 20,000L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines' performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements. Copyright © 2014 Elsevier B.V. All rights reserved.

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

DOE Office of Scientific and Technical Information (OSTI.GOV)

Medina, D.; Oborn, C.J.; Li, M.L.

1987-09-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appearedmore » to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.« less

EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step

PubMed Central

Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki; Kamiya, Yukiko; Kato, Koichi; Horimoto, Satoshi; Ishikawa, Tokiro; Takeda, Shunichi; Sakuma, Tetsushi; Yamamoto, Takashi

2014-01-01

Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) in which Htm1-mediated mannose trimming from the oligosaccharide Man8GlcNAc2 to Man7GlcNAc2 is the rate-limiting step in yeast. In contrast, the roles of the three Htm1 homologues (EDEM1/2/3) in mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or as lectins. We therefore conducted transcription activator-like effector nuclease–mediated gene knockout analysis in human cell line and found that all endogenous EDEMs possess mannosidase activity. Mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 is performed mainly by EDEM3 and to a lesser extent by EDEM1. Most surprisingly, the upstream mannose trimming from Man9GlcNAc2 to Man8GlcNAc2 is conducted mainly by EDEM2, which was previously considered to lack enzymatic activity. Based on the presence of two rate-limiting steps in mammalian gpERAD, we propose that mammalian cells double check gpERAD substrates before destruction by evolving EDEM2, a novel-type Htm1 homologue that catalyzes the first mannose trimming step from Man9GlcNAc2. PMID:25092655

Molecular and Bioenergetic Differences between Cells with African versus European Inherited Mitochondrial DNA Haplogroups: Implications for Population Susceptibility to Diseases

PubMed Central

Kenney, M. Cristina; Chwa, Marilyn; Atilano, Shari R.; Falatoonzadeh, Payam; Ramirez, Claudio; Malik, Deepika; Tarek, Mohamed; Cáceres del Carpio, Javier; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Vawter, Marquis P.; Jazwinski, S. Michal; Miceli, Michael V.; Wallace, Douglas C.; Udar, Nitin

2015-01-01

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP turnover rates and lower levels of ROS production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases. PMID:24200652

Regulation by CD45 of the tyrosine phosphorylation of high affinity IgE receptor beta- and gamma-chains.

PubMed

Adamczewski, M; Numerof, R P; Koretzky, G A; Kinet, J P

1995-04-01

Previous studies using tyrosine phosphatase inhibitors have implicated tyrosine phosphatases in the signal transduction pathway initiated by aggregation of Fc epsilon RI, the high affinity receptor for IgE. To define more precisely a role for the tyrosine phosphatase CD45 in Fc epsilon RI-mediated signaling, we have transfected the three subunits of Fc epsilon RI into wild-type Jurkat and a CD45-deficient Jurkat derivative. Here we demonstrate that CD45 is necessary for the initiation of calcium flux through the transfected Fc epsilon RI. In contrast to the effect of phosphatase inhibitors, the tyrosine phosphorylation levels of beta and gamma after aggregation of Fc epsilon RI are surprisingly reduced, relative to wild-type Jurkat, in the CD45-deficient cells. After reconstitution of the CD45-deficient cells with a chimeric molecule containing the cytoplasmic phosphatase domains of CD45, both the base line and activation-induced tyrosine phosphorylation levels are increased. By examining Lck autophosphorylation, we find that Fc epsilon RI aggregation induces an increase in Lck enzymatic activity only in wild-type Jurkat and the CD45-deficient Jurkat reconstituted with chimeric CD45. This regulation of src-family tyrosine kinase activity may be the means by which CD45 controls aggregation-induced receptor phosphorylation.

Antitumor activity of JS-K [O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] and related O2-aryl diazeniumdiolates in vitro and in vivo.

PubMed

Shami, Paul J; Saavedra, Joseph E; Bonifant, Challice L; Chu, Jingxi; Udupi, Vidya; Malaviya, Swati; Carr, Brian I; Kar, Siddhartha; Wang, Meifeng; Jia, Lee; Ji, Xinhua; Keefer, Larry K

2006-07-13

The literature provides evidence that metabolic nitric oxide (NO) release mediates the cytotoxic activities (against human leukemia and prostate cancer xenografts in mice) of JS-K, a compound of structure R(2)N-N(O)=NO-Ar for which R(2)N is 4-(ethoxycarbonyl)piperazin-1-yl and Ar is 2,4-dinitrophenyl. Here we present comparative data on the potencies of JS-K and 41 other O(2)-arylated diazeniumdiolates as inhibitors of HL-60 human leukemia cell proliferation, as well as in the NCI 51-cell-line screen for six of them. The data show JS-K to be the most potent of the 42 in both screens and suggest that other features of its structure and metabolism besides NO release may contribute importantly to its activity. Results with control compounds implicate JS-K's arylating ability, and the surprisingly low IC(50) value of the N-(ethoxycarbonyl)piperazine byproduct of NO release suggests a role for the R(2)N moiety. In addition to the above-mentioned in vivo activities, JS-K is shown here to be carcinostatic in a rat liver cancer model.

Adventitious viruses in insect cell lines used for recombinant protein expression.

PubMed

Geisler, Christoph; Jarvis, Donald L

2018-04-01

Insect cells are widely used for recombinant protein expression, typically as hosts for recombinant baculovirus vectors, but also for plasmid-mediated transient transfection or stable genetic transformation. Insect cells are used to express proteins for research, as well as to manufacture biologicals for human and veterinary medicine. Recently, several insect cell lines used for recombinant protein expression were found to be persistently infected with adventitious viruses. This has raised questions about how these infections might affect research performed using those cell lines. Furthermore, these findings raised serious concerns about the safety of biologicals produced using those cell lines. In response, new insect cell lines lacking adventitious viruses have been isolated for use as improved research tools and safer biological manufacturing platforms. Here, we review the scientific and patent literature on adventitious viruses found in insect cell lines, affected cell lines, and new virus-free cell lines. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines.

PubMed

Almanaa, Taghreed N; Geusz, Michael E; Jamasbi, Roudabeh J

2012-10-24

Many cancers contain cell subpopulations that display characteristics of stem cells. Because these cancer stem cells (CSCs) appear to provide resistance to chemo-radiation therapy, development of therapeutic agents that target CSCs is essential. Curcumin is a phytochemical agent that is currently used in clinical trials to test its effectiveness against cancer. However, the effect of curcumin on CSCs is not well established. The current study evaluated curcumin-induced cell death in six cancer cell lines derived from human esophageal squamous cell carcinomas. Moreover, these cell lines and the ones established from cells that survived curcumin treatments were characterized. Cell loss was assayed after TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2 cells were exposed to 20-80 μM curcumin for 30 hrs. Cell lines surviving 40 or 60 μM curcumin were established from these six original lines. The stem cell markers aldehyde dehydrogenase-1A1 (ALDH1A1) and CD44 as well as NF-κB were used to compare CSC-like subpopulations within and among the original lines as well as the curcumin-surviving lines. YES-2 was tested for tumorsphere-forming capabilities. Finally, the surviving lines were treated with 40 and 60 μM curcumin to determine whether their sensitivity was different from the original lines. The cell loss after curcumin treatment increased in a dose-dependent manner in all cell lines. The percentage of cells remaining after 60 μM curcumin treatment varied from 10.9% to 36.3% across the six lines. The cell lines were heterogeneous with respect to ALDH1A1, NF-κB and CD44 expression. KY-5 and YES-1 were the least sensitive and had the highest number of stem-like cells whereas TE-1 had the lowest. The curcumin-surviving lines showed a significant loss in the high staining ALDH1A1 and CD44 cell populations. Tumorspheres formed from YES-2 but were small and rare in the YES-2 surviving line. The curcumin-surviving lines showed a small but significant decrease in sensitivity to curcumin when compared with the original lines. Our results suggest that curcumin not only eliminates cancer cells but also targets CSCs. Therefore, curcumin may be an effective compound for treating esophageal and possibly other cancers in which CSCs can cause tumor recurrence.

Cytoplasmic sequestration of the tumor suppressor p53 by a heat shock protein 70 family member, mortalin, in human colorectal adenocarcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gestl, Erin E., E-mail: egestl@wcupa.edu; Anne Boettger, S., E-mail: aboettger@wcupa.edu

2012-06-29

Highlights: Black-Right-Pointing-Pointer Eight human colorectal cell lines were evaluated for p53 and mortalin localization. Black-Right-Pointing-Pointer Six cell lines displayed cytoplasmic sequestration of the tumor suppressor p53. Black-Right-Pointing-Pointer Direct interaction between mortalin and p53 was shown in five cell lines. Black-Right-Pointing-Pointer Cell lines positive for p53 sequestration yielded elevated p53 expression levels. Black-Right-Pointing-Pointer This study yields the first evidence of cytoplasmic sequestration p53 by mortalin. -- Abstract: While it is known that cytoplasmic retention of p53 occurs in many solid tumors, the mechanisms responsible for this retention have not been positively identified. Since heatshock proteins like mortalin have been associated withmore » p53 inactivation in other tumors, the current study sought to characterize this potential interaction in never before examined colorectal adenocarcinoma cell lines. Six cell lines, one with 3 different fractions, were examined to determine expression of p53 and mortalin and characterize their cellular localization. Most of these cell lines displayed punctate p53 and mortalin localization in the cell cytoplasm with the exception of HCT-8 and HCT116 379.2 cells, where p53 was not detected. Nuclear p53 was only observed in HCT-116 40-16, LS123, and HT-29 cell lines. Mortalin was only localized in the cytoplasm in all cell lines. Co-immunoprecipitation and immunohistochemistry revealed that p53 and mortalin were bound and co-localized in the cytoplasmic fraction of four cell lines, HCT-116 (40-16 and 386; parental and heterozygous fractions respectively of the same cell line), HT-29, LS123 and LoVo, implying that p53 nuclear function is limited in those cell lines by being restricted to the cytoplasm. Mortalin gene expression levels were higher than gene expression levels of p53 in all cell lines. Cell lines with cytoplasmic sequestration of p53, however, also displayed elevated p53 gene expression levels compared to cell lines without p53 sequestration. Our data reveal the characteristic cytoplasmic sequestration of p53 by the heat shock protein mortalin in human colorectal adenocarcinoma cell lines, as is the case for other cancers, such as glioblastomas and hepatocellular carcinomas.« less

Development of a new canine osteosarcoma cell line.

PubMed

Séguin, B; Zwerdling, T; McCallan, J L; DeCock, H E V; Dewe, L L; Naydan, D K; Young, A E; Bannasch, D L; Foreman, O; Kent, M S

2006-12-01

Establishing a canine osteosarcoma (OSA) cell line can be useful to develop in vivo and in vitro models of OSA. The goal of this study was to develop, characterize and authenticate a new canine OSA cell line and a clone. A cell line and a clone were developed with standard cell culture techniques from a naturally occurring OSA in a dog. The clonal cell line induced a tumour after injection in RAG 1-deficient mouse. Histology was consistent with OSA. The original tumour from the dog and the tumour induced in the mouse were both reactive with vimentin and osteonectin (ON). The parent cell line and clonal cell line were reactive with ON, osteocalcin and alkaline phosphatase. Loss of heterozygosity was found in the same three microsatellite markers in the parent and clonal cell lines, and the tumour tissue grown in the mouse.

Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines

PubMed Central

Bock, Christoph; Kiskinis, Evangelos; Verstappen, Griet; Gu, Hongcang; Boulting, Gabriella; Smith, Zachary D.; Ziller, Michael; Croft, Gist F.; Amoroso, Mackenzie W.; Oakley, Derek H.; Gnirke, Andreas; Eggan, Kevin; Meissner, Alexander

2011-01-01

SUMMARY The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines. PMID:21295703

Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Felthaus, O.; Department of Oral and Maxillofacial Surgery, University of Regensburg; Ettl, T.

2011-04-01

Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simplemore » method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.« less

Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

PubMed Central

Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

2014-01-01

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

HIV integration sites in latently infected cell lines: evidence of ongoing replication.

PubMed

Symons, Jori; Chopra, Abha; Malatinkova, Eva; De Spiegelaere, Ward; Leary, Shay; Cooper, Don; Abana, Chike O; Rhodes, Ajantha; Rezaei, Simin D; Vandekerckhove, Linos; Mallal, Simon; Lewin, Sharon R; Cameron, Paul U

2017-01-13

Assessing the location and frequency of HIV integration sites in latently infected cells can potentially inform our understanding of how HIV persists during combination antiretroviral therapy. We developed a novel high throughput sequencing method to evaluate HIV integration sites in latently infected cell lines to determine whether there was virus replication or clonal expansion in these cell lines observed as multiple integration events at the same position. We modified a previously reported method using random DNA shearing and PCR to allow for high throughput robotic processing to identify the site and frequency of HIV integration in latently infected cell lines. Latently infected cell lines infected with intact virus demonstrated multiple distinct HIV integration sites (28 different sites in U1, 110 in ACH-2 and 117 in J1.1 per 150,000 cells). In contrast, cell lines infected with replication-incompetent viruses (J-Lat cells) demonstrated single integration sites. Following in vitro passaging of the ACH-2 cell line, we observed a significant increase in the frequency of unique HIV integration sites and there were multiple mutations and large deletions in the proviral DNA. When the ACH-2 cell line was cultured with the integrase inhibitor raltegravir, there was a significant decrease in the number of unique HIV integration sites and a transient increase in the frequency of 2-LTR circles consistent with virus replication in these cells. Cell lines latently infected with intact HIV demonstrated multiple unique HIV integration sites indicating that these cell lines are not clonal and in the ACH-2 cell line there was evidence of low level virus replication. These findings have implications for the use of latently infected cell lines as models of HIV latency and for the use of these cells as standards.

Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.

PubMed

Corral-Vázquez, C; Aguilar-Quesada, R; Catalina, P; Lucena-Aguilar, G; Ligero, G; Miranda, B; Carrillo-Ávila, J A

2017-06-01

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

Mycobacterial biofilms: a greasy way to hold it together.

PubMed

Zambrano, María Mercedes; Kolter, Roberto

2005-12-02

Microorganisms growing on surfaces can form biofilms under certain conditions. In this issue of Cell, Ojha et al. (2005) investigate biofilm formation in mycobacteria. They identify new cell-wall components that are required for the formation of architecturally complex mature biofilms in these bacteria and the surprising involvement of a chaperone protein in this process.

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis).

PubMed

Jager, Martine J; Magner, J Antonio Bermudez; Ksander, Bruce R; Dubovy, Sander R

2016-08-01

To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines.

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis)

PubMed Central

Jager, Martine J.; Magner, J. Antonio Bermudez; Ksander, Bruce R.; Dubovy, Sander R.

2016-01-01

Purpose To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Methods Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Results Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. Conclusions All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines. PMID:28018010

Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

PubMed Central

Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

2015-01-01

Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed. PMID:26136685

Lung Cancer Cell Lines as Tools for Biomedical Discovery and Research

PubMed Central

Girard, Luc; Lockwood, William W.; Lam, Wan L.; Minna, John D.

2010-01-01

Lung cancer cell lines have made a substantial contribution to lung cancer translational research and biomedical discovery. A systematic approach to initiating and characterizing cell lines from small cell and non–small cell lung carcinomas has led to the current collection of more than 200 lung cancer cell lines, a number that exceeds those for other common epithelial cancers combined. The ready availability and widespread dissemination of the lines to investigators worldwide have resulted in more than 9000 citations, including multiple examples of important biomedical discoveries. The high (but not perfect) genomic similarities between lung cancer cell lines and the lung tumor type from which they were derived provide evidence of the relevance of their use. However, major problems including misidentification or cell line contamination remain. Ongoing studies and new approaches are expected to reveal the full potential of the lung cancer cell line panel. PMID:20679594

Cell lines derived from feline fibrosarcoma display unstable chromosomal aneuploidy and additionally centrosome number aberrations.

PubMed

von Erichsen, J; Hecht, W; Löhberg-Gruene, C; Reinacher, M

2012-07-01

The purpose of the study was to evaluate clonality and presence of numerical chromosomal and centrosomal aberrations in 5 established feline fibrosarcoma cell lines and in a fetal dermal fibroblast cell line as a control. The clonality of all cell lines was examined using limited-dilution cloning. The number of chromosomes was counted in metaphase spreads. The immunocytochemical analysis of centrosome numbers was performed by indirect immunofluorescence using a monoclonal antibody that targets γ-tubulin, a well-characterized component of centrosomes. Monoclonal cell populations could be established from all cell lines. In all feline fibrosarcoma cell lines, the number of chromosomes deviated abnormally from the normal feline chromosome number of 2n = 38, ranging from 19 to 155 chromosomes per cell. Centrosome hyperamplification was observed in all 5 feline fibrosarcoma cell lines with a proportion of cells (5.7 to 15.2%) having more than 2 centrosomes. In the control cell line, only 0.6% of the cells had more than 2 centrosomes. In conclusion, the examinations revealed that centrosome hyperamplification occurs in feline fibrosarcoma cell lines. The feline fibrosarcoma cell lines possessed 10 to 25 times as many cells with centrosome hyperamplification as the control cell line. These observations suggest an association of numerical centrosome aberrations with karyotype instability by increasing the frequency of chromosome missegregation. The results of this study may be helpful for further characterization of feline fibrosarcomas and may contribute to the knowledge of cytogenetic factors that may be important for the pathogenesis of feline fibrosarcomas.

Discovery of an X-ray Violently Variable Broad Absorption Line Quasar

NASA Technical Reports Server (NTRS)

Ghosh, Kajal K.; Gutierrez, Carlos M.; Punsly, Brian; Chevallier, Loic; Goncalves, Anabela C.

2006-01-01

In this letter, we report on a quasar that is violently variable in the X-rays, XVV. It is also a broad absorption line quasar (BALQSO) that exhibits both high ionization and low ionization UV absorption lines (LoBALQSO). It is very luminous in the X-rays (approximately 10(exp 46) ergs s(sup -l) over the entire X-ray band). Surprisingly, this does not over ionize the LoBAL outflow. The X-rays vary by a factor of two within minutes in the quasar rest frame, which is shorter than 1/30 of the light travel time across a scale length equal to the black hole radius. We concluded that the X-rays are produced in a relativistic jet beamed toward earth in which variations in the Doppler enhancement produce the XVV behavior.

Attempt to develop taste bud models in three-dimensional culture.

PubMed

Nishiyama, Miyako; Yuki, Saori; Fukano, Chiharu; Sako, Hideyuki; Miyamoto, Takenori; Tomooka, Yasuhiro

2011-09-01

Taste buds are the end organs of taste located in the gustatory papillae, which occur on the surface of the oral cavity. The goal of the present study was to establish a culture model mimicking the lingual taste bud of the mouse. To this end, three cell lines were employed: taste bud-derived cell lines (TBD cell lines), a lingual epithelial cell-derived cell line (20A cell line), and a mesenchymal cell-derived cell line (TMD cell line). TBD cells embedded in collagen gel formed three-dimensional clusters, which had an internal cavity equipped with a tight junction-like structure, a microvilluslike structure, and a laminin-positive layer surrounding the cluster. The cells with this epitheliumlike morphology expressed marker proteins of taste cells: gustducin and NCAM. TBD cells formed a monolayer on collagen gel when they were co-cultured with TMD cells. TBD, 20A, and TMD cell lines were maintained in a triple cell co-culture, in which TBD cells were pre-seeded as aggregates or in suspension on the collagen gel containing TMD cells, and 20A cells were laid over the TBD cells. TBD cells in the triple cell co-culture expressed NCAM. This result suggests that co-cultured TBD cells exhibited a characteristic of Type III taste cells. The culture model would be useful to study morphogenesis and functions of the gustatory organ.

Enhancement of Radiation Response in Osteosarcoma and Rhabomyosarcoma Cell Lines by Histone Deacetylase Inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blattmann, Claudia, E-mail: claudia.blattmann@med.uni-heidelberg.d; Oertel, Susanne; Ehemann, Volker

2010-09-01

Purpose: Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. Methods and Materials: Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS-24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. Results: SAHA induced anmore » inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). Conclusion: Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs.« less

Generation of genome-modified Drosophila cell lines using SwAP.

PubMed

Franz, Alexandra; Brunner, Erich; Basler, Konrad

2017-10-02

The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.

Fluoroorotic acid-selected Nicotiana plumbaginifolia cell lines with a stable thymine starvation phenotype have lost the thymine-regulated transcriptional program.

PubMed

Santoso, D; Thornburg, R

2000-08-01

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines.

Fluoroorotic Acid-Selected Nicotiana plumbaginifolia Cell Lines with a Stable Thymine Starvation Phenotype Have Lost the Thymine-Regulated Transcriptional Program1

PubMed Central

Santoso, Djoko; Thornburg, Robert

2000-01-01

We have selected 143 independent Nicotiana plumbaginifolia cell lines that survive in the presence of 5-fluoroorotic acid. These lines show several diverse phenotypes. The majority of these cell lines showed reduced levels of UMP synthase. However, one particular phenotype, which represents 14% of the total independent lines (20 cell lines), showed an unexpected, high level of UMP synthase and was therefore analyzed in detail. The selected cell lines showed no differences with wild-type cells with respect to uptake of orotic acid, affinity of UMP synthase for its substrates, or UMP synthase gene-copy number. Alternative detoxification mechanisms were also excluded. The elevated enzyme activity was correlated with elevated UMP synthase protein levels as well as elevated UMP synthase mRNA levels. In contrast to wild-type cell lines, the fluoroorotic acid-selected cell lines did not respond to thymine or to other biochemicals that affect thymine levels. In addition, there was also a concomitant up-regulation of aspartate transcarbamoylase, however, dihydroorotase and dihydroorotate dehydrogenase are not up-regulated in these cell lines. PMID:10938367

Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

PubMed

Kniss, Douglas A; Summerfield, Taryn L

2014-08-01

Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

Fatty acid binding proteins (FABPs) in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma

PubMed Central

2011-01-01

Background Fatty acid binding proteins (FABP) play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines. Methods Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome. Results General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome. Conclusions Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the same cancer. The FABP patterns in the cell lines do not always reflect the real situation in the tumours. These facts have to be considered in functional studies concerning the different FABPs. PMID:21767383

Influence of Impurities and Filling Protocol on the Aluminum Fixed Point

NASA Astrophysics Data System (ADS)

Renaot, E.; Valin, M. H.; Elgourdou, M.

2008-06-01

To improve the uncertainty of the aluminum fixed point, a study was launched by LNE-INM/CNAM in the framework of the EUROMET Project 732 “Toward more accurate temperature fixed points” (Coordinating laboratory: LNE-INM/CNAM, 17 partner countries). A new open cell was filled with aluminum of 99.99995% purity. A French laboratory carried out elemental analysis of the sample using glow discharge-mass spectrometry (GD-MS). The values of the equilibrium distribution coefficient k and of the derivative {δ T_{{l}}/δ ci_{{l}}} of the temperature of the liquidus line with respect to the concentration of impurity i will be obtained through collaboration with a French physical and chemical laboratory. In the past, some aluminum cells were opened after several melts and freezes. The aluminum ingot was sticking to the graphite crucible, indicating that physicochemical reactions had likely occurred between Al and C. To avoid this reaction, an effort was made to draw benefit from the Al2O3 film that appears immediately on the surface of the aluminum ingot when it is exposed to oxygen. The open aluminum cell was tested in different furnaces and with different thermal insulator arrangements inside the fixed-point assembly. The observed drifts of the plateaux were always larger than the expected values. The cell was opened to inspect the aluminum ingot. The ingot was extracted easily, since no sticking to the crucible had occurred. The aluminum showed a very bright surface, but the presence of many “craters” throughout the thickness of the ingot was surprising. In some cases, the thermometer well was even apparent.

C 60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russ, K. A.; Elvati, P.; Parsonage, T. L.

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C 60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. Themore » evidence suggests marginal uptake of C 60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Compu-tational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal defor-mation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. Lastly, the surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.« less

C 60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

DOE PAGES

Russ, K. A.; Elvati, P.; Parsonage, T. L.; ...

2016-01-01

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C 60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. Themore » evidence suggests marginal uptake of C 60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Compu-tational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal defor-mation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. Lastly, the surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.« less

Integrating temperature with odor processing in the olfactory bulb.

PubMed

Kludt, Eugen; Okom, Camille; Brinkmann, Alexander; Schild, Detlev

2015-05-20

Temperature perception has long been classified as a somesthetic function solely. However, in recent years several studies brought evidence that temperature perception also takes place in the olfactory system of rodents. Temperature has been described as an effective stimulus for sensory neurons of the Grueneberg ganglion located at the entrance of the nose. Here, we investigate whether a neuronal trace of temperature stimulation can be observed in the glomeruli and mitral cells of the olfactory bulb, using calcium imaging and fast line-scanning microscopy. We show in the Xenopus tadpole system that the γ-glomerulus, which receives input from olfactory neurons, is highly sensitive to temperature drops at the olfactory epithelium. We observed that thermo-induced activity in the γ-glomerulus is conveyed to the mitral cells innervating this specific neuropil. Surprisingly, a substantial number of thermosensitive mitral cells were also chemosensitive. Moreover, we report another unique feature of the γ-glomerulus: it receives ipsilateral and contralateral afferents. The latter fibers pass through the contralateral bulb, cross the anterior commissure, and then run to the ipsilateral olfactory bulb, where they target the γ-glomerulus. Temperature drops at the contralateral olfactory epithelium also induced responses in the γ-glomerulus and in mitral cells. Temperature thus appears to be a relevant physiological input to the Xenopus olfactory system. Each olfactory bulb integrates and codes temperature signals originating from receptor neurons of the ipsilateral and contralateral nasal cavities. Finally, temperature and chemical information is processed in shared cellular networks. Copyright © 2015 the authors 0270-6474/15/357892-11$15.00/0.

Utility of human embryonic kidney cell line HEK-293 for rapid isolation of fixed and street rabies viruses: comparison with Neuro-2a and BHK-21 cell lines.

PubMed

Madhusudana, Shampur Narayan; Sundaramoorthy, Subha; Ullas, Padinjaremattatthil Thankappan

2010-12-01

A confirmatory rabies diagnosis can be achieved by rapid virus isolation in cell culture using brain tissue from the suspect animal. Several cell lines have been used for this purpose and the murine neuroblastoma cell line Neuro-2a has been found to be the most sensitive. The human embryonic kidney cell line HEK-293 is known to express several neuronal proteins and is believed to be of neuronal origin. We hypothesized that this cell line could be susceptible to rabies virus, which is highly neurotropic. First we tested the sensitivity of HEK-293 cells to the laboratory strain, challenge virus standard (CVS). We then tested 120 brain samples from different animals and humans suspected to have died of rabies by fluorescent antibody test (FAT). Both FAT-positive and FAT-negative brains were tested for virus isolation using Neuro-2a, BHK-21, and HEK-293 cell lines and also by mouse inoculation. There was 100% correlation between FAT, virus isolation in Neuro-2a and HEK-293 cells, and mouse inoculation. However, the rate of virus isolation in the BHK-21 cell line was only 28% when compared to the other cell lines. The sensitivity of HEK-293 to CVS strain of virus was similar to that of Neuro-2a. We conclude that the HEK-293 cell line is as sensitive as the Neuro-2a cell line for the rapid isolation of rabies virus and may serve as an alternative cell line for rabies diagnosis and future research. Copyright © 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

PubMed

Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

2013-10-01

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

[Establishment of Z-HL16C cell line.].

PubMed

Chen, J P; Li, J; Zhao, S L; Tian, J Y; Ye, F

2006-09-01

To establish and study the nature and the application of Z-HL16C cell line. The cell line was continuously passed, frozen stored and recovered. Its application was expanded and the cell type was identified. The cell line had an epithelial-cell-like shape, the size appeared uniform, the cell boundary was distinct. It has been continuously passed, frozen stored and recovered for ten years. Its recovery rate was about 90%. It has been proved to be sensitive to the tested viruses which were enteroviruses (Polio, Cox, Echo), influenza viruses, parainfluenzaviruses, adenoviruses, measles virus. This cell line has been identified as a cancerization cell. The cell line Z-HL16C has been stably established, it has a broad spectrum in sensitivity for culturing viruses.

Development and characterization of a cell line WAF from freshwater shark Wallago attu.

PubMed

Dubey, Akhilesh; Goswami, Mukunda; Yadav, Kamalendra; Sharma, Bhagwati S

2014-02-01

A new epithelial cell line, WAF was developed from caudal fin of freshwater shark, Wallago attu. The cell line was optimally maintained at 28 °C in Leibovitz-15 (L-15) medium supplemented with 20 % fetal bovine serum. The cell line was characterized by various cytogenetic and molecular markers. The cytogenetic analysis revealed a diploid count of 86 chromosomes at different passages. The origin of the cell lines was confirmed by the amplification of 547 and 654 bp sequences of 16S rRNA and cytochrome oxidase subunit I genes of mitochondrial DNA, respectively. WAF cells were characterized for their growth characteristics at different temperature and serum concentration. Epithelial morphology of the cell line was confirmed using immunocytochemistry. Further cell plating efficiency, transfection efficiency and viability of cryopreserved WAF cells was also determined. Cytotoxicity and genotoxicity assessment of cadmium salts on WAF cells by MTT, NR and comet assay illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The cell line will be further useful for studying oxidative stress markers against aquatic pollutants.

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

PubMed

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, K.; Chubb, C.; Huberman, E.

High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteinsmore » were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.« less

Neurotox-98 Neuroprotection by Progesterones Through Stimulation of Mitochondrial Gene Expression

DTIC Science & Technology

2006-06-01

excitotoxicity that leads to neuronal cell loss. However, surprisingly little is known of the mechanism for this effect . The working hypothesis for the...regulation that is independent of energy status of the cell . Although not included in the publication, we tested for effects of progesterone on... effects (Goodman et al., 1996); however, high levels of progesterone did nothing to protect the animals from seizures or neuronal cell death. In summary

Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

USDA-ARS?s Scientific Manuscript database

The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

Making maxillary barbels with a proximal-distal gradient of Wnt signals in matrix-bound mesenchymal cells

PubMed Central

Figueroa, Francisco; Singer, Susan S.; LeClair, Elizabeth E.

2015-01-01

The evolution of specific appendages is made possible by the ontogenetic deployment of general cell signaling pathways. Many fishes, amphibians and reptiles have unique skin appendages known as barbels, which are poorly understood at the cellular and molecular level. In this study, we examine the cell arrangements, cell division patterns, and gene expression profiles associated with the zebrafish maxillary barbel, or ZMB. The earliest cellular organization of the ZMB is an internal whorl of mesenchymal cells in the dermis of the maxilla; there is no epithelial placode, nor any axially-elongated epithelial cells as expected of an apical ectodermal ridge (AER). As the ZMB develops, cells in S-phase are at first distributed randomly throughout the appendage, gradually transitioning to a proliferative population concentrated at the distal end. By observing ZMB ontogenetic stages in a Wnt-responsive transgenic reporter line, TCFsiam, we identified a strongly fluorescent mesenchymal cell layer within these developing appendages. Using an in vitro explant culture technique on developing barbel tissues, we co-localized the fluorescent label in these cells with the mitotic marker EdU. Surprisingly, TCF+ cells showed little proliferation, indicating a slow-cycling subpopulation. Transmission electron microscopy of the ZMB located the TCF+ cells in a single, circumferential layer within the barbel’s matrix core. Morphologically, these cells resemble fibroblasts or osteoblasts; in addition to their matrix-bound location, they are identified by their pancake-shaped nuclei, abundant rough endoplasmic reticulum, and cytoplasmic extensions into the surrounding extracellular matrix. Taken together, these features define a novel mesenchymal cell population in zebrafish, the ‘TCF+ core cells.’ A working model of barbel development is proposed, in which these minimally mitotic mesodermal cells produce collagenous matrix in response to ectodermally-derived Wnt signals deployed in a proximal-distal gradient along the appendage. This documents a novel mechanism of vertebrate appendage outgrowth. Similar genetic signals and cell behaviors may be responsible for the independent and repeated evolution of barbel structures in other fish species. PMID:26492827

Penetration and differentiation of cephalic neural crest-derived cells in the developing mouse telencephalon.

PubMed

Yamanishi, Emiko; Takahashi, Masanori; Saga, Yumiko; Osumi, Noriko

2012-12-01

Neural crest (NC) cells originate from the neural folds and migrate into the various embryonic regions where they differentiate into multiple cell types. A population of cephalic neural crest-derived cells (NCDCs) penetrates back into the developing forebrain to differentiate into microvascular pericytes, but little is known about when and how cephalic NCDCs invade the telencephalon and differentiate into pericytes. Using a transgenic mouse line in which NCDCs are genetically labeled with enhanced green fluorescent protein (EGFP), we observed that NCDCs started to invade the telencephalon together with endothelial cells from embryonic day (E) 9.5. A majority of NCDCs located in the telencephalon expressed pericyte markers, that is, PDGFRβ and NG2, and differentiated into pericytes around E11.5. Surprisingly, many of the NC-derived pericytes express p75, an undifferentiated NCDC marker at E11.5, as well as NCDCs in the mesenchyme. At the same time, a minor population of NCDCs that located separately from blood vessels in the telencephalon were NG2-negative and some of these NCDCs also expressed p75. Proliferation and differentiation of pericytes appeared to occur in a specific mesenchymal region where blood vessels penetrated into the telencephalon. These results indicate that (i) NCDCs penetrate back into the telencephalon in parallel with angiogenesis, (ii) many NC-derived pericytes may be still in pre-mature states even though after differentiation into pericytes in the early developing stages, (iii) a small minority of NCDCs may retain undifferentiated states in the developing telencephalon, and (iv) a majority of NCDCs proliferate and differentiate into pericytes in the mesenchyme around the telencephalon. © 2012 The Authors Development, Growth & Differentiation © 2012 Japanese Society of Developmental Biologists.

Complex Actions of Thyroid Hormone Receptor Antagonist NH-3 on Gene Promoters in Different Cell Lines

PubMed Central

Shah, Vanya; Nguyen, Phuong; Nguyen, Ngoc-Ha; Togashi, Marie; Scanlan, Thomas S.; Baxter, John D.; Webb, Paul

2014-01-01

It is desirable to obtain new antagonists for thyroid hormone (TRs) and other nuclear receptors (NRs). We previously used X-ray structural models of TR ligand binding domains (LBDs) to design compounds, such as NH-3, that impair coactivator binding to activation function 2 (AF-2) and block thyroid hormone (triiodothyronine, T3) actions. However, TRs bind DNA and are transcriptionally active without ligand. Thus, NH-3 could modulate TR activity via effects on other coregulator interaction surfaces, such as activation function (AF-1) and corepressor binding sites. Here, we find that NH-3 blocks TR-LBD interactions with coactivators and corepressors and also inhibits activities of AF-1 and AF-2 in transfections. While NH-3 lacks detectable agonist activity at T3-activated genes in GC pituitary cells it nevertheless activates spot 14 (S14) in HTC liver cells with the latter effect accompanied by enhanced histone H4 acetylation and coactivator recruitment at the S14 promoter. Surprisingly, T3 promotes corepressor recruitment to target promoters. NH-3 effects vary; we observe transient recruitment of N-CoR to S14 in GC cells and dismissal and rebinding of N-CoR to the same promoter in HTC cells. We propose that NH-3 will generally behave as an antagonist by blocking AF-1 and AF-2 but that complex effects on coregulator recruitment may result in partial/mixed agonist effects that are independent of blockade of T3 binding in some contexts. These properties could ultimately be utilized in drug design and development of new selective TR modulators. PMID:18930112

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants.

PubMed

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; Nguyen, Thuy N; Gidda, Satinder K; Watt, Samantha C; Yurchenko, Olga; Park, Sunjung; Sturtevant, Drew; Mullen, Robert T; Dyer, John M; Chapman, Kent D

2017-07-01

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER-vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

PD-1 or PD-L1 Blockade Restores Antitumor Efficacy Following SSX2 Epitope-Modified DNA Vaccine Immunization.

PubMed

Rekoske, Brian T; Smith, Heath A; Olson, Brian M; Maricque, Brett B; McNeel, Douglas G

2015-08-01

DNA vaccines have demonstrated antitumor efficacy in multiple preclinical models, but low immunogenicity has been observed in several human clinical trials. This has led to many approaches seeking to improve the immunogenicity of DNA vaccines. We previously reported that a DNA vaccine encoding the cancer-testis antigen SSX2, modified to encode altered epitopes with increased MHC class I affinity, elicited a greater frequency of cytolytic, multifunctional CD8(+) T cells in non-tumor-bearing mice. We sought to test whether this optimized vaccine resulted in increased antitumor activity in mice bearing an HLA-A2-expressing tumor engineered to express SSX2. We found that immunization of tumor-bearing mice with the optimized vaccine elicited a surprisingly inferior antitumor effect relative to the native vaccine. Both native and optimized vaccines led to increased expression of PD-L1 on tumor cells, but antigen-specific CD8(+) T cells from mice immunized with the optimized construct expressed higher PD-1. Splenocytes from immunized animals induced PD-L1 expression on tumor cells in vitro. Antitumor activity of the optimized vaccine could be increased when combined with antibodies blocking PD-1 or PD-L1, or by targeting a tumor line not expressing PD-L1. These findings suggest that vaccines aimed at eliciting effector CD8(+) T cells, and DNA vaccines in particular, might best be combined with PD-1 pathway inhibitors in clinical trials. This strategy may be particularly advantageous for vaccines targeting prostate cancer, a disease for which antitumor vaccines have demonstrated clinical benefit and yet PD-1 pathway inhibitors alone have shown little efficacy to date. ©2015 American Association for Cancer Research.

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

DOE PAGES

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; ...

2017-01-18

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less

RNA interference suppression of MUC1 reduces the growth rate and metastatic phenotype of human pancreatic cancer cells.

PubMed

Tsutsumida, Hideaki; Swanson, Benjamin J; Singh, Pankaj K; Caffrey, Thomas C; Kitajima, Shinichi; Goto, Masamichi; Yonezawa, Suguru; Hollingsworth, Michael A

2006-05-15

MUC1 is a highly glycosylated, type I transmembrane protein expressed by normal ductal epithelial cells of the pancreas, breast, lung, and gastrointestinal tract, and overexpressed in many cases of adenocarcinoma. We down-regulated MUC1 expression by RNA interference and investigated the effects on malignant and metastatic potential of a human pancreatic cancer cell line, S2-013. MUC1-suppressed clones, S2-013.MTII.C1 and S2-013.MTII.C2, were established by targeting a sequence 3,151 bp from the initiation codon and characterized in vitro for proliferation, invasion, and adhesion. We evaluated the effects of MUC1 suppression in vivo on tumor growth and metastatic properties following implantation into the cecum or pancreas of athymic mice. MUC1-suppressed clones showed significantly decreased proliferation in vitro and in vivo. Global gene expression was evaluated by oligonucleotide microarray analysis. Surprisingly, genes predicted to increase doubling times (cyclin B1 and cyclin D3) were overexpressed in MUC1-suppressed clones. There were alterations in expression of several genes that may affect the malignant properties of pancreatic cancer. Adhesion of MUC1-suppressed cells in vitro to type IV collagen and fibronectin was slightly increased, and adhesion was slightly decreased to type I collagen and laminin. Results of implantation to cecum and pancreas showed significant reduction of metastasis to lymph nodes, lung, or peritoneal sites compared with S2-013.gfp-neo control cells. These results support the hypothesis that MUC1 contributes significantly to growth and metastasis, and that down-regulation of MUC1 protein expression decreases the metastatic potential of pancreatic adenocarcinoma.

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less

Cell-intrinsic determinants of ibrutinib-induced apoptosis in Chronic Lymphocytic Leukemia

PubMed Central

Amin, Nisar A.; Balasubramanian, Sriram; Saiya-Cork, Kamlai; Shedden, Kerby; Hu, Nan; Malek, Sami N.

2016-01-01

Purpose Ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed CLL and CLL with del17p. Mechanistically, ibrutinib interferes with BCR signaling as well as multiple CLL cell to microenvironment interactions. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance is warranted. Experimental Design We studied 48 longitudinally sampled paired CLL samples, 42 of which were procured before and after standard CLL chemotherapies, and characterized them for well-studied CLL molecular traits as well as by whole exome sequencing and SNP 6.0 array profiling. We exposed these samples to 0.25 μM – 5 μM of ibrutinib ex vivo and measured apoptosis fractions as well as BCR signaling by immunoblotting. We disrupted TP53 in HG3, PGA1 and PG-EBV cell lines and measured BCR signaling and ibrutinib responses. Results CLL samples demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib with IC50 values ranging from 0.4 μM – 9.7 μM. Unmutated IGVH status, elevated ZAP70 expression and trisomy 12 were associated with heightened sensitivity to ibrutinib treatment. Five CLL samples were substantially more resistant to ibrutinib following relapse from chemotherapy; of these, three had acquired a del17p/TP53 mutated status. A validation sample of 15 CLL carrying TP53 mutations, of which 13 carried both del17p and a TP53 mutation confirmed substantially less sensitivity to ibrutinib-induced apoptosis. Conclusions This study identifies that CLL harboring del17p/TP53 mutated cells are substantially less sensitive to ibrutinib-induced apoptosis than del17p/TP53 wild type cells. PMID:27535981

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines

PubMed Central

ASADA, Hajime; TOMIYASU, Hirotaka; GOTO-KOSHINO, Yuko; FUJINO, Yasuhito; OHNO, Koichi; TSUJIMOTO, Hajime

2015-01-01

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS. PMID:25715778

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines.

PubMed

Asada, Hajime; Tomiyasu, Hirotaka; Goto-Koshino, Yuko; Fujino, Yasuhito; Ohno, Koichi; Tsujimoto, Hajime

2015-06-01

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.

Genetic Profiling Reveals Cross-Contamination and Misidentification of 6 Adenoid Cystic Carcinoma Cell Lines: ACC2, ACC3, ACCM, ACCNS, ACCS and CAC2

PubMed Central

Phuchareon, Janyaporn; Ohta, Yoshihito; Woo, Jonathan M.; Eisele, David W.; Tetsu, Osamu

2009-01-01

Adenoid cystic carcinoma (ACC) is the second most common malignant neoplasm of the salivary glands. Most patients survive more than 5 years after surgery and postoperative radiation therapy. The 10 year survival rate, however, drops to 40%, due to locoregional recurrences and distant metastases. Improving long-term survival in ACC requires the development of more effective systemic therapies based on a better understanding of the biologic behavior of ACC. Much preclinical research in this field involves the use of cultured cells and, to date, several ACC cell lines have been established. Authentication of these cell lines, however, has not been reported. We performed DNA fingerprint analysis on six ACC cell lines using short tandem repeat (STR) examinations and found that all six cell lines had been contaminated with other cells. ACC2, ACC3, and ACCM were determined to be cervical cancer cells (HeLa cells), whereas the ACCS cell line was composed of T24 urinary bladder cancer cells. ACCNS and CAC2 cells were contaminated with cells derived from non-human mammalian species: the cells labeled ACCNS were mouse cells and the CAC2 cells were rat cells. These observations suggest that future studies using ACC cell lines should include cell line authentication to avoid the use of contaminated or non-human cells. PMID:19557180

THE UNCOVERING OF A NOVEL REGULATORY MECHANISM FOR PLD2: FORMATION OF A TERNARY COMPLEX WITH PROTEIN TYROSINE PHOSPHATASE PTP1B AND GROWTH FACTOR RECEPTOR-BOUND PROTEIN GRB2

PubMed Central

Horn, Jeff; Lopez, Isabel; Miller, Mill; Gomez-Cambronero, Julian

2011-01-01

The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2 and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase. PMID:15896299

Models in search of a brain.

PubMed

Love, Bradley C; Gureckis, Todd M

2007-06-01

Mental localization efforts tend to stress the where more than the what. We argue that the proper targets for localization are well-specified cognitive models. We make this case by relating an existing cognitive model of category learning to a learning circuit involving the hippocampus, perirhinal, and prefrontal cortices. Results from groups varying in function along this circuit (e.g., infants, amnesics, and older adults) are successfully simulated by reducing the model's ability to form new clusters in response to surprising events, such as an error in supervised learning or an unfamiliar stimulus in unsupervised learning. Clusters in the model are akin to conjunctive codes that are rooted in an episodic experience (the surprising event) yet can develop to resemble abstract codes as they are updated by subsequent experiences. Thus, the model holds that the line separating episodic and semantic information can become blurred. Dissociations (categorization vs. recognition) are explained in terms of cluster recruitment demands.

Continuous human cell lines and method of making same

DOEpatents

Stampfer, Martha R.

1989-01-01

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

Establishment of dermal sheath cell line from Cashmere goat and characterizing cytokeratin 13 as its novel biomarker.

PubMed

Zhu, Bing; Guo, Zhili; Jin, Muzi; Bai, Yujuan; Yang, Wenliang; Hui, Lihua

2018-05-01

To establish a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from Cashmere goat and clarify the similarities and differences among them. We established a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from the pelage skin hair follicles of Cashmere goat. The growth rate of dermal sheath cells was intermediate between that of dermal papilla cells and outer root sheath cells. Immunofluorescence experiments and reverse transcription-polymerase chain reaction analysis showed that at both the transcriptional and translational levels, the dermal sheath cells were alpha-smooth muscle actin (α-SMA) + /cytokeratin 13 + , while the dermal papilla cells were α-SMA + /cytokeratin 13 - and the outer root sheath cells were α-SMA - /cytokeratin 13 + . Patterns of cytokeratin 13 expression could distinguish the dermal sheath cells from the dermal papilla cells. These results suggest that cytokeratin 13 could serve as a novel biomarker for dermal sheath cells of Cashmere goat, and should prove useful for researchers investigating dermal stem cells or interaction of different types of cells during hair cycle.

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities.

PubMed

Lapointe, Jason F; Dunphy, Gary B; Giannoulis, Paschalis; Mandato, Craig A; Nardi, James B; Gharib, Osama H; Niven, Donald F

2011-11-01

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell-cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions. Copyright © 2011 Elsevier Inc. All rights reserved.

Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032

PubMed Central

2010-01-01

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity. PMID:20406486

Molecular characterization of breast cancer cell lines through multiple omic approaches.

PubMed

Smith, Shari E; Mellor, Paul; Ward, Alison K; Kendall, Stephanie; McDonald, Megan; Vizeacoumar, Frederick S; Vizeacoumar, Franco J; Napper, Scott; Anderson, Deborah H

2017-06-05

Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated. We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer. The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated in common or unique ways. These two new kinase or "Kin-OMIC" analyses add another dimension of important data about these frequently used breast cancer cell lines. This will assist researchers in selecting the most appropriate cell lines to use for breast cancer studies and provide context for the interpretation of the emerging results.

Development and characterization of two cell lines PDF and PDH from Puntius denisonii (Day 1865).

PubMed

Lakra, Wazir S; Goswami, M; Yadav, Kamalendra; Gopalakrishnan, A; Patiyal, R S; Singh, M

2011-02-01

The Puntius denisonii colloquially and more popularly referred to as Miss Kerala is a subtropical fish belonging to the genus Puntius (Barb) and family Cyprinidae. Two cell lines PDF and PDH were developed from the caudal fin and heart of P. denisonii, respectively. The cell lines were optimally maintained at 26°C in Leibovitz-15 medium supplemented with 10% fetal bovine serum. A diploid count of 50 chromosomes at passage 50 was observed in both the cell lines. The high growth potential of the cell lines was reflected from the cell doubling time of 28 and 30 h of PDF and PDH cell lines, respectively. The viability of the PDF and PDH cell lines was 70% and 76%, respectively, after 4 mo of storage in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 653 bp fragments of cytochrome oxidase subunit I of mitochondrial DNA genes.

COLD CO GAS IN THE DISK OF THE YOUNG ERUPTIVE STAR EX LUP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kóspál, Á.; Ábrahám, P.; Moór, A.

EX Lupi-type objects (EXors) form a sub-class of T Tauri stars, defined by sudden sporadic flare-ups of 1–5 mag at optical wavelengths. These eruptions are attributed to enhanced mass accretion from the circumstellar disk to the star, and may constitute important events in shaping the structure of the inner disk and the forming planetary system. Although disk properties must play a fundamental role in driving the outbursts, they are surprisingly poorly known. In order to characterize the dust and gas components of EXor disks, here we report on observations of the {sup 12}CO J = 3−2 and 4–3 lines, and themore » {sup 13}CO 3–2 line in EX Lup, the prototype of the EXor class. We reproduce the observed line fluxes and profiles with a line radiative transfer model and compare the obtained parameters with corresponding ones of other T Tauri disks.« less

Fibroblast Growth Factor Receptors Are Components of Autocrine Signaling Networks in Head and Neck Squamous Cell Carcinoma Cells

PubMed Central

Marshall, Marianne E.; Hinz, Trista K.; Kono, Scott A.; Singleton, Katherine R.; Bichon, Brady; Ware, Kathryn E.; Marek, Lindsay; Frederick, Barbara A.; Raben, David; Heasley, Lynn E.

2011-01-01

Purpose We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC). Experimental Design FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and EGFR-dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR. Results FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed while only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and ERK signaling in three HNSCC cell lines while three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR TKIs with no contribution from FGFR pathways. Conclusions FGFR signaling was dominant or co-dominant with EGFR in six HNSCC lines while three lines exhibited little or no role for FGFRs and were highly EGFR-dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. PMID:21673064

Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines

PubMed Central

Wen, Jiayu; Mohammed, Jaaved; Bortolamiol-Becet, Diane; Tsai, Harrison; Robine, Nicolas; Westholm, Jakub O.; Ladewig, Erik; Dai, Qi; Okamura, Katsutomo; Flynt, Alex S.; Zhang, Dayu; Andrews, Justen; Cherbas, Lucy; Kaufman, Thomas C.; Cherbas, Peter; Siepel, Adam; Lai, Eric C.

2014-01-01

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage. PMID:24985917

Molluscan cells in culture: primary cell cultures and cell lines

PubMed Central

Yoshino, T. P.; Bickham, U.; Bayne, C. J.

2013-01-01

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

'Where' depends on 'what': a differential functional anatomy for position discrimination in one- versus two-dimensions.

PubMed

Fink, G R; Marshall, J C; Weiss, P H; Shah, N J; Toni, I; Halligan, P W; Zilles, K

2000-01-01

Line bisection is widely used as a clinical test of spatial cognition in patients with left visuospatial neglect after right hemisphere lesion. Surprisingly, many neglect patients who show severe impairment on marking the center of horizontal lines can accurately mark the center of squares. That these patients with left neglect are also typically poor at judging whether lines are correctly prebisected implies that the deficit can be perceptual rather than motoric. These findings suggest a differential neural basis for one- and two-dimensional visual position discrimination that we investigated with functional neuroimaging (fMRI). Normal subjects judged whether, in premarked lines or squares, the mark was placed centrally. Line center judgements differentially activated right parietal cortex, while square center judgements differentially activated the lingual gyrus bilaterally. These distinct neural bases for one- and two-dimensional visuospatial judgements help explain the observed clinical dissociations by showing that as a stimulus becomes a better, more 'object-like' gestalt, the ventral visuoperceptive route assumes more responsibility for assessing position within the object.

Chronic Suppression of Acetyl-CoA Carboxylase 1 in β-Cells Impairs Insulin Secretion via Inhibition of Glucose Rather Than Lipid Metabolism*

PubMed Central

Ronnebaum, Sarah M.; Joseph, Jamie W.; Ilkayeva, Olga; Burgess, Shawn C.; Lu, Danhong; Becker, Thomas C.; Sherry, A. Dean; Newgard, Christopher B.

2008-01-01

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60–80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to β-cells suppressed [14C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy. PMID:18381287

Chronic suppression of acetyl-CoA carboxylase 1 in beta-cells impairs insulin secretion via inhibition of glucose rather than lipid metabolism.

PubMed

Ronnebaum, Sarah M; Joseph, Jamie W; Ilkayeva, Olga; Burgess, Shawn C; Lu, Danhong; Becker, Thomas C; Sherry, A Dean; Newgard, Christopher B

2008-05-23

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen

PubMed Central

Chan, Kok-Fei; Oveissi, Sara; Jackson, Heather M.; Dimopoulos, Nektaria; Guillaume, Philippe; Knights, Ashley J.; Lowen, Tamara; Robson, Neil C.; Russell, Sarah E.; Scotet, Emmanuel; Davis, Ian D.; Maraskovsky, Eugene; Cebon, Jonathan; Luescher, Immanuel F.; Chen, Weisan

2012-01-01

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8+ T cell epitope, NY-ESO-188–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-188–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1157–165. On the other hand, NY-ESO-1157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A26–35; whereas NY-ESO-188–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-188–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-188–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8+ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed. PMID:22970293

Trifluorothymidine resistance is associated with decreased thymidine kinase and equilibrative nucleoside transporter expression or increased secretory phospholipase A2.

PubMed

Temmink, Olaf H; Bijnsdorp, Irene V; Prins, Henk-Jan; Losekoot, Nienke; Adema, Auke D; Smid, Kees; Honeywell, Richard J; Ylstra, Bauke; Eijk, Paul P; Fukushima, Masakazu; Peters, Godefridus J

2010-04-01

Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism. Cells were exposed either continuously or intermittently to TFT, resulting in H630-cTFT and H630-4TFT, respectively. Cells were analyzed for cross-resistance, cell cycle, protein expression, and activity of thymidine phosphorylase (TP), thymidine kinase (TK), thymidylate synthase (TS), equilibrative nucleoside transporter (hENT), gene expression (microarray), and genomic alterations. Both cell lines were cross-resistant to 2'-deoxy-5-fluorouridine (>170-fold). Exposure to IC(75)-TFT increased the S/G(2)-M phase of H630 cells, whereas in the resistant variants, no change was observed. The two main target enzymes TS and TP remained unchanged in both TFT-resistant variants. In H630-4TFT cells, TK protein expression and activity were decreased, resulting in less activated TFT and was most likely the mechanism of TFT resistance. In H630-cTFT cells, hENT mRNA expression was decreased 2- to 3-fold, resulting in a 5- to 10-fold decreased TFT-nucleotide accumulation. Surprisingly, microarray-mRNA analysis revealed a strong increase of secretory phospholipase-A2 (sPLA2; 47-fold), which was also found by reverse transcription-PCR (RT-PCR; 211-fold). sPLA2 inhibition reversed TFT resistance partially. H630-cTFT had many chromosomal aberrations, but the exact role of sPLA2 in TFT resistance remains unclear. Altogether, resistance induction to TFT can lead to different mechanisms of resistance, including decreased TK protein expression and enzyme activity, decreased hENT expression, as well as (phospho)lipid metabolism. Mol Cancer Ther; 9(4); 1047-57. (c)2010 AACR.

The use of human tumour cell lines in the discovery of new cancer chemotherapeutic drugs.

PubMed

Baguley, Bruce C; Marshall, Elaine S

2008-02-01

Human tumour cell lines have played a major role in anticancer drug discovery, but cell lines may model only some aspects of tumour behaviour in cancer patients. Growing evidence supports a theory that stem cells with self-renewing properties sustain tumours. This review considers the extent to which a deeper understanding of the origin and properties of tumour cell lines might lead to new strategies for anticancer drug discovery. Recent literature on normal and tumour stem cells is reviewed and placed in the context of a discussion on the derivation and properties of tumour cell lines. Early-passage cell lines may model the more rapidly proliferating cells in human tumours and, thus, retain some of the properties of tumour stem cells. The effects of anticancer drugs on cell lines should be considered not only with regards to the induction of apoptosis, but also to the induction of senescence or other pathways that lead to host immune and inflammatory responses.

Replication of Heliothis virescens ascovirus in insect cell lines.

PubMed

Asgari, S

2006-09-01

Ascoviruses (AVs) infect larvae of various insect pests belonging to the family Noctuidae. The result of AV infection in the hosts is cleavage of infected cells into vesicles, a unique feature of AV infection. Since insect cell lines facilitate the study of virus life cycles, attempts were made to analyze Heliothis virescens AV (HvAV3e) infection in several cell lines and compare cell pathology to larval infection. In this study, replication and cytopathological effects of HvAV3e on four different cell lines were investigated. HvAV3e replication was confirmed in three noctuid cell lines from Spodoptera frugiperda (Sf9) and Helicoverpa zea (BCIRL-Hz-AM1 and FB33). However, the virus did not replicate in the non-noctuid insect cell line from Pieris rapae (Pieridae). Despite replication of the virus in the three permissive cell lines, the cytopathological effects of the virus were significantly different from that of larval infection.

Transcriptional Inducers of Acetylcholinesterase Expression as Novel Antidotes for Protection Against Chemical Warfare Agents

DTIC Science & Technology

2005-10-01

neuroblastoma cell line , P19 and a human neuroblastoma cell line SH - SY5Y (data not shown). Effect of trichostatin A on...mouse neuroblastoma P19 cell line and a human neuroblastoma cell line SH - SY5Y . More experiments are needed to prove the potential of AChE expression in...treatment of nerve agent exposure. MATERIALS AND METHODS Neuronal cell lines and

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines

PubMed Central

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

2009-01-01

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines. PMID:18927105

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

PubMed

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

2009-01-01

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

Generating mammalian stable cell lines by electroporation.

PubMed

A Longo, Patti; Kavran, Jennifer M; Kim, Min-Sung; Leahy, Daniel J

2013-01-01

Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr-Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method. Copyright © 2013 Elsevier Inc. All rights reserved.

A Double Selection Approach to Achieve Specific Expression of Toxin Genes for Ovarian Cancer Gene Therapy

DTIC Science & Technology

2007-11-01

cells ( gray fill), cells preincubated with PBS and infected with virus (solid line), and cells preincubated with recombinant knob protein and incubated...U118-hCAR-tailless cells (b). Gray line¼ cells alone, solid line¼ cells+Ad5Luc1-CK1, dashed line¼ cells+Ad5Luc1. Figure 5 Ad5Luc1-CK1CAR-independent...line, whereas U118MG-hCAR-tailless stably expresses the extracellular domain of human CAR. Cells were infected with Ad5Luc1 ( gray bar) and Ad5-r1 (black

Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

PubMed Central

Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

2013-01-01

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

Assembly And Initial Characterization Of A Panel Of 85 Genomically Validated Cell Lines From Diverse Head And Neck Tumor Sites

PubMed Central

Zhao, Mei; Sano, Daisuke; Pickering, Curtis R.; Jasser, Samar A.; Henderson, Ying C.; Clayman, Gary L.; Sturgis, Erich M.; Ow, Thomas J.; Lotan, Reuben; Carey, Thomas E.; Sacks, Peter G.; Grandis, Jennifer R.; Sidransky, David; Heldin, Nils Erik; Myers, Jeffrey N.

2011-01-01

Purpose Human cell lines are useful for studying cancer biology and pre-clinically modeling cancer therapy, but can be misidentified and cross contamination is unfortunately common. The purpose of this study was to develop a panel of validated head and neck cell lines representing the spectrum of tissue sites and histologies that could be used for studying the molecular, genetic, and phenotypic diversity of head and neck cancer. Methods A panel of 122 clinically and phenotypically diverse head and neck cell lines from head and neck squamous cell carcinoma (HNSCC), thyroid cancer, cutaneous squamous cell carcinoma, adenoid cystic carcinoma, oral leukoplakia, immortalized primary keratinocytes, and normal epithelium, was assembled from the collections of several individuals and institutions. Authenticity was verified by performing short tandem repeat (STR) analysis. Human papillomavirus (HPV) status and cell morphology were also determined. Results Eighty-five of the 122 cell lines had unique genetic profiles. HPV-16 DNA was detected in 2 cell lines. These 85 cell lines included cell lines from the major head and neck primary tumor sites, and close examination demonstrates a wide range of in vitro phenotypes. Conclusion This panel of 85 genomically validated head and neck cell lines represents a valuable resource for the head and neck cancer research community that can help advance understanding of the disease by providing a standard reference for cell lines that can be utilized for biological as well as preclinical studies. PMID:21868764

Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation.

PubMed

Fukai, Katsuhiko; Morioka, Kazuki; Yamada, Manabu; Nishi, Tatsuya; Yoshida, Kazuo; Kitano, Rie; Yamazoe, Reiko; Kanno, Toru

2015-07-01

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-α(v)β(6) have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-α(v)β(6) cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-α(v)β(6) cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same. © 2015 The Author(s).

JPRS Report, Science & Technology, Europe

DTIC Science & Technology

1989-07-12

April. This is consistent with the policy suggested in the "report of the four advisors " that was submitted to the governments concerned last year and...surprising situation, considering that Peugeot, at Soch- aux, has adopted the opposite strategy by entirely robo - tizing its painting operations for body...At present, 20 persons per team are working on each of the two lacquering lines. "One of the main advantages of robotization will be to eliminate

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines

PubMed Central

Yu, Channing; Mannan, Aristotle M.; Yvone, Griselda Metta; Ross, Kenneth N.; Zhang, Yan-Ling; Marton, Melissa A.; Taylor, Bradley R.; Crenshaw, Andrew; Gould, Joshua Z.; Tamayo, Pablo; Weir, Barbara A.; Tsherniak, Aviad; Wong, Bang; Garraway, Levi A.; Shamji, Alykhan F.; Palmer, Michelle A.; Foley, Michael A.; Winckler, Wendy; Schreiber, Stuart L.; Kung, Andrew L.; Golub, Todd R.

2016-01-01

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control1-4. Here, we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM displayed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo. PMID:26928769

Development, characterization and application of a new epithelial cell line from caudal fin of Pangasianodon hypophthalmus (Sauvage 1878).

PubMed

Soni, Pankaj; Pradhan, Pravata K; Swaminathan, T R; Sood, Neeraj

2018-06-01

A cell line, designated as PHF, has been established from caudal fin of Pangasianodon hypophthalmus. The cell line was developed using explant method and PHF cells have been subcultured for more than 72 passages over a period of 14 months. The cells were able to grow at temperatures between 24 and 32° C, with an optimum temperature of 28° C. The growth rate of PHF cells was directly proportional to FBS concentration, with optimum growth observed at 20% FBS concentration. On the basis of immunophenotyping assay, PHF cells were confirmed to be of epithelial type. Karyotyping of PHF cells revealed diploid number of chromosomes (2n = 60) at 39th and 65th passage, which indicated that the developed cell line is chromosomally stable. The origin of the cell line was confirmed by amplification and sequencing of cytochrome oxidase c subunit I and 16S rRNA genes. The cell line was tested for Mycoplasma contamination and found to be negative. The cells were successfully transfected with GFP reporter gene suggesting that the developed cell line could be utilized for gene expression studies in future. The cell line could be successfully employed for evaluating the cytotoxicity of heavy metals, namely mercuric chloride and sodium arsenite suggesting that PHF cell line can be potential surrogate for whole fish for studying the cytotoxicity of water soluble compounds. The result of virus susceptibility to tilapia lake virus (TiLV) revealed that PHF cells were refractory to TiLV virus. The newly established cell line would be a useful tool for investigating disease outbreaks particularly of viral etiology, transgenic as well as cytotoxicity studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency.

PubMed

de Sá Rodrigues, L C; Holmes, K E; Thompson, V; Newton, M A; Stein, T J

2017-03-01

An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells. © 2015 John Wiley & Sons Ltd.

The transcriptional diversity of 25 Drosophila cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cherbas, Lucy; Willingham, Aarron; Zhang, Dayu

2010-12-22

Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signalingmore » pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what those patterns reveal about the origins of the lines and the stability of spatial expression patterns. In addition, we offer an initial analysis of previously unannotated transcripts in the cell lines.« less

BHD Tumor Cell Line and Renal Cell Carcinoma Line | NCI Technology Transfer Center | TTC

Cancer.gov

Scientists at the National Cancer Institute  have developed a novel renal cell carcinoma (RCC) cell line designated UOK257, which was derived from the surgical kidney tissue of a patient with hereditary Birt-Hogg-Dube''''(BHD) syndrome and companion cell line UOK257-2 in which FLCN expression has been restored by lentivirus infection. The NCI Urologic Oncology Branch seeks parties interested in licensing or collaborative research to co-develop, evaluate, or commercialize kidney cancer tumor cell lines.

Macrophage cell lines derived from major histocompatibility complex II-negative mice

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1998-01-01

Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

Xenobiotic metabolism in the fish hepatic cell lines Hepa-E1 and RTH-149, and the gill cell lines RTgill-W1 and G1B: Biomarkers of CYP450 activity and oxidative stress.

PubMed

Franco, Marco E; Sutherland, Grace E; Lavado, Ramon

2018-04-01

The use of fish cell cultures has proven to be an effective tool in the study of environmental and aquatic toxicology. Valuable information can be obtained from comparisons between cell lines from different species and organs. In the present study, specific chemicals were used and biomarkers (e.g. 7-Ethoxyresorufin-O-deethylase (EROD) activity and reactive oxygen species (ROS)) were measured to assess the metabolic capabilities and cytotoxicity of the fish hepatic cell lines Hepa-E1 and RTH-149, and the fish gill cell lines RTgill-W1 and G1B. These cell lines were exposed to β-naphthoflavone (BNF) and benzo[a]pyrene (BaP), the pharmaceutical tamoxifen (TMX), and the organic peroxide tert-butylhydroperoxide (tBHP). Cytotoxicity in gill cell lines was significantly higher than in hepatic cells, with BNF and TMX being the most toxic compounds. CYP1-like associated activity, measured through EROD activity, was only detected in hepatic cells; Hepa-E1 cells showed the highest activity after exposure to both BNF and BaP. Significantly higher levels of CYP3A-like activity were also observed in Hepa-E1 cells exposed to TMX, while gill cell lines presented the lowest levels. Measurements of ROS and antioxidant enzymes indicated that peroxide levels were higher in gill cell lines in general. However, levels of superoxide were significantly higher in RTH-149 cells, where no distinctive increase of superoxide-related antioxidants was observed. The present study demonstrates the importance of selecting adequate cell lines in measuring specific metabolic parameters and provides strong evidence for the fish hepatocarcinoma Hepa-E1 cells to be an excellent alternative in assessing metabolism of xenobiotics, and in expanding the applicability of fish cell lines for in vitro studies. Copyright © 2018 Elsevier Inc. All rights reserved.

An Online Compendium of CHO RNA-Seq Data Allows Identification of CHO Cell Line-specific Transcriptomic Signatures.

PubMed

Singh, Ankita; Kildegaard, Helene F; Andersen, Mikael R

2018-05-15

Chinese hamster ovary (CHO) cell lines can fold, assemble and modify proteins post-translationally to produce human-like proteins; as a consequence, it is the single most common expression systems for industrial production of recombinant therapeutic proteins. A thorough knowledge of cultivation conditions of different CHO cell lines has been developed over the last decade, but comprehending gene or pathway-specific distinctions between CHO cell lines at transcriptome level remains a challenge. To address these challenges, we compiled a compendium of 23 RNA-Seq studies from public and in-house data on CHO cell lines, i.e. CHO-S, CHO-K1 and DG44. Significantly differentially expressed (DE) genes particularly related to subcellular structure and macromolecular categories were used to identify differences between the cell lines. A R-based web application was developed specifically for CHO cell lines to further visualize expression values across different cell lines, and make available the normalized full CHO data set graphically as a CHO research community resource. This study quantitatively categorizes CHO cell lines based on patterns at transcriptomic level and detects gene and pathway specific key distinctions among sibling cell lines. Studies such as this can be used to select desired characteristics across various CHO cell lines. Furthermore, the availability of the data as an internet-based application can be applied to broad range of CHO engineering applications. This article is protected by copyright. All rights reserved.

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

PubMed

Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

2015-12-01

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

Continuous human cell lines and method of making same

DOEpatents

Stampfer, M.R.

1985-07-01

Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines.

PubMed Central

Cheng, J; Haas, M

1990-01-01

Human T-cell leukemia and T-cell acute lymphoblastic leukemia cell lines were studied for alterations in the p53 tumor suppressor gene. Southern blot analysis of 10 leukemic T-cell lines revealed no gross genomic deletions or rearrangements. Reverse transcription-polymerase chain reaction analysis of p53 mRNA indicated that all 10 lines produced p53 mRNA of normal size. By direct sequencing of polymerase chain reaction-amplified cDNA, we detected 11 missense and nonsense point mutations in 5 of the 10 leukemic T-cell lines studied. The mutations are primarily located in the evolutionarily highly conserved regions of the p53 gene. One of the five cell lines in which a mutation was detected possesses a homozygous point mutation in both p53 alleles, while the other four cell lines harbor from two to four different point mutations. An allelic study of two of the lines (CEM, A3/Kawa) shows that the two missense mutations found in each line are located on separate alleles, thus both alleles of the p53 gene may have been functionally inactivated by two different point mutations. Since cultured leukemic T-cell lines represent a late, fully tumorigenic stage of leukemic T cells, mutation of both (or more) alleles of the p53 gene may reflect the selection of cells possessing an increasingly tumorigenic phenotype, whether the selection took place in vivo or in vitro. Previously, we have shown that the HSB-2 T-cell acute lymphoblastic leukemia cell line had lost both alleles of the retinoblastoma tumor suppressor gene. Taken together, our data show that at least 6 of 10 leukemic T-cell lines examined may have lost the normal function of a known tumor suppressor gene, suggesting that this class of genes serves a critical role in the generation of fully tumorigenic leukemic T cells. Images PMID:2144611

Numerical solution of the Hele-Shaw equations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whitaker, N.

1987-04-01

An algorithm is presented for approximating the motion of the interface between two immiscible fluids in a Hele-Shaw cell. The interface is represented by a set of volume fractions. We use the Simple Line Interface Calculation method along with the method of fractional steps to transport the interface. The equation of continuity leads to a Poisson equation for the pressure. The Poisson equation is discretized. Near the interface where the velocity field is discontinuous, the discretization is based on a weak formulation of the continuity equation. Interpolation is used on each side of the interface to increase the accuracy ofmore » the algorithm. The weak formulation as well as the interpolation are based on the computed volume fractions. This treatment of the interface is new. The discretized equations are solved by a modified conjugate gradient method. Surface tension is included and the curvature is computed through the use of osculating circles. For perturbations of small amplitude, a surprisingly good agreement is found between the numerical results and linearized perturbation theory. Numerical results are presented for the finite amplitude growth of unstable fingers. 62 refs., 13 figs.« less

BIM expression in treatment naïve cancers predicts responsiveness to kinase inhibitors

PubMed Central

Faber, Anthony; Corcoran, Ryan B.; Ebi, Hiromichi; Sequist, Lecia V.; Waltman, Belinda A.; Chung, Euiheon; Incio, Joao; Digumarthy, Subba R.; Pollack, Sarah F.; Song, Youngchul; Muzikansky, Alona; Lifshits, Eugene; Roberge, Sylvie; Coffman, Erik J.; Benes, Cyril; Gómez, Henry; Baselga, Jose; Arteaga, Carlos L.; Rivera, Miguel N.; Dias-Santagata, Dora; Jain, Rakesh K.; Engelman, Jeffrey A.

2011-01-01

Cancers with specific genetic mutations are susceptible to selective kinase inhibitors. However, there is wide spectrum of benefit among cancers harboring the same sensitizing genetic mutations. Herein, we measured apoptotic rates among cell lines sharing the same driver oncogene following treatment with the corresponding kinase inhibitor. There was a wide range of kinase inhibitor-induced apoptosis despite comparable inhibition of the target and associated downstream signaling pathways. Surprisingly, pre-treatment RNA levels of the BH3-only pro-apoptotic BIM strongly predicted the capacity of EGFR, HER2, and PI3K inhibitors to induce apoptosis in EGFR mutant, HER2 amplified, and PIK3CA mutant cancers, respectively, but BIM levels did not predict responsiveness to standard chemotherapies. Furthermore, BIM RNA levels in EGFR mutant lung cancer specimens predicted response and duration of clinical benefit from EGFR inhibitors. These findings suggest assessment of BIM levels in treatment naïve tumor biopsies may indicate the degree of benefit from single-agent kinase inhibitors in multiple oncogene-addiction paradigms. PMID:22145099

Adaptive Molecular Evolution for 13,000 Phage Generations

PubMed Central

Wichman, Holly A.; Millstein, Jack; Bull, J. J.

2005-01-01

Bacteriophage φX174 was evolved on a continuous supply of sensitive hosts for 180 days (∼13,000 phage generations). The average rate of nucleotide substitution was nearly 0.2% (11 substitutions)/20 days, and, surprisingly, substitutions accumulated in a clock-like manner throughout the study, except for a low rate during the first 20 days. Rates of silent and missense substitutions varied over time and among genes. Approximately 40% of the 71 missense changes and 25% of the 58 silent changes have been observed in previous adaptations; the rate of parallel substitution was highest in the early phase of the evolution, but 7% of the later changes had evolved in previous studies of much shorter duration. Several lines of evidence suggest that most of the changes were adaptive, even many of the silent substitutions. The sustained, high rate of adaptive evolution for 180 days defies a model of adaptation to a constant environment. We instead suggest that continuing molecular evolution reflects a potentially indefinite arms race, stemming from high levels of co-infection and the resulting conflict among genomes competing within the same cell. PMID:15687276

Biosynthesis, structural, and functional attributes of tocopherols in planta; past, present, and future perspectives.

PubMed

Hussain, Nazim; Irshad, Faiza; Jabeen, Zahra; Shamsi, Imran Haider; Li, Zhilan; Jiang, Lixi

2013-07-03

Tocopherols are lipophilic molecules, ubiquitously synthesized in all photosynthetic organisms. Being a group of vitamin E compounds, they play an essential role in human nutrition and health. Despite their structural and functional attributes as important antioxidants in plants, it would be misleading to ignore the potential roles of tocopherols beyond their antioxidant properties in planta. Detailed characterization of mutants and transgenic plants, including Arabidopsis (vte1, vte2, vte4, and so on), maize (sxd1) mutants, and transgenic potato and tobacco lines altered in tocopherol biosynthesis and contents, has led to surprising outcomes regarding the additional functions of these molecules. Thus, the aim of this review is to highlight the past and present research findings on tocopherols' structural, biosynthesis, and functional properties in plants. Special emphasis is given to their suggested functions in planta, such as cell signaling, hormonal interactions, and coordinated response of tocopherols to other antioxidants under abiotic stresses. Moreover, some important questions about possible new functions of tocopherols will be discussed as future prospects to stimulate further research.

Neuroimmune Interface in the Comorbidity between Alcohol Use Disorder and Major Depression

PubMed Central

Neupane, Sudan Prasad

2016-01-01

Bidirectional communication links operate between the brain and the body. Afferent immune-to-brain signals are capable of inducing changes in mood and behavior. Chronic heavy alcohol drinking, typical of alcohol use disorder (AUD), is one such factor that provokes an immune response in the periphery that, by means of circulatory cytokines and other neuroimmune mediators, ultimately causes alterations in the brain function. Alcohol can also directly impact the immune functions of microglia, the resident immune cells of the central nervous system (CNS). Several lines of research have established the contribution of specific inflammatory mediators in the development and progression of depressive illness. Much of the available evidence in this field stems from cross-sectional data on the immune interactions between isolated AUD and major depression (MD). Given their heterogeneity as disease entities with overlapping symptoms and shared neuroimmune correlates, it is no surprise that systemic and CNS inflammation could be a critical determinant of the frequent comorbidity between AUD and MD. This review presents a summary and analysis of the extant literature on neuroimmune interface in the AUD–MD comorbidity. PMID:28082989

Fourier analysis of the cell shape of paired human urothelial cell lines of the same origin but of different grades of transformation.

PubMed

Ostrowski, K; Dziedzic-Goclawska, A; Strojny, P; Grzesik, W; Kieler, J; Christensen, B; Mareel, M

1986-01-01

The rationale of the present investigation is the observations made by many authors of changes in the molecular structure of the cell surface during the multistep process of malignant transformation. These changes may influence cell-matrix and cell-cell interactions and thereby cause changes in cell adhesiveness and cell shape. The aim of the present work was to investigate whether the development of various grades of transformation in vivo and in vitro of human urothelial cells is accompanied by significant changes in cell shape as measured by Fourier analysis. The following transformation grades (TGr) have been defined (Christensen et al. 1984; Kieler 1984): TGr I = nonmalignant, mortal cell lines that grow independently of fibroblasts and have a prolonged life span. TGr II = nonmalignant cell lines with an infinite life span. TGr III = malignant and immortal cell lines that grow invasively in co-cultures with embryonic chick heart fragments and possess tumorigenic properties after s.c. injection into nude mice. Comparisons of 4 pairs of cell lines were performed; each pair was of the same origin. Two pairs--each including a TGr I cell line (Hu 961b and Hu 1703S) compared to a TGr III cell line (Hu 961a or Hu 1703He)--were derived from two transitional cell carcinomas (TCC) containing a heterogeneous cell population. Two additional cell lines classified as TGr II (HCV-29 and Hu 609) were compared to two TGr III sublines (HCV-29T and Hu 609T, respectively) which arose by "spontaneous" transformation during propagation in vitro of the respective maternal TGr II-cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Exploitation of the host cell ubiquitin machinery by microbial effector proteins.

PubMed

Lin, Yi-Han; Machner, Matthias P

2017-06-15

Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu ( J. Cell Sci. 130 , 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' ( J. Cell Sci. 130 , 1981-1983). © 2017. Published by The Company of Biologists Ltd.

Mycoplasma Infection Alters Cancer Stem Cell Properties in Vitro.

PubMed

Gedye, Craig; Cardwell, Tracy; Dimopoulos, Nektaria; Tan, Bee Shin; Jackson, Heather; Svobodová, Suzanne; Anaka, Matthew; Behren, Andreas; Maher, Christopher; Hofmann, Oliver; Hide, Winston; Caballero, Otavia; Davis, Ian D; Cebon, Jonathan

2016-02-01

Cancer cell lines can be useful to model cancer stem cells. Infection with Mycoplasma species is an insidious problem in mammalian cell culture. While investigating stem-like properties in early passage melanoma cell lines, we noted poorly reproducible results from an aliquot of a cell line that was later found to be infected with Mycoplasma hyorhinis. Deliberate infection of other early passage melanoma cell lines aliquots induced variable and unpredictable effects on expression of putative cancer stem cell markers, clonogenicity, proliferation and global gene expression. Cell lines established in stem cell media (SCM) were equally susceptible. Mycoplasma status is rarely reported in publications using cultured cells to study the cancer stem cell hypothesis. Our work highlights the importance of surveillance for Mycoplasma infection while using any cultured cells to interrogate tumor heterogeneity.

The influence of lightning induced voltage on the distribution power line polymer insulators.

PubMed

Izadi, Mahdi; Abd Rahman, Muhammad Syahmi; Ab-Kadir, Mohd Zainal Abidin; Gomes, Chandima; Jasni, Jasronita; Hajikhani, Maryam

2017-01-01

Protection of medium voltage (MV) overhead lines against the indirect effects of lightning is an important issue in Malaysia and other tropical countries. Protection of these lines against the indirect effects of lightning is a major concern and can be improved by several ways. The choice of insulator to be used for instance, between the glass, ceramic or polymer, can help to improve the line performance from the perspective of increasing the breakdown strength. In this paper, the electrical performance of a 10 kV polymer insulator under different conditions for impulse, weather and insulator angle with respect to a cross-arm were studied (both experimental and modelling) and the results were discussed accordingly. Results show that the weather and insulator angle (with respect to the cross-arm) are surprisingly influenced the values of breakdown voltage and leakage current for both negative and positive impulses. Therefore, in order to select a proper protection system for MV lines against lightning induced voltage, consideration of the local information concerning the weather and also the insulator angles with respect to the cross-arm are very useful for line stability and performance.

The influence of lightning induced voltage on the distribution power line polymer insulators

PubMed Central

Ab-Kadir, Mohd Zainal Abidin; Gomes, Chandima; Jasni, Jasronita; Hajikhani, Maryam

2017-01-01

Protection of medium voltage (MV) overhead lines against the indirect effects of lightning is an important issue in Malaysia and other tropical countries. Protection of these lines against the indirect effects of lightning is a major concern and can be improved by several ways. The choice of insulator to be used for instance, between the glass, ceramic or polymer, can help to improve the line performance from the perspective of increasing the breakdown strength. In this paper, the electrical performance of a 10 kV polymer insulator under different conditions for impulse, weather and insulator angle with respect to a cross-arm were studied (both experimental and modelling) and the results were discussed accordingly. Results show that the weather and insulator angle (with respect to the cross-arm) are surprisingly influenced the values of breakdown voltage and leakage current for both negative and positive impulses. Therefore, in order to select a proper protection system for MV lines against lightning induced voltage, consideration of the local information concerning the weather and also the insulator angles with respect to the cross-arm are very useful for line stability and performance. PMID:28234930

Widom Lines in Binary Mixtures of Supercritical Fluids.

PubMed

Raju, Muralikrishna; Banuti, Daniel T; Ma, Peter C; Ihme, Matthias

2017-06-08

Recent experiments on pure fluids have identified distinct liquid-like and gas-like regimes even under supercritical conditions. The supercritical liquid-gas transition is marked by maxima in response functions that define a line emanating from the critical point, referred to as Widom line. However, the structure of analogous state transitions in mixtures of supercritical fluids has not been determined, and it is not clear whether a Widom line can be identified for binary mixtures. Here, we present first evidence for the existence of multiple Widom lines in binary mixtures from molecular dynamics simulations. By considering mixtures of noble gases, we show that, depending on the phase behavior, mixtures transition from a liquid-like to a gas-like regime via distinctly different pathways, leading to phase relationships of surprising complexity and variety. Specifically, we show that miscible binary mixtures have behavior analogous to a pure fluid and the supercritical state space is characterized by a single liquid-gas transition. In contrast, immiscible binary mixture undergo a phase separation in which the clusters transition separately at different temperatures, resulting in multiple distinct Widom lines. The presence of this unique transition behavior emphasizes the complexity of the supercritical state to be expected in high-order mixtures of practical relevance.

X-ray line coincidence photopumping in a solar flare

DOE PAGES

Keenan, F. P.; Poppenhaeger, K.; Mathioudakis, M.; ...

2017-11-23

Line coincidence photopumping is a process where the electrons of an atomic or molecular species are radiatively excited through the absorption of line emission from another species at a coincident wavelength. There are many instances of line coincidence photopumping in astrophysical sources at optical and ultraviolet wavelengths, with the most famous example being Bowen fluorescence (pumping of O III 303.80 Å by He II), but none to our knowledge in X-rays. However, here we report on a scheme where a He-like line of Ne IX at 11.000 Å is photopumped by He-like Na X at 11.003 Å, which predicts significantmore » intensity enhancement in the Ne IX 82.76 Å transition under physical conditions found in solar flare plasmas. A comparison of our theoretical models with published X-ray observations of a solar flare obtained during a rocket flight provides evidence for line enhancement, with the measured degree of enhancement being consistent with that expected from theory, a truly surprising result. Observations of this enhancement during flares on stars other than the Sun would provide a powerful new diagnostic tool for determining the sizes of flare loops in these distant, spatially unresolved, astronomical sources.« less

X-ray line coincidence photopumping in a solar flare

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keenan, F. P.; Poppenhaeger, K.; Mathioudakis, M.

Line coincidence photopumping is a process where the electrons of an atomic or molecular species are radiatively excited through the absorption of line emission from another species at a coincident wavelength. There are many instances of line coincidence photopumping in astrophysical sources at optical and ultraviolet wavelengths, with the most famous example being Bowen fluorescence (pumping of O III 303.80 Å by He II), but none to our knowledge in X-rays. However, here we report on a scheme where a He-like line of Ne IX at 11.000 Å is photopumped by He-like Na X at 11.003 Å, which predicts significantmore » intensity enhancement in the Ne IX 82.76 Å transition under physical conditions found in solar flare plasmas. A comparison of our theoretical models with published X-ray observations of a solar flare obtained during a rocket flight provides evidence for line enhancement, with the measured degree of enhancement being consistent with that expected from theory, a truly surprising result. Observations of this enhancement during flares on stars other than the Sun would provide a powerful new diagnostic tool for determining the sizes of flare loops in these distant, spatially unresolved, astronomical sources.« less

X-ray line coincidence photopumping in a solar flare

NASA Astrophysics Data System (ADS)

Keenan, F. P.; Poppenhaeger, K.; Mathioudakis, M.; Rose, S. J.; Flowerdew, J.; Hynes, D.; Christian, D. J.; Nilsen, J.; Johnson, W. R.

2018-03-01

Line coincidence photopumping is a process where the electrons of an atomic or molecular species are radiatively excited through the absorption of line emission from another species at a coincident wavelength. There are many instances of line coincidence photopumping in astrophysical sources at optical and ultraviolet wavelengths, with the most famous example being Bowen fluorescence (pumping of O III 303.80 Å by He II), but none to our knowledge in X-rays. However, here we report on a scheme where a He-like line of Ne IX at 11.000 Å is photopumped by He-like Na X at 11.003 Å, which predicts significant intensity enhancement in the Ne IX 82.76 Å transition under physical conditions found in solar flare plasmas. A comparison of our theoretical models with published X-ray observations of a solar flare obtained during a rocket flight provides evidence for line enhancement, with the measured degree of enhancement being consistent with that expected from theory, a truly surprising result. Observations of this enhancement during flares on stars other than the Sun would provide a powerful new diagnostic tool for determining the sizes of flare loops in these distant, spatially unresolved, astronomical sources.

The HepaRG cell line: biological properties and relevance as a tool for cell biology, drug metabolism, and virology studies.

PubMed

Marion, Marie-Jeanne; Hantz, Olivier; Durantel, David

2010-01-01

Liver progenitor cells may play an important role in carcinogenesis in vivo and represent therefore useful cellular materials for in vitro studies. The HepaRG cell line, which is a human bipotent progenitor cell line capable to differentiate toward two different cell phenotypes (i.e., biliary-like and hepatocyte-like cells), has been established from a liver tumor associated with chronic hepatitis C. This cell line represents a valuable alternative to ex vivo cultivated primary human hepatocytes (PHH), as HepaRG cells share some features and properties with adult hepatocytes. The cell line is particularly useful to evaluate drugs and perform drug metabolism studies, as many detoxifying enzymes are expressed and functional. It is also an interesting tool to study some aspect of progenitor biology (e.g., differentiation process), carcinogenesis, and the infection by some pathogens for which the cell line is permissive (e.g., HBV infection). Overall, this chapter gives a concise overview of the biological properties and potential applications of this cell line.

[Expression of Chemokine receptor CXCR6 and its significance in breast cancer cell lines].

PubMed

Cheng, Hao; Chen, Nian-yong

2014-05-01

To detect the expression of Chemokine receptor CXCR6 in invasive breast cancer cell lines and normal mammary epithelial cell line, and assess the relationship between CXCR6 expression and malignant behavior of breast cancer cells. Expression level of CXCR6 in different invasive breast cancer cell lines (SK-BR-3, MCF-7, MDA-MB-231) and normal mammary epithelial cell line (MCF-10A)was detected by real time reverse transcription-polymerase chain reaction (real time-PCR) and Western blot. Lentivirus was employed to interfere CXCR6 expression in MDA-MB-231. MTT assay and transwell chamber were used to study proliferative and invasive ability of those cells respectively. Vascular enothelial growth factor (VEGF) expression was detected to study the role of CXCR6 in angiogenesis. At both mRNA level and protein level, normal mammary epithelial cell line MCF-10A showed the weakest CXCR6 expression. The breast cancer cell lines expressed CXCR6 in different levels, the expression level of CXCR6 in highly invasive cell line MDA-MB-231 was significantly higher than that in two low-invasive cell lines SK-BR-3 and MCF-7 (P < 0.05). Silencing CXCR6 gene by Lentivirus-mediated RNA interference in MDA-MB-231 inhibited its proliferation ability, invasion ability and angiogenesis ability in vitro (P < 0.05). Different invasive breast cancer cell lines express CXCR6 at different levels, positively correlated with its invasive ability.

Cytogenetics of small cell carcinoma of the lung.

PubMed

Wurster-Hill, D H; Cannizzaro, L A; Pettengill, O S; Sorenson, G D; Cate, C C; Maurer, L H

1984-12-01

Nineteen cell lines derived from various malignant tissues of 15 patients with small cell carcinoma of the lung (SCCL) have been studied. The results showed heterogeneity in all cell lines, with no one consistent abnormality among them. Cell lines from 11 of the patients had minute and double minute chromosomes, and cell lines from 2 patients had abnormally banding regions, designated as ABRs, as distinguished from homogeneously staining regions (HSRs). The latter 2 and several of the former cell lines were derived from specimens taken before the patients were placed on therapy. All but 2 of the cell lines had a constant marker load, consisting of 24%-35% of the complement. Some markers remained stable through months and years of culture life, while other markers came and went. Chromosomes #1, #6 and #11 were most frequently involved in marker formation in the cell lines, and these were compared to similar markers in direct bone marrow preparations. Chromosome #1 markers were of variable structure, whereas #6 and #11 most often took the form of 6q- and 11p+ markers, with breakpoints most frequently at 6q23-25 and 11p11-12. A 3p- marker was found in a minority of cell lines. All of these markers were also found in direct marrow preparations from some patients with SCCL. Nonmonoclonal tumors arose from inoculation of bimodal cell lines into nude mice, but population selection by undetermined mechanism was evident. Cytogenetic parameters showed no positive correlation with hormone production by these cell lines.

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

PubMed

Zwollo, Patty; Ray, Jocelyn C; Sestito, Michael; Kiernan, Elizabeth; Wiens, Gregory D; Kaattari, Steve; StJacques, Brittany; Epp, Lidia

2015-01-01

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1β, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout. Copyright © 2014 Elsevier Ltd. All rights reserved.

Forced neuronal interactions cause poor communication.

PubMed

Krzisch, Marine; Toni, Nicolas

2017-01-01

Post-natal hippocampal neurogenesis plays a role in hippocampal function, and neurons born post-natally participate to spatial memory and mood control. However, a great proportion of granule neurons generated in the post-natal hippocampus are eliminated during the first 3 weeks of their maturation, a mechanism that depends on their synaptic integration. In a recent study, we examined the possibility of enhancing the synaptic integration of neurons born post-natally, by specifically overexpressing synaptic cell adhesion molecules in these cells. Synaptic cell adhesion molecules are transmembrane proteins mediating the physical connection between pre- and post-synaptic neurons at the synapse, and their overexpression enhances synapse formation. Accordingly, we found that overexpressing synaptic adhesion molecules increased the synaptic integration and survival of newborn neurons. Surprisingly, the synaptic adhesion molecule with the strongest effect on new neurons' survival, Neuroligin-2A, decreased memory performances in a water maze task. We present here hypotheses explaining these surprising results, in the light of the current knowledge of the mechanisms of synaptic integration of new neurons in the post-natal hippocampus.

Cytotoxic components of Pereskia bleo (Kunth) DC. (Cactaceae) leaves.

PubMed

Malek, Sri Nurestri Abdul; Shin, Sim Kae; Wahab, Norhanom Abdul; Yaacob, Hashim

2009-05-06

Dihydroactinidiolide (1) and a mixture of sterols [campesterol (2), stigmasterol (3) and beta-sitosterol (4)], together with the previously isolated individual compounds beta-sitosterol (4), 2,4-di-tert-butylphenol (5), alpha-tocopherol (6), phytol (7) were isolated from the active ethyl acetate fraction of Pereskia bleo (Kunth) DC. (Cactaceae) leaves. Cytotoxic activities of the above mentioned compounds against five human carcinoma cell lines, namely the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), human hormone-dependent breast carcinoma cell line (MCF7) and human lung carcinoma cell line (A549); and non-cancer human fibroblast cell line (MRC-5) were investigated. Compound 5 possessed very remarkable cytotoxic activity against KB cells, with an IC(50 )value of 0.81microg/mL. This is the first report on the cytotoxic activities of the compounds isolated from Pereskia bleo.

Innate responses to gene knockouts impact overlapping gene networks and vary with respect to resistance to viral infection.

PubMed

Liu, Yonghong; Liu, Yuanyuan; Wu, Jiaming; Roizman, Bernard; Zhou, Grace Guoying

2018-04-03

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: ( i ) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. ( ii ) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

Comparative study of the photodynamic effect in tumor and nontumor animal cell lines

NASA Astrophysics Data System (ADS)

Stoykova, Elena V.; Alexandrova, R.; Shurulinkov, Stanislav; Sabotinov, O.; Minchev, Georgi

2004-09-01

In this study we evaluate the cytotoxicity of two photosensitisers with absorption peaks in the green and red part of the spectrum on animal cell lines. The cytotoxicity assessment was performed for a tumor cell line LSCC-SF-Mc29, obtained from a transplantable chicken hepatoma induced by the myelocytomatosis virus Mc29, a tumor line LSR-SF-SR, obtained from a transplantable sarcoma in rat induced by Rous sarcoma virus strain Schmidt-Ruppin and for normal mouse and bovine cell lines. Up to now the effect of the photodynamic therapy on virus-induced cancers has not been clarified. The cells were treated with 5,10,15,20 - tetra (4-sulfophenyl) porphyrin with main absorption peak at 519 nm and a dye activated with a red light. The cells were seeded in 96-well plates at 2 x 104 cells/well. The cells were exposed to irradiation from a pulsed CuBr vapor laser at 510.6 nm and 578.2 nm and exposure rate 50 mW/cm2, from an Ar-ion laser at 514 nm and 1 mW/cm2 and to 655 nm-irradiation from a semiconductor laser at 10 mW/cm2. The biological activity of the tested compounds was measured by the neutral red uptake cytotoxicity test. The light dose-response curves and light exposures that ensure 50% drop in the treated cells viability in comparison with the cells grown in non-modified medium were obtained for each cell line. The cytotoxic effect of both photosensitisers is most distinguished for the tumor line LSCC-SF-Mc29. The 2-4 times higher viability of the normal cell lines in comparison with the tumor lines is established. The bovine cell lines are more vulnerable than the mouse lines.

Establishment and characterization of Asian oral cancer cell lines as in vitro models to study a disease prevalent in Asia.

PubMed

Hamid, Sharifah; Lim, Kue Peng; Zain, Rosnah Binti; Ismail, Siti Mazlipah; Lau, Shin Hin; Mustafa, Wan Mahadzir Wan; Abraham, M Thomas; Nam, Noor Akmar; Teo, Soo-Hwang; Cheong, Sok Ching

2007-03-01

We have established 3 cell lines ORL-48, -115 and -136 from surgically resected specimens obtained from untreated primary human oral squamous cell carcinomas of the oral cavity. The in vitro growth characteristics, epithelial origin, in vitro anchorage independency, human papilloma-virus (HPV) infection, microsatellite instability status, karyotype and the status of various cell cycle regulators and gatekeepers of these cell lines were investigated. All 3 cell lines grew as monolayers with doubling times ranging between 26.4 and 40.8 h and were immortal. Karyotyping confirmed that these cell lines were of human origin with multiple random losses and gains of entire chromosomes and regions of chromosomes. Immunohistochemistry staining of cytokeratins confirmed the epithelial origin of these cell lines, and the low degree of anchorage independency expressed by these cell lines suggests non-transformed phenotypes. Genetic analysis identified mutations in the p53 gene in all cell lines and hypermethylation of p16INK4a in ORL-48 and -136. Analysis of MDM2 and EGFR expression indicated MDM2 overexpression in ORL-48 and EGFR overexpression in ORL-136 in comparison to the protein levels in normal oral keratinocytes. Analysis of the BAT-26 polyadenine repeat sequence and MLH-1 and MSH-2 repair enzymes demonstrated that all 3 cell lines were microsatellite stable. The role of HPV in driving carcinogenesis in these tumours was negated by the absence of HPV. Finally, analysis of the tissues from which these cell lines were derived indicated that the cell lines were genetically representative of the tumours, and, therefore, are useful tools in the understanding of the molecular changes associated with oral cancers.

Bio-Physicochemical Interactions of Engineered Nanomaterials in In Vitro Cell Culture Model

DTIC Science & Technology

2012-08-14

viz. human hepatocarcinoma cell line (Hep G2), human adinocarcinoma cell line (A549), human embryonic kidney cell line (HEK 293), human neuroblastoma...Glutamine, 1% Na-Pyruvate and 10 ml/L antibiotic solution at 37oC under a humidified atmosphere of 5% CO2/95% air.  Human hepatocarcinoma cell line

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

9 CFR 113.52 - Requirements for cell lines used for production of biologics.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A cell...

Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland).

PubMed

Goswami, M; Sharma, B S; Tripathi, A K; Yadav, Kamalendra; Bahuguna, S N; Nagpure, N S; Lakra, W S; Jena, J K

2012-05-25

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies. Copyright © 2012 Elsevier B.V. All rights reserved.

Planetary Building Blocks Found in Surprising Place

NASA Technical Reports Server (NTRS)

2005-01-01

[figure removed for brevity, see original site] Figure 1

This graph of data from NASA's Spitzer Space Telescope shows that an extraordinarily low-mass brown dwarf, or 'failed star,' is circled by a disc of planet-building dust. The brown dwarf, called OTS 44, is only 15 times the mass of Jupiter, making it the smallest known brown dwarf to host a planet-forming disc.

Spitzer was able to see this unusual disc by measuring its infrared brightness. Whereas a brown dwarf without a disc (red dashed line) radiates infrared light at shorter wavelengths, a brown dwarf with a disc (orange line) gives off excess infrared light at longer wavelengths. This surplus light comes from the disc itself and is represented here as a yellow dotted line. Actual data points from observations of OTS 44 are indicated with orange dots. These data were acquired using Spitzer's infrared array camera.

A Comparison of Observed Abundances in Five Well-Studied Planetary Nebulae

NASA Astrophysics Data System (ADS)

Tanner, Jolene; Balick, B.; Kwitter, K. B.

2013-01-01

We have assembled data and derived abundances in several recent careful studies for five bright planetary nebulae (PNe) of low, moderate, and high ionization and relatively simple morphology. Each of the studies employ different apertures, aperture placement, and facilities for the observations. Various methods were used to derive total abundances. All used spectral windows that included [OII]3727 in the UV through Argon lines in the red. Our ultimate goal is to determine the extent to which the derived abundances are consistent. We show that the reddening-corrected line ratios are surprisingly similar despite the different modes of observation and that the various abundance analysis methods yield generally consistent results for He/H, N/H, O/H, and Ne/H (within 50% with a few larger deviations). In addition we processed the line ratios from the different sources using a common abundance derivation method (ELSA) to search for clues of systematic methodological inconsistencies. None were uncovered.

Subclones in B-lymphoma cell lines: isogenic models for the study of gene regulation

PubMed Central

Quentmeier, Hilmar; Pommerenke, Claudia; Ammerpohl, Ole; Geffers, Robert; Hauer, Vivien; MacLeod, Roderick AF; Nagel, Stefan; Romani, Julia; Rosati, Emanuela; Rosén, Anders; Uphoff, Cord C; Zaborski, Margarete; Drexler, Hans G

2016-01-01

Genetic heterogeneity though common in tumors has been rarely documented in cell lines. To examine how often B-lymphoma cell lines are comprised of subclones, we performed immunoglobulin (IG) heavy chain hypermutation analysis. Revealing that subclones are not rare in B-cell lymphoma cell lines, 6/49 IG hypermutated cell lines (12%) consisted of subclones with individual IG mutations. Subclones were also identified in 2/284 leukemia/lymphoma cell lines exhibiting bimodal CD marker expression. We successfully isolated 10 subclones from four cell lines (HG3, SU-DHL-5, TMD-8, U-2932). Whole exome sequencing was performed to molecularly characterize these subclones. We describe in detail the clonal structure of cell line HG3, derived from chronic lymphocytic leukemia. HG3 consists of three subclones each bearing clone-specific aberrations, gene expression and DNA methylation patterns. While donor patient leukemic cells were CD5+, two of three HG3 subclones had independently lost this marker. CD5 on HG3 cells was regulated by epigenetic/transcriptional mechanisms rather than by alternative splicing as reported hitherto. In conclusion, we show that the presence of subclones in cell lines carrying individual mutations and characterized by sets of differentially expressed genes is not uncommon. We show also that these subclones can be useful isogenic models for regulatory and functional studies. PMID:27566572

The Cellosaurus, a Cell-Line Knowledge Resource

PubMed Central

Bairoch, Amos

2018-01-01

The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server (https://web.expasy.org/cellosaurus/) and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences. PMID:29805321

Expression and rearrangement of the ROS1 gene in human glioblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Birchmeier, C.; Sharma, S.; Wigler, M.

1987-12-01

The human ROS1 gene, which possibly encodes a growth factor receptor, was found to be expressed in human tumor cell lines. In a survey of 45 different human cell lines, the authors found ROS1 to be expressed in glioblastoma-derived cell lines at high levels and not to be expressed at all, or expressed at very low levels, in the remaining cell lines. The ROS1 gene was present in normal copy numbers in all cell lines that expressed the gene. However, in one particular glioblastoma line, they detected a potentially activating mutation at the ROS1 locus.

Establishment and characterization of two new cell lines from the mosquito Armigeres subalbatus (Coquillett) (Diptera: Culicidae).

PubMed

Hoshino, Keita; Isawa, Haruhiko; Kuwata, Ryusei; Tajima, Shigeru; Takasaki, Tomohiko; Iwabuchi, Kikuo; Sawabe, Kyoko; Kobayashi, Mutsuo; Sasaki, Toshinori

2015-08-01

Armigeres subalbatus (Coquillett) is a medically important mosquito and a model species for immunology research. We successfully established two cell lines from the neonate larvae of A. subalbatus using two different media. To our knowledge, this is the first report of an established Armigeres mosquito cell line. The cell lines, designated as Ar-3 and Ar-13, consist of adherent and diploid cells with compact colonies. Both these cell lines grow slowly after passage at a split ratio of 1:5 and a population doubling time of 2.7 and 3.0 d, respectively. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to confirm that these lines correspond to the species of origin and are clearly distinct from seven other insect cell lines. Furthermore, reverse-transcription PCR was used to demonstrate that the Ar-3 cell line is susceptible to the Japanese encephalitis virus and two insect flaviviruses associated with Culex and Aedes mosquitoes but relatively insensitive to dengue virus. These data indicate that the newly established cell lines are cellular models of A. subalbatus as well as beneficial tools for the propagation of viruses associated with the Armigeres mosquito.

Deleterious CHEK2 1100delC and L303X mutants identified among 38 human breast cancer cell lines.

PubMed

Wasielewski, Marijke; Hanifi-Moghaddam, Pejman; Hollestelle, Antoinette; Merajver, Sofia D; van den Ouweland, Ans; Klijn, Jan G M; Ethier, Stephen P; Schutte, Mieke

2009-01-01

The CHEK2 protein plays a major role in the regulation of DNA damage response pathways. Mutations in the CHEK2 gene, in particular 1100delC, have been associated with increased cancer risks, but the precise function of CHEK2 mutations in carcinogenesis is not known. Human cancer cell lines with CHEK2 mutations are therefore of main interest. Here, we have sequenced 38 breast cancer cell lines for mutations in the CHEK2 gene and identified two cell lines with deleterious CHEK2 mutations. Cell line UACC812 has a nonsense truncating mutation in the CHEK2 kinase domain (L303X) and cell line SUM102PT has the well-known oncogenic CHEK2 1100delC founder mutation. Immunohistochemical analysis revealed that the two CHEK2 mutant cell lines expressed neither CHEK2 nor P-Thr(68) CHEK2 proteins, implying abrogation of normal CHEK2 DNA repair functions. Cell lines UACC812 and SUM102PT thus are the first human CHEK2 null cell lines reported and should therefore be a major help in further unraveling the function of CHEK2 mutations in carcinogenesis.

The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

DTIC Science & Technology

1996-08-01

J-4030 TITLE: The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle PRINCIPAL...The In Vivo DNA Binding Properties of 5. FUNDING NUMBERS Wild-Type and Mutant p53 Proteins in Mammary Cell Lines DAMD17-94-J-4030 During the Course of...ABSTRACT (Maximum 200 Using a pair of murine cell lines, one lacking p53 and a derivative cell line containing temperature sensitive p53 val 135

Tofacitinib induces G1 cell-cycle arrest and inhibits tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells.

PubMed

Ando, Shotaro; Kawada, Jun-Ichi; Watanabe, Takahiro; Suzuki, Michio; Sato, Yoshitaka; Torii, Yuka; Asai, Masato; Goshima, Fumi; Murata, Takayuki; Shimizu, Norio; Ito, Yoshinori; Kimura, Hiroshi

2016-11-22

Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

HuH-7 reference genome profile: complex karyotype composed of massive loss of heterozygosity.

PubMed

Kasai, Fumio; Hirayama, Noriko; Ozawa, Midori; Satoh, Motonobu; Kohara, Arihiro

2018-05-17

Human cell lines represent a valuable resource as in vitro experimental models. A hepatoma cell line, HuH-7 (JCRB0403), has been used extensively in various research fields and a number of studies using this line have been published continuously since it was established in 1982. However, an accurate genome profile, which can be served as a reliable reference, has not been available. In this study, we performed M-FISH, SNP microarray and amplicon sequencing to characterize the cell line. Single cell analysis of metaphases revealed a high level of heterogeneity with a mode of 60 chromosomes. Cytogenetic results demonstrated chromosome abnormalities involving every chromosome in addition to a massive loss of heterozygosity, which accounts for 55.3% of the genome, consistent with the homozygous variants seen in the sequence analysis. We provide empirical data that the HuH-7 cell line is composed of highly heterogeneous cell populations, suggesting that besides cell line authentication, the quality of cell lines needs to be taken into consideration in the future use of tumor cell lines.

Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.

PubMed

Hollingsworth, M A; Strawhecker, J M; Caffrey, T C; Mack, D R

1994-04-15

We examined the steady-state expression levels of mRNA for the MUC1, MUC2, MUC3 and MUC4 gene products in 12 pancreatic tumor cell lines, 6 colon tumor cell lines, and one ileocecal tumor cell line. The results showed that 10 of 12 pancreatic tumor cell lines expressed MUC1 mRNA and that 7 of these 12 lines also expressed relatively high levels of MUC4 mRNA. In contrast, MUC2 mRNA was expressed at only low levels and MUC3 was not detected in the pancreatic tumor cell lines. All 7 intestinal tumor cell lines examined expressed MUC2, and 5 of 7 expressed MUC3; however only one expressed significant levels of MUC1 and 2 expressed low levels of MUC4 mRNA. This report of high levels of MUC4 mRNA expression by pancreatic tumor cells raises the possibility that mucin carbohydrate epitopes defined by antibodies such as DuPan 2 may be expressed on a second mucin core protein produced by pancreatic tumor cells.

Expression of apoptosis-regulatory genes in lung tumour cell lines: relationship to p53 expression and relevance to acquired drug resistance.

PubMed Central

Reeve, J. G.; Xiong, J.; Morgan, J.; Bleehen, N. M.

1996-01-01

As a first step towards elucidating the potential role(s) of bcl-2 and bcl-2-related genes in lung tumorigenesis and therapeutic responsiveness, the expression of these genes has been examined in a panel of lung cancer cell lines derived from untreated and treated patients, and in cell lines selected in vitro for multidrug resistance. Bcl-2 was hyperexpressed in 15 of 16 small-cell lung cancer (SCLC) cell lines and two of five non-small-cell lung cancer (NSCLC) lines compared with normal lung and brain, and hyperexpression was not chemotherapy related. Bcl-x was hyperexpressed in the majority of SCLC and NSCLC cell lines as compared with normal tissues, and all lung tumour lines preferentially expressed bcl-x1-mRNA, the splice variant form that inhibits apoptosis. Bax gene transcripts were hyperexpressed in most SCLC and NSCLC cell lines examined compared with normal adult tissues. Mutant p53 gene expression was detected in the majority of the cell lines and no relationship between p53 gene expression and the expression of either bcl-2, bcl-x or bax was observed. No changes in bcl-2, bcl-x and bax gene expression were observed in multidrug-resistant cell lines compared with their drug-sensitive counterparts. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8630278

Establishment and characterization of a new fish cell line from head kidney of half-smooth tongue sole (Cynoglossus semilaevis).

PubMed

Zheng, Yuan; Wang, Na; Xie, Ming-Shu; Sha, Zhen-Xia; Chen, Song-Lin

2012-12-01

A new cell line (TSHKC) derived from half-smooth tongue sole (Cynoglossus semilaevis) head kidney was developed. The cell line was subcultured for 40 passages over a period of 360 days. The cell line was optimally maintained in minimum essential medium supplemented with HEPES, antibiotics, fetal bovine serum, 2-Mercaptoethanol (2-Me), sodium pyruvate and basic fibroblast growth factor. The suitable growth temperature for TSHKC cells was 24 °C, and microscopically, TSHKC cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TSHKC cell line had a normal diploid karyotype with 2n = 42, contained the heterogametic W chromosome. The TSHKC cell line was found to be susceptible to lymphocystis disease virus. The fluorescent signals were observed in TSHKC when the cells were transfected with green fluorescent protein and red fluorescent protein reporter plasmids.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment.

PubMed

Eguchi, Takanori; Sogawa, Chiharu; Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-Ichi; Okamoto, Kuniaki; Calderwood, Stuart K

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment

PubMed Central

Okusha, Yuka; Uchibe, Kenta; Iinuma, Ryosuke; Ono, Kisho; Nakano, Keisuke; Murakami, Jun; Itoh, Manabu; Arai, Kazuya; Fujiwara, Toshifumi; Namba, Yuri; Murata, Yoshiki; Ohyama, Kazumi; Shimomura, Manami; Okamura, Hirohiko; Takigawa, Masaharu; Nakatsura, Tetsuya; Kozaki, Ken-ichi; Okamoto, Kuniaki; Calderwood, Stuart K.

2018-01-01

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression. PMID:29415026

Cytogenetic damage, oncogenic transformation and p53 induction in human epithelial cells in response to irradiation

NASA Astrophysics Data System (ADS)

Armitage, Mark

Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or mutant conformation in all the above cells lines and two other control lines HOS (a human osteosarcoma cell line) and H Tori-3 (SV40 immortalised thyroid epithelial cells).

Genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia cell line

PubMed Central

STOCZYNSKA-FIDELUS, EWELINA; PIASKOWSKI, SYLWESTER; PAWLOWSKA, ROZA; SZYBKA, MALGORZATA; PECIAK, JOANNA; HULAS-BIGOSZEWSKA, KRYSTYNA; WINIECKA-KLIMEK, MARTA; RIESKE, PIOTR

2016-01-01

Thorough examination of genetic heterogeneity of cell lines is uncommon. In order to address this issue, the present study analyzed the genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia (T-ALL) cell line. For this purpose, traditional techniques such as fluorescence in situ hybridization and immunocytochemistry were used, in addition to more advanced techniques, including cell sorting, Sanger sequencing and massive parallel sequencing. The results indicated that the RPMI-8402 cell line consists of several genetically different cell subpopulations. Furthermore, massive parallel sequencing of RPMI-8402 provided insight into the evolution of T-ALL carcinogenesis, since this cell line exhibited the genetic heterogeneity typical of T-ALL. Therefore, the use of cell lines for drug testing in future studies may aid the progress of anticancer drug research. PMID:26870252

The effect of syndecan-4 and glypican-1 knockdown on the proliferation and differentiation of turkey satellite cells differing in age and growth rates.

PubMed

Velleman, Sandra G; Clark, Daniel L; Tonniges, Jeffrey R

2018-09-01

Posthatch skeletal muscle growth requires myogenic satellite cells and the dynamic expression of cell membrane-associated proteins. The membrane associated heparan sulfate proteoglycans, syndecan-4 and glypican-1, link the satellite cell niche to the intracellular environment. Sydnecan-4 and glypican-1 are differentially expressed with age in turkey satellite cells and their over-expression impacts both satellite cell proliferation and differentiation, but their effect on satellite cells from lines with different growth potentials is not known. The objective of the current study was to determine if syndecan-4 and glypican-1 regulation of satellite cell proliferation and differentiation is affected by age and growth selection. Pectoralis major satellite cells isolated at 1 d, 7 and 16-wk of age from a Randombred Control 2 (RBC2) line and a 16-wk body weight (F) line selected from the RBC2 line turkeys were studied. Syndecan-4 and glypican-1 expression was knocked down in both lines. The F-line cells proliferated faster than RBC2 line cells regardless of age, while differentiation tended to be greater in RBC2 line cells than F-line cells at each age. Syndecan-4 knockdown decreased proliferation at 7- and 16-wk but not 1 d cells, and increased differentiation at 1 d and 7 wk but not 16 wk cells. Glypican-1 knockdown differentially affected proliferation depending on cell age, whereas differentiation was decreased for 7- and 16-wk but not 1 d cells. These data suggest syndecan-4 and glypican-1 differentially affected satellite cell function in an age-dependent manner, but had little impact on differences in proliferation and differentiation due to growth selection. Copyright © 2018. Published by Elsevier Inc.

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

PubMed Central

Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.

1974-01-01

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225

History of leukemia-lymphoma cell lines.

PubMed

Drexler, Hans G; Macleod, Roderick A F

2010-08-01

We outline the near 50-year history of leukemia-lymphoma (LL) cell lines - a key model system in biomedicine. Due to the detailed documentation of their oncogenomic and transcriptional alterations via recent advances in molecular medicine, LL cell lines may be fitted to parent tumors with a degree of precision unattainable in other cancers. We have surveyed the corpus of published LL cell lines and found 637 examples that meet minimum standards of authentication and characterization. Alarmingly, the rate of establishment of new LL cell lines has plummeted over the last decade. Although the main hematopoietic developmental cell types are represented by cell lines, some LL categories stubbornly resist establishment in vitro. The advent of engineering techniques for immortalizing primary human cells that maintain differentiation means the time is ripe for renewed search for in vitro models from un(der)represented hematologic entities. Given their manifold applications in biomedicine, there is little doubt that LL-derived cell lines will continue to play a vital part well into the next half-century as well. © 2010 The Authors. Human Cell © 2010 Japan Human Cell Society.

Molecular characterization of immortalized normal and dysplastic oral cell lines.

PubMed

Dickman, Christopher T D; Towle, Rebecca; Saini, Rajan; Garnis, Cathie

2015-05-01

Cell lines have been developed for modeling cancer and cancer progression. The molecular background of these cell lines is often unknown to those using them to model disease behaviors. As molecular alterations are the ultimate drivers of cell phenotypes, having an understanding of the molecular make-up of these systems is critical for understanding the disease biology modeled. Six immortalized normal, one immortalized dysplasia, one self-immortalized dysplasia, and two primary normal cell lines derived from oral tissues were analyzed for DNA copy number changes and changes in both mRNA and miRNA expression using SMRT-v.2 genome-wide tiling comparative genomic hybridization arrays, Agilent Whole Genome 4x44k expression arrays, and Exiqon V2.M-RT-PCR microRNA Human panels. DNA copy number alterations were detected in both normal and dysplastic immortalized cell lines-as well as in the single non-immortalized dysplastic cell line. These lines were found to have changes in expression of genes related to cell cycle control as well as alterations in miRNAs that are deregulated in clinical oral squamous cell carcinoma tissues. Immortal lines-whether normal or dysplastic-had increased disruption in expression relative to primary lines. All data are available as a public resource. Molecular profiling experiments have identified DNA, mRNA, and miRNA alterations for a panel of normal and dysplastic oral tissue cell lines. These data are a valuable resource to those modeling diseases of the oral mucosa, and give insight into the selection of model cell lines and the interpretation of data from those lines. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Prioritization of anticancer drugs against a cancer using genomic features of cancer cells: A step towards personalized medicine

PubMed Central

Gupta, Sudheer; Chaudhary, Kumardeep; Kumar, Rahul; Gautam, Ankur; Nanda, Jagpreet Singh; Dhanda, Sandeep Kumar; Brahmachari, Samir Kumar; Raghava, Gajendra P. S.

2016-01-01

In this study, we investigated drug profile of 24 anticancer drugs tested against a large number of cell lines in order to understand the relation between drug resistance and altered genomic features of a cancer cell line. We detected frequent mutations, high expression and high copy number variations of certain genes in both drug resistant cell lines and sensitive cell lines. It was observed that a few drugs, like Panobinostat, are effective against almost all types of cell lines, whereas certain drugs are effective against only a limited type of cell lines. Tissue-specific preference of drugs was also seen where a drug is more effective against cell lines belonging to a specific tissue. Genomic features based models have been developed for each anticancer drug and achieved average correlation between predicted and actual growth inhibition of cell lines in the range of 0.43 to 0.78. We hope, our study will throw light in the field of personalized medicine, particularly in designing patient-specific anticancer drugs. In order to serve the scientific community, a webserver, CancerDP, has been developed for predicting priority/potency of an anticancer drug against a cancer cell line using its genomic features (http://crdd.osdd.net/raghava/cancerdp/). PMID:27030518

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells.

PubMed

Fan, F; Bellister, S; Lu, J; Ye, X; Boulbes, D R; Tozzi, F; Sceusi, E; Kopetz, S; Tian, F; Xia, L; Zhou, Y; Bhattacharya, R; Ellis, L M

2015-02-03

Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs. Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment. None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not. PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

Nonlinear mixed effects dose response modeling in high throughput drug screens: application to melanoma cell line analysis.

PubMed

Ding, Kuan-Fu; Petricoin, Emanuel F; Finlay, Darren; Yin, Hongwei; Hendricks, William P D; Sereduk, Chris; Kiefer, Jeffrey; Sekulic, Aleksandar; LoRusso, Patricia M; Vuori, Kristiina; Trent, Jeffrey M; Schork, Nicholas J

2018-01-12

Cancer cell lines are often used in high throughput drug screens (HTS) to explore the relationship between cell line characteristics and responsiveness to different therapies. Many current analysis methods infer relationships by focusing on one aspect of cell line drug-specific dose-response curves (DRCs), the concentration causing 50% inhibition of a phenotypic endpoint (IC 50 ). Such methods may overlook DRC features and do not simultaneously leverage information about drug response patterns across cell lines, potentially increasing false positive and negative rates in drug response associations. We consider the application of two methods, each rooted in nonlinear mixed effects (NLME) models, that test the relationship relationships between estimated cell line DRCs and factors that might mitigate response. Both methods leverage estimation and testing techniques that consider the simultaneous analysis of different cell lines to draw inferences about any one cell line. One of the methods is designed to provide an omnibus test of the differences between cell line DRCs that is not focused on any one aspect of the DRC (such as the IC 50 value). We simulated different settings and compared the different methods on the simulated data. We also compared the proposed methods against traditional IC 50 -based methods using 40 melanoma cell lines whose transcriptomes, proteomes, and, importantly, BRAF and related mutation profiles were available. Ultimately, we find that the NLME-based methods are more robust, powerful and, for the omnibus test, more flexible, than traditional methods. Their application to the melanoma cell lines reveals insights into factors that may be clinically useful.

A Novel Inhibitor Of Topoisomerase I is Selectively Toxic For A Subset of Non-Small Cell Lung Cancer Cell Lines | Office of Cancer Genomics

Cancer.gov

SW044248, identified through a screen for chemicals that are selectively toxic for NSCLC cell lines, was found to rapidly inhibit macromolecular synthesis in sensitive, but not in insensitive cells. SW044248 killed approximately 15% of a panel of 74 NSCLC cell lines and was non-toxic to immortalized human bronchial cell lines.

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Establishment and characterization of five immortalized human scalp dermal papilla cell lines.

PubMed

Kwack, Mi Hee; Yang, Jung Min; Won, Gong Hee; Kim, Moon Kyu; Kim, Jung Chul; Sung, Young Kwan

2018-02-05

Dermal papilla (DP) regulates the growth and cycling of hair follicles. Cultured DP cells are useful for the study of their role in relation to hair growth and regeneration. However, cultivation of human DP cells is tedious and difficult. In addition, cultured DP cells possess a relatively short replicative life span, requiring immortalized human DP cell lines. We previously established an immortalized human DP cell line, SV40T-hTERT-DPC, by introducing human telomerase reverse transcriptase (hTERT) gene into the transformed cell line, SV40T-DPC. In this study, we co-transfected the simian virus 40 large T antigen (SV40T-Ag) and hTERT into DP cells from scalp hair follicles from a male with androgenetic alopecia and established five immortalized DP cell lines and named KNU-101, KNU-102, KNU-103, KNU-201 and KNU-202. We then evaluated tumorigenicity, expression of DP markers, responses to androgen, Wnt3a and BMP4, and expression of DP signature genes. These cell lines displayed early passage morphology and maintained responses to androgen, Wnt and BMP. Furthermore, these cell lines expressed DP markers and DP signature genes. KNU cell lines established in this study are potentially useful sources for hair research. Copyright © 2018 Elsevier Inc. All rights reserved.

76 FR 42678 - Submission for OMB Review; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-19

... collection). Burden Hours: 250. Number of Respondents: 100 (15 cell line limit). Average Hours per Response: 2 hours and 30 minutes (10 minutes/cell line x 15 cell lines). Needs and Uses: The NIST Biochemical...: National Institute of Standards and Technology (NIST) Title: Identification of Human Cell Lines Project...

Heterogeneity in cancer cells: variation in drug response in different primary and secondary colorectal cancer cell lines in vitro.

PubMed

Arul, Melanie; Roslani, April Camilla; Cheah, Swee Hung

2017-05-01

Tumor heterogeneity may give rise to differential responses to chemotherapy drugs. Therefore, unraveling tumor heterogeneity has an implication for biomarker discovery and cancer therapeutics. To test this phenomenon, we investigated the differential responses of three secondary colorectal cancer cell lines of different origins (HCT116, HT29, and SW620 cells) and four novel primary cell lines obtained from different colorectal cancer patients to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP) and explored the differences in gene expression among the primary cell lines in response to exposure to cytotoxic drugs. Cells were exposed to different doses of 5-FU and L-OHP separately or in combinations of equitoxic drug or equimolar drug ratios (median effect of Chou-Talalay principle). Cell viability was assessed using MTT assay and the respective IC 50 values were determined. Changes in gene expression in primary cell lines after exposure to the same drug doses were compared using real-time PCR array. The sensitivities (IC 50 ) of different cell lines, both secondary and primary, to 5-FU and L-OHP were significantly different, whether in monotherapy or combined treatment. Primary cell lines needed higher doses to reach IC 50 . There were variations in gene expression among the primary cell lines of different chemosensitivities to the challenge of the same combined dose of 5-FU and L-OHP. The results confirm the heterogeneous nature of colorectal cancer cells from different patient tumors. Studies using primary cancer cells established from patient's tumors rather than secondary cell lines will more closely reflect the actual character of the disease.

Oxygen and Cell Fate Decisions

DTIC Science & Technology

2009-05-27

mesenchymal stem cells may still be able to commit to chondro- genic differentiation under hypoxic conditions in vivo. Surprisingly, Malladi et al. have...are induced to undergo differentiation in vitro at 2% O2 ( Malladi et al. 2006). Nonetheless, targeted deletion of HIF-1α in adipose-derived adult... Malladi et al. 2007). These dis- crepancies could potentially be appreciated from at least two perspectives. (1) Chondrogenic dif- ferentiation is

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination.

PubMed

Korch, Christopher; Spillman, Monique A; Jackson, Twila A; Jacobsen, Britta M; Murphy, Susan K; Lessey, Bruce A; Jordan, V Craig; Bradford, Andrew P

2012-10-01

Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models. Copyright © 2012 Elsevier Inc. All rights reserved.

Effect of capping agents on the cytotoxicity of silver nanoparticles in human normal and cancer skin cell lines

NASA Astrophysics Data System (ADS)

Netchareonsirisuk, Ponsawan; Puthong, Songchan; Dubas, Stephan; Palaga, Tanapat; Komolpis, Kittinan

2016-11-01

Silver nanoparticles (AgNPs) are among the most widely used nanomaterials in medical and consumer products. However, safety in the uses of AgNPs is still controversial. The toxicity of AgNPs toward various cell types has been reported to depend on the surface properties of the nanoparticles. In this study, the effect of AgNPs with the average size of 5-15 nm on the viability of the CCD-986SK human normal skin fibroblast cell line and A375 human malignant melanoma cell line was evaluated. Comparative toxicity studies, based on MTT assay, were performed by using either sodium alginate or poly (4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA) as capping agent in the nanoparticle preparation. The cytotoxicity tests revealed that AgNO3 alone was highly toxic to both cell types while both alginate and PSSMA alone were not toxic. AgNPs capped with alginate were selectively toxic to the cancer cell line but not to the normal cell line while AgNPs capped with PSSMA were toxic to both cancer and normal cell lines. Judging from the 50 % inhibition concentration (IC50), it was found that the cancer cell line was more sensitive to AgNPs than the normal cell line. Study on the mode of cell death by annexin V and propidium iodide staining revealed that AgNPs induced more apoptotic cell death (84-90 %) than necrosis (8-12 %) in the skin cancer cell line. These results suggest that the toxicity of AgNPs depended on the type of capping agent and the type of cell line.

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

PubMed Central

van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

The combination effect of sodium butyrate and 5-Aza-2'-deoxycytidine on radiosensitivity in RKO colorectal cancer and MCF-7 breast cancer cell lines.

PubMed

Cho, Hang Joo; Kim, Sin Young; Kim, Kee Hwan; Kang, Won Kyung; Kim, Ji Il; Oh, Seong Tack; Kim, Jeong Soo; An, Chang Hyeok

2009-05-21

The overall level of chromatin compaction is an important mechanism of radiosensitivity, and modification of DNA methylation and histone deacetylation may increase radiosensitivity by altering chromatin compaction. In this study, we investigated the effect of a demethylating agent, a histone deacetylase(HDAC) inhibitor, and the two agents combined on radiosensitivity in human colon and breast cancer cell lines. In this study, we used RKO colorectal cancer cell line and MCF-7 breast cancer cell lines and normal colon cell lines. On each of the cell lines, we used three different agents: the HDAC inhibitor sodium butyrate(SB), the demethylating agent 5-Aza-2'-deoxycytidine(5-aza-DC), and radiation. We then estimated the percentage of the cell survival using the XTT method and experimented to determine if there was an augmentation in the therapeutic effect by using different combinations of the two or three of the treatment methods. After treatment of each cell lines with 5-aza-DC, SB and 6 grays of radiation, we observed that the survival fraction was lower after the treatment with 5-aza-DC or SB than with radiation alone in RKO and MCF-7 cell lines(p < 0.001). The survival fraction was lowest when the two agents, 5-aza-DC and SB were combined with radiation in both RKO and MCF-cell lines. In conclusion, 5-aza-DC and SB can enhance radiosensitivity in both MCF-7 and RKO cell lines. The combination effect of a demethylating agent and an HDAC inhibitor is more effective than that of single agent treatment in both breast and colon cancer cell lines.

[The characters and specific features of new human embryonic stem cells lines].

PubMed

Krylova, T A; Kol'tsova, A M; Zenin, V V; Gordeeva, O F; Musorina, A S; Goriachaia, T S; Shlykova, S A; Kamenetskaia, Iu K; Pinaev, G P; Polianskaia, G G

2009-01-01

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives

PubMed Central

Dumont, Jennifer; Euwart, Don; Mei, Baisong; Estes, Scott; Kshirsagar, Rashmi

2016-01-01

Abstract Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins. PMID:26383226

Newly established human retinoblastoma cell lines exhibit an "immortalized" but not an invasive phenotype in vitro.

PubMed

Griegel, S; Hong, C; Frötschl, R; Hülser, D F; Greger, V; Horsthemke, B; Rajewsky, M F

1990-07-15

Retinoblastoma (RB), an intraocular childhood tumor occurring in a hereditary (mostly bilateral) or non-hereditary (unilateral) form, is associated with the inactivation of both alleles of a putative tumor suppressor gene (RB-I) located on chromosome 13q14. Both the process of RB development and the biological characteristics of RB cells are as yet poorly understood. We have established 7 new RBL lines (RBL13, RBL14, RBL18 and RBL30, derived from unilateral RB; and RBL7, RBL15 and RBL20, derived from bilateral RB). Southern blot analyses of restriction fragment length polymorphisms in DNA samples from 6 cell lines revealed loss of constitutional heterozygosity at one or several polymorphic loci on chromosome 13 in 4 cases. Gross deletions involving the RB-I locus and amplification of the N-myc gene were not detected in any of the RBL lines. The phenotypic properties of the RBL lines were analyzed in comparison with cells from the original RB tumors, with 4 RB lines established by others (RB383, RB355, RB247C3 and Y79) and with the adenovirus-EIA-transformed human retinoblast line HER-Xhol-CC2. It was found that RB tumors consist of phenotypically heterogeneous cell subpopulations with varying nutrient requirements and differentiation potential in vitro. All cell lines showed the typical characteristics of established ("immortalized") cells. In some cases, cells from original RB tumors or cell lines were able to form colonies when cell aggregates of 2-10 cells were suspended in semi-solid agar medium; however, anchorage-independent colonies never developed from single cells. Cell lines RBL13, RBL18, RB247C3, RB355, RB383 and Y79 were tested for invasion into embryonic chick heart fragments in vitro and found to be non-invasive. None of the RBL or RB lines were tumorigenic in nu/nu (T-) mice. Y79 cells (propagated in culture for many years) exhibited properties distinctly different from those of the other cell lines, and thus cannot be considered phenotypically representative of RB cells.

DNA Methylation as an Epigenetic Factor in the Development and Progression of Polycythemia Vera

DTIC Science & Technology

2008-10-01

vera and idiopathic myelofibrosis. Pathol Biol ( Paris ). 2001;49:164-166. 2. Spivak JL. Diagnosis of the myeloproliferative disorders: resolving...leukemia cell lines with different cellular origin (myeloid cell lines KG1, KG1a, HEL, K562, and TF1; T lymphoid cell lines CEM and JTAg; and B lymphoid...in the cell lines of lymphoid origin versus myeloid leukemia cell lines and a GM-CSF- Services Email this article to a friend Download to

Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)

PubMed Central

Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko

2012-01-01

Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD. PMID:23818740

Establishment and proteomic characterization of a novel synovial sarcoma cell line, NCC-SS2-C1.

PubMed

Oyama, Rieko; Kito, Fusako; Sakumoto, Marimu; Shiozawa, Kumiko; Toki, Shunichi; Endo, Makoto; Yoshida, Akihiko; Kawai, Akira; Kondo, Tadashi

2018-05-01

Synovial sarcoma is an aggressive mesenchymal tumor, characterized by the presence of unique transfusion gene, SS18-SSX. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of SS18-SSX in relevant cellular contexts. We report the establishment and proteomic characterization of a novel synovial sarcoma cell line. Primary tissue culture was performed using tumor tissue of synovial sarcoma. The established cell line was authenticated by assessing its DNA microsatellite short tandem repeat analysis and characterized by in vitro assay. Proteomic study was achieved by mass spectrometry, and the results were analyzed by treemap. The cell line NCC-SS2-C1 was established from a primary tumor tissue of a synovial sarcoma patient. The cell line has grown well for 11 mo and has been subcultured more than 15 times. The established cells were authenticated by assessing their short tandem repeat pattern comparing with that of original tumor tissue. The cells showed polygonal in shape and formed spheroid when seeded on the low-attachment dish. Proteomic analysis revealed the molecular pathways which are unique to the original tumor tissue or the established cell line. In conclusion, a novel synovial sarcoma cell line NCC-SS2-C1 was successfully established from the primary tumor tissue. The cell line has characteristic transfusion SS18-SSX and poses aggressive in vitro growth and capability of spheroid formation. Thus, NCC-SS2-C1 cell line will be a useful tool for investigation of the mechanisms of disease and the biological role of fusion gene.

Anticancer drugs are synergistic with freezing in induction of apoptosis in HCC cells.

PubMed

Yuan, FangJun; Zhou, Wenbo; Zhang, Jifa; Zhang, Zhiyun; Zou, Can; Huang, Ling; Zhang, YouShun; Dai, Zongqing

2008-08-01

Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from -15 to -80 degrees C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.

Participation of an abnormality in the transforming growth factor-beta signaling pathway in resistance of malignant glioma cells to growth inhibition induced by that factor.

PubMed

Zhang, Lei; Sato, Eiji; Amagasaki, Kenichi; Nakao, Atsuhito; Naganuma, Hirofumi

2006-07-01

Malignant glioma cells secrete and activate transforming growth factor-beta (TGFbeta) and are resistant to growth inhibition by that factor. Nevertheless, the mechanism underlying this effect remains poorly understood. In this study, the mechanism of the resistance to growth inhibition induced by TGFbeta was investigated. The authors examined the expression of downstream components of the TGFbeta receptor, including Smad2, Smad3, Smad4, and Smad7, and the effect of TGFbeta1 treatment on the phosphorylation of Smad2 and the nuclear translocation of Smad2 and Smad3 by using 10 glioma cell lines and the A549 cell line, which is sensitive to TGFbeta-mediated growth inhibition. The expression of two transcriptional corepressor proteins, SnoN and Ski, and the effect of TGFbeta1 treatment on the expression of the SnoN protein and the cell cycle regulators p21, p15, cyclin-dependent kinase-4 (CDK4), and cyclin D1 were also examined. Expression of the Smad2 and Smad3 proteins was lower in the glioma cell lines than in the A549 cell line and in normal astrocytes. In particular, Smad3 expression was low or very low in nine of the 10 malignant glioma cell lines. Expression of Smad4 was low in four glioma cell lines, and expression of the Smad7 protein was similar when compared with protein expression in the A549 cell line and in normal astrocytes. The levels of Smad2 phosphorylation after TGFbeta1 treatment were lower in glioma cell lines than in the A549 cell line, except for one glioma cell line. Seven of the 10 glioma cell lines exhibited lower levels of nuclear translocation of Smad2 and Smad3, and two cell lines that expressed very low levels of Smad3 protein showed no nuclear translocation. All glioma cell lines expressed the SnoN protein and its expression was unaltered by treatment with TGFbeta1. Three glioma cell lines expressed high levels of the Ski protein. The expression of the p21(cip1), p15(INK4B), CDK4, and cyclin D1 proteins was not altered by TGFbeta1, treatment, except in one cell line that displayed a slight increase in p21 protein. Overall, the expression of the Smad2 and Smad3 proteins was low in the glioma cell lines, the phosphorylation and nuclear translocation of Smad2 and Smad3 were impaired, and the TGFbeta receptor signal did not affect the expression of the SnoN, p21, p15, cyclin D1, and CDK4 proteins. These results suggest that the ability to resist TGFbeta-mediated growth inhibition in malignant glioma cells is due to abnormalities in the TGFbeta signaling pathway.

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

PubMed

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1. Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines, but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation, cytokine release); and (iii) whether coculture experiments are included.

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line.

PubMed

Islam, M Q; Ringe, J; Reichmann, E; Migotti, R; Sittinger, M; da S Meirelles, L; Nardi, N B; Magnusson, P; Islam, K

2006-10-01

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.

Lung cancer cell lines: Useless artifacts or invaluable tools for medical science?

PubMed Central

Gazdar, Adi F.; Gao, Boning; Minna, John D.

2011-01-01

Multiple cell lines (estimated at 300–400) have been established from human small cell (SCLC) and non-small cell lung cancers (NSCLC). These cell lines have been widely dispersed to and used by the scientific community worldwide, with over 8000 citations resulting from their study. However, there remains considerable skepticism on the part of the scientific community as to the validity of research resulting from their use. These questions center around the genomic instability of cultured cells, lack of differentiation of cultured cells and absence of stromal–vascular–inflammatory cell compartments. In this report we discuss the advantages and disadvantages of the use of cell lines, address the issues of instability and lack of differentiation. Perhaps the most important finding is that every important, recurrent genetic and epigenetic change including gene mutations, deletions, amplifications, translocations and methylation-induced gene silencing found in tumors has been identified in cell lines and vice versa. These “driver mutations” represented in cell lines offer opportunities for biological characterization and application to translational research. Another potential shortcoming of cell lines is the difficulty of studying multistage pathogenesis in vitro.To overcome this problem, we have developed cultures from central and peripheral airways that serve as models for the multistage pathogenesis of tumors arising in these two very different compartments. Finally the issue of cell line contamination must be addressed and safeguarded against. A full understanding of the advantages and shortcomings of cell lines is required for the investigator to derive the maximum benefit from their use. PMID:20079948

Low-dose non-targeted radiation effects in human esophageal adenocarcinoma cell lines.

PubMed

Hanu, Christine; Wong, Raimond; Sur, Ranjan K; Hayward, Joseph E; Seymour, Colin; Mothersill, Carmel

2017-02-01

To investigate non-targeted radiation effects in esophageal adenocarcinoma cell lines (OE19 and OE33) using human keratinocyte and colorectal cancer cell reporters following γ-ray exposure. Both clonogenic assays and ratiometric calcium endpoints were used to check for the occurrence of bystander signals in reporter cells. We report data suggesting that γ-irradiation increases cell killing over the expected linear quadratic (LQ) model levels in the OE19 cell line exposed to doses below 1 Gy, i.e. which may be suggestive to be a low hyper-radiosensitive (HRS) response to direct irradiation. Both EAC cell lines (OE19 and OE33) have the ability to produce bystander signals when irradiated cell conditioned medium (ICCM) is placed onto human keratinocyte reporters, but do not seem to be capable of responding to bystander signals when placed on their autologous reporters. Further work with human keratinocyte reporter models showed statistically significant intracellular calcium fluxes following exposure of the reporters to ICCM harvested from both EAC cell lines exposed to 0.5 Gy. These experiments suggest that the OE19 and OE33 cell lines produce bystander signals in human keratinocyte reporter cells. However, the radiosensitivity of the EAC cell lines used in this study cannot be enhanced by the bystander response since both cell lines could not respond to bystander signals.

Effect of leukemia inhibitory factor and forskolin on establishment of rat embryonic stem cell lines.

PubMed

Hirabayashi, Masumi; Goto, Teppei; Tamura, Chihiro; Sanbo, Makoto; Hara, Hiromasa; Hochi, Shinichi

2014-03-07

This study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (LIF) and/or forskolin would support establishment of germline-competent rat embryonic stem (ES) cell lines. Due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without LIF contributed to the higher establishment efficiency of ES cell lines in the WDB strain. Germline transmission competency of the chimeric rats was not influenced by the profile of ES cell lines until their establishment. When the LIF/forskolin-supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the WI strain, was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, all lines were found to be germline-competent by a progeny test in chimeric rats. In conclusion, both LIF and forskolin are not essential but can play a beneficial role in the establishment of "genuine" rat ES cell lines.

Cellular characteristics of primary and immortal canine embryonic fibroblast cells.

PubMed

You, Seungkwon; Moon, Jai-Hee; Kim, Tae-Kyung; Kim, Sung-Chan; Kim, Jai-Woo; Yoon, Du-Hak; Kwak, Sungwook; Hong, Ki-Chang; Choi, Yun-Jaie; Kim, Hyunggee

2004-08-31

Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.

B7-H4 as a Target for Breast Cancer Immunotherapy

DTIC Science & Technology

2012-06-01

lymphoma and leukemia cell lines. CEM, Karpas 299, and TLBR -1, cell lines derived from acute T-cell lymphoblastic leukemia, large cell anaplastic...Accomplishments  Generation of human B7-H4-Fc fusion protein (antigen).  Discovery of a B7-H4 receptor on CEM, Karpas 299, and TLBR -1 cell lines...CEM Karpas 299 TLBR -1 Jurkat B7-H4R Figure 3. B7-H4 binding to human T-cell lymphoma cell lines. Red

Novel Tissue Protective Agents for the Treatment of Acute Radiation-induced BMF

DTIC Science & Technology

2013-03-01

induced apoptosis in the following hematopoietic cell lines: HL-60, NB-4 cells, 32Dc13 and EML cell line. Experimental design and methods: HL-60, a...et al., 1999). EML Cell line (ATCC CRL-11691), a bone marrow cell line obtained by immortalizing bone marrow cells from male BDF1 mice with a...Membrane preparations were made from HL-60, NB-4, 32Dc13 and EML cells attempts were made to co-immunoprecipitate the CD131 molecule with EPOR in

A Reasoned Response to Nimby Opposition to Incineration of Chemical Weapon Stockpiles

DTIC Science & Technology

1993-04-01

retrograde operation on July 23, 1990.194 Greenpeace International and the Sierra Club. Legal Defense Fund each submitted extensive comments to the SSEIS .1...95 Since the ROD based its conclusion on the SSEIS , it came as no surprise when Greenpeace sought a temporary restraining order in August 1990.196...6 The supporting site- specific EIS ( SSEIS ) published in May fell in line with the PFEIS and endorsed JACADS reverse assembly incineration technology