PLOS ONE: [sortOrder=DATE_NEWEST_FIRST, from=editorLink, sort=Relevance, q=editor:"Mikhail V Blagosklonny"]PLOShttps://journals.plos.org/plosone/webmaster@plos.orgaccelerating the publication of peer-reviewed sciencehttps://journals.plos.org/plosone/search/feed/atom?sortOrder=DATE_NEWEST_FIRST&unformattedQuery=editor:%22Mikhail%20V%20Blagosklonny%22&from=editorLink&sort=RelevanceAll PLOS articles are Open Access.https://journals.plos.org/plosone/resource/img/favicon.icohttps://journals.plos.org/plosone/resource/img/favicon.ico2024-03-28T23:09:34ZTargeted Delivery of Chemotherapy Agents Using a Liver Cancer-Specific AptamerLing MengLiu YangXiangxuan ZhaoLucy ZhangHaizhen ZhuChen LiuWeihong Tan10.1371/journal.pone.00334342012-04-25T14:00:00Z2012-04-25T14:00:00Z<p>by Ling Meng, Liu Yang, Xiangxuan Zhao, Lucy Zhang, Haizhen Zhu, Chen Liu, Weihong Tan</p>
Background <p>Using antibody/aptamer-drug conjugates can be a promising method for decreasing toxicity, while increasing the efficiency of chemotherapy.</p> Methodology/Principal Findings <p>In this study, the antitumor agent Doxorubicin (Dox) was incorporated into the modified DNA aptamer TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. Cell viability tests demonstrated that the TLS11a-GC-Dox conjugates exhibited both potency and target specificity. Importantly, intercalating Dox into the modified aptamer inhibited nonspecific uptake of membrane-permeable Dox to the non-target cell line. Since the conjugates are selective for cells that express higher amounts of target proteins, both criteria noted above are met, making TLS11a-GC-Dox conjugates potential candidates for targeted delivery to liver cancer cells.</p> Conclusions/Significance <p>Considering the large number of available aptamers that have specific targets for a wide variety of cancer cells, this novel aptamer-drug intercalation method will have promising implications for chemotherapeutics in general.</p>Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1α Protein under Hypoxic ConditionsAlexander LaemmleAntje LechleiterVincent RohChrista SchwarzSimone PortmannCynthia FurerAdrian KeoghMario P. TschanDaniel CandinasStephan A. VorburgerDeborah Stroka10.1371/journal.pone.00334332012-03-30T14:00:00Z2012-03-30T14:00:00Z<p>by Alexander Laemmle, Antje Lechleiter, Vincent Roh, Christa Schwarz, Simone Portmann, Cynthia Furer, Adrian Keogh, Mario P. Tschan, Daniel Candinas, Stephan A. Vorburger, Deborah Stroka</p>
Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models <i>in vitro</i> and in <i>in vivo</i> models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.Activation Status of Wnt/ß-Catenin Signaling in Normal and Neoplastic Breast Tissues: Relationship to HER2/neu Expression in Human and MouseSara KhalilGrace A. TanDilip D. GiriXi Kathy ZhouLouise R. Howe10.1371/journal.pone.00334212012-03-23T14:00:00Z2012-03-23T14:00:00Z<p>by Sara Khalil, Grace A. Tan, Dilip D. Giri, Xi Kathy Zhou, Louise R. Howe</p>
Wnt/ß-catenin signaling is strongly implicated in neoplasia, but the role of this pathway in human breast cancer has been controversial. Here, we examined Wnt/ß-catenin pathway activation as a function of breast cancer progression, and tested for a relationship with HER2/neu expression, using a human tissue microarray comprising benign breast tissues, ductal carcinoma <i>in situ</i> (DCIS), and invasive carcinomas. Cores were scored for membranous ß-catenin, a key functional component of adherens junctions, and for nucleocytoplasmic ß-catenin, a hallmark of Wnt/ß-catenin pathway activation. Only 82% of benign samples exhibited membrane-associated ß-catenin, indicating a finite frequency of false-negative staining. The frequency of membrane positivity was similar in DCIS samples, but was significantly reduced in carcinomas (45%, <i>P</i><0.001), consistent with loss of adherens junctions during acquisition of invasiveness. Negative membrane status in cancers correlated with higher grade (<i>P</i> = 0.04) and estrogen receptor-negative status (<i>P</i> = 0.03), both indices of poor prognosis. Unexpectedly, a substantial frequency of nucleocytoplasmic ß-catenin was observed in benign breast tissues (36%), similar to that in carcinomas (35%). Positive-staining basal nuclei observed in benign breast may identify putative stem cells. An increased frequency of nucleocytoplasmic ß-catenin was observed in DCIS tumors (56%), suggesting that pathway activation may be an early event in human breast neoplasia. A correlation was observed between HER2/neu expression and nucleocytoplasmic ß-catenin in node-positive carcinomas (<i>P</i> = 0.02). Furthermore, cytoplasmic ß-catenin was detected in HER2/neu-induced mouse mammary tumors. The <i>Axin2NLSlacZ</i> mouse strain, a previously validated reporter of mammary Wnt/ß-catenin signaling, was utilized to define <i>in vivo</i> transcriptional consequences of HER2/neu-induced ß-catenin accumulation. Discrete hyperplastic foci observed in mammary glands from bigenic MMTV/<i>neu</i>, <i>Axin2NLSlacZ</i> mice, highlighted by robust ß-catenin/TCF signaling, likely represent the earliest stage of mammary intraepithelial neoplasia in MMTV/<i>neu</i> mice. Our study thus provides provocative evidence for Wnt/ß-catenin signaling as an early, HER2/neu-inducible event in breast neoplasia.Daf-2 Signaling Modifies Mutant SOD1 Toxicity in <i>C. elegans</i>Marco BoccittoTodd LamitinaRobert G. Kalb10.1371/journal.pone.00334942012-03-20T14:00:00Z2012-03-20T14:00:00Z<p>by Marco Boccitto, Todd Lamitina, Robert G. Kalb</p>
The DAF-2 Insulin/IGF-1 signaling (IIS) pathway is a strong modifier of <i>Caenorhabditis elegans</i> longevity and healthspan. As aging is the greatest risk factor for developing neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), we were interested in determining if DAF-2 signaling modifies disease pathology in mutant superoxide dismutase 1 (SOD1) expressing <i>C. elegans</i>. Worms with pan-neuronal G85R SOD1 expression demonstrate significantly impaired locomotion as compared to WT SOD1 expressing controls and they develop insoluble SOD1 aggregates. Reductions in DAF-2 signaling, either through a hypomorphic allele or neuronally targeted RNAi, decreases the abundance of aggregated SOD1 and results in improved locomotion in a DAF-16 dependant manner. These results suggest that manipulation of the DAF-2 Insulin/IGF-1 signaling pathway may have therapeutic potential for the treatment of ALS.Mechanism of RPE Cell Death in α-Crystallin Deficient Mice: A Novel and Critical Role for MRP1-Mediated GSH EffluxParameswaran G. SreekumarChristine SpeeStephen J. RyanSusan P. C. ColeRam KannanDavid R. Hinton10.1371/journal.pone.00334202012-03-19T14:00:00Z2012-03-19T14:00:00Z<p>by Parameswaran G. Sreekumar, Christine Spee, Stephen J. Ryan, Susan P. C. Cole, Ram Kannan, David R. Hinton</p>
Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Interacts with the Ku70 Subunit of the DNA-Dependent Protein Kinase ComplexPhilip J. JensikJodi I. HuggenvikMichael W. Collard10.1371/journal.pone.00334042012-03-19T14:00:00Z2012-03-19T14:00:00Z<p>by Philip J. Jensik, Jodi I. Huggenvik, Michael W. Collard</p>
Deformed Epidermal Autoregulatory Factor 1 (DEAF1) is a transcription factor linked to suicide, cancer, autoimmune disorders and neural tube defects. To better understand the role of DEAF1 in protein interaction networks, a GST-DEAF1 fusion protein was used to isolate interacting proteins in mammalian cell lysates, and the XRCC6 (Ku70) and the XRCC5 (Ku80) subunits of DNA dependent protein kinase (DNA-PK) complex were identified by mass spectrometry, and the DNA-PK catalytic subunit was identified by immunoblotting. Interaction of DEAF1 with Ku70 and Ku80 was confirmed to occur within cells by co-immunoprecipitation of epitope-tagged proteins, and was mediated through interaction with the Ku70 subunit. Using <i>in vitro</i> GST-pulldowns, interaction between DEAF1 and the Ku70 subunit was mapped to the DEAF1 DNA binding domain and the C-terminal Bax-binding region of Ku70. In transfected cells, DEAF1 and Ku70 colocalized to the nucleus, but Ku70 could not relocalize a mutant cytoplasmic form of DEAF1 to the nucleus. Using an <i>in vitro</i> kinase assay, DEAF1 was phosphorylated by DNA-PK in a DNA-independent manner. Electrophoretic mobility shift assays showed that DEAF1 or Ku70/Ku80 did not interfere with the DNA binding of each other, but DNA containing DEAF1 binding sites inhibited the DEAF1-Ku70 interaction. The data demonstrates that DEAF1 can interact with the DNA-PK complex through interactions of its DNA binding domain with the carboxy-terminal region of Ku70 that contains the Bax binding domain, and that DEAF1 is a potential substrate for DNA-PK.miR-181a Post-Transcriptionally Downregulates Oncogenic RalA and Contributes to Growth Inhibition and Apoptosis in Chronic Myelogenous Leukemia (CML)Jia FeiYumin LiXuejiao ZhuXiaochuang Luo10.1371/journal.pone.00328342012-03-19T14:00:00Z2012-03-19T14:00:00Z<p>by Jia Fei, Yumin Li, Xuejiao Zhu, Xiaochuang Luo</p>
MicroRNAs (miRNAs) are a class of short RNAs that regulate gene expression through either translational repression or mRNA cleavage. miRNA-181a (miR-181a), one of the many miRNAs conserved among vertebrates, is differentially expressed in a variety of leukemia. However, its function in leukemia, particularly chronic myelogenous leukemia (CML), is poorly understood. Here we have reported the identification of miR-181a targets by combining TargetScan software prediction and expression profiling through overexpression of miR-181a mimic in leukemic K562 cells. Four overlapping genes were found to be the likely targets of miR-181a. Among the four genes, RalA is a downstream molecule of bcr-abl fusion protein in ras signaling pathway. However, its role in CML remains elusive. Luciferase reporter and Western blot assays confirmed that RalA is a direct target of miR-181a. overexpression of miR-181a effectively suppresses cell growth and induces G2-phase arrest and apoptosis partially by targeting RalA in leukemic K562 cells. Using the KEGG database combined with recent publications, downstream signaling pathway of RalA was graphed by cytoscape software. Therefore, our study is the first to report that RalA is directly regulated by miR-181a and plays an important role in CML. The approach of computational prediction combined with expression profiling might be valuable for the identification of miRNA targets in animal.Androgen Receptor Drives Cellular SenescenceYelena MirochnikDorina VeliceasaLatanya WilliamsKelly MaxwellAlexander YemelyanovIrina BudunovaOlga V. Volpert10.1371/journal.pone.00310522012-03-05T14:00:00Z2012-03-05T14:00:00Z<p>by Yelena Mirochnik, Dorina Veliceasa, Latanya Williams, Kelly Maxwell, Alexander Yemelyanov, Irina Budunova, Olga V. Volpert</p>
The accepted androgen receptor (AR) role is to promote proliferation and survival of prostate epithelium and thus prostate cancer progression. While growth-inhibitory, tumor-suppressive AR effects have also been documented, the underlying mechanisms are poorly understood. Here, we for the first time link AR anti-cancer action with cell senescence <i>in vitro</i> and <i>in vivo</i>. First, AR-driven senescence was p53-independent. Instead, AR induced p21, which subsequently reduced ΔN isoform of p63. Second, AR activation increased reactive oxygen species (ROS) and thereby suppressed Rb phosphorylation. Both pathways were critical for senescence as was proven by p21 and Rb knock-down and by quenching ROS with N-Acetyl cysteine and p63 silencing also mimicked AR-induced senescence. The two pathways engaged in a cross-talk, likely via PML tumor suppressor, whose localization to senescence-associated chromatin foci was increased by AR activation. All these pathways contributed to growth arrest, which resolved in senescence due to concomitant lack of p53 and high mTOR activity. This is the first demonstration of senescence response caused by a nuclear hormone receptor.GUCY2C Opposes Systemic Genotoxic Tumorigenesis by Regulating AKT-Dependent Intestinal Barrier IntegrityJieru Egeria LinAdam Eugene SnookPeng LiBrian Arthur StoeckerGilbert Won KimMichael Sullivan MageeAlex Vladimir Mejia GarciaMichael Anthony ValentinoTerry HyslopStephanie SchulzScott Arthur Waldman10.1371/journal.pone.00316862012-02-22T14:00:00Z2012-02-22T14:00:00Z<p>by Jieru Egeria Lin, Adam Eugene Snook, Peng Li, Brian Arthur Stoecker, Gilbert Won Kim, Michael Sullivan Magee, Alex Vladimir Mejia Garcia, Michael Anthony Valentino, Terry Hyslop, Stephanie Schulz, Scott Arthur Waldman</p>
The barrier separating mucosal and systemic compartments comprises epithelial cells, annealed by tight junctions, limiting permeability. GUCY2C recently emerged as an intestinal tumor suppressor coordinating AKT1-dependent crypt-villus homeostasis. Here, the contribution of GUCY2C to barrier integrity opposing colitis and systemic tumorigenesis is defined. Mice deficient in GUCY2C (<i>Gucy2c−/−</i>) exhibited barrier hyperpermeability associated with reduced junctional proteins. Conversely, activation of GUCY2C in mice reduced barrier permeability associated with increased junctional proteins. Further, silencing GUCY2C exacerbated, while activation reduced, chemical barrier disruption and colitis. Moreover, eliminating GUCY2C amplified, while activation reduced, systemic oxidative DNA damage. This genotoxicity was associated with increased spontaneous and carcinogen-induced systemic tumorigenesis in <i>Gucy2c−/−</i> mice. GUCY2C regulated barrier integrity by repressing AKT1, associated with increased junction proteins occludin and claudin 4 in mice and Caco2 cells in vitro. Thus, GUCY2C defends the intestinal barrier, opposing colitis and systemic genotoxicity and tumorigenesis. The therapeutic potential of this observation is underscored by the emerging clinical development of oral GUCY2C ligands, which can be used for chemoprophylaxis in inflammatory bowel disease and cancer.Protection from Intracellular Oxidative Stress by Cytoglobin in Normal and Cancerous Oesophageal CellsFiona E. McRonaldJanet M. RiskNikolas J. Hodges10.1371/journal.pone.00305872012-02-16T14:00:00Z2012-02-16T14:00:00Z<p>by Fiona E. McRonald, Janet M. Risk, Nikolas J. Hodges</p>
Cytoglobin is an intracellular globin of unknown function that is expressed mostly in cells of a myofibroblast lineage. Possible functions of cytoglobin include buffering of intracellular oxygen and detoxification of reactive oxygen species. Previous work in our laboratory has demonstrated that cytoglobin affords protection from oxidant-induced DNA damage when over expressed <i>in vitro</i>, but the importance of this in more physiologically relevant models of disease is unknown. Cytoglobin is a candidate for the tylosis with oesophageal cancer gene, and its expression is strongly down-regulated in non-cancerous oesophageal biopsies from patients with TOC compared with normal biopsies. Therefore, oesophageal cells provide an ideal experimental model to test our hypothesis that downregulation of cytoglobin expression sensitises cells to the damaging effects of reactive oxygen species, particularly oxidative DNA damage, and that this could potentially contribute to the TOC phenotype. In the current study, we tested this hypothesis by manipulating cytoglobin expression in both normal and oesophageal cancer cell lines, which have normal physiological and no expression of cytoglobin respectively. Our results show that, in agreement with previous findings, over expression of cytoglobin in cancer cell lines afforded protection from chemically-induced oxidative stress but this was only observed at non-physiological concentrations of cytoglobin. In addition, down regulation of cytoglobin in normal oesophageal cells had no effect on their sensitivity to oxidative stress as assessed by a number of end points. We therefore conclude that normal physiological concentrations of cytoglobin do not offer cytoprotection from reactive oxygen species, at least in the current experimental model.Necdin, a p53-Target Gene, Is an Inhibitor of p53-Mediated Growth ArrestJulie LafontaineFrancis RodierVéronique OuelletAnne-Marie Mes-Masson10.1371/journal.pone.00319162012-02-15T14:00:00Z2012-02-15T14:00:00Z<p>by Julie Lafontaine, Francis Rodier, Véronique Ouellet, Anne-Marie Mes-Masson</p>
<i>In vitro</i>, cellular immortalization and transformation define a model for multistep carcinogenesis and current ongoing challenges include the identification of specific molecular events associated with steps along this oncogenic pathway. Here, using NIH3T3 cells, we identified transcriptionally related events associated with the expression of Polyomavirus Large-T antigen (PyLT), a potent viral oncogene. We propose that a subset of these alterations in gene expression may be related to the early events that contribute to carcinogenesis. The proposed tumor suppressor Necdin, known to be regulated by p53, was within a group of genes that was consistently upregulated in the presence of PyLT. While Necdin is induced following p53 activation with different genotoxic stresses, Necdin induction by PyLT did not involve p53 activation or the Rb-binding site of PyLT. Necdin depletion by shRNA conferred a proliferative advantage to NIH3T3 and PyLT-expressing NIH3T3 (NIHLT) cells. In contrast, our results demonstrate that although overexpression of Necdin induced a growth arrest in NIH3T3 and NIHLT cells, a growing population rapidly emerged from these arrested cells. This population no longer showed significant proliferation defects despite high Necdin expression. Moreover, we established that Necdin is a negative regulator of p53-mediated growth arrest induced by nutlin-3, suggesting that Necdin upregulation could contribute to the bypass of a p53-response in p53 wild type tumors. To support this, we characterized Necdin expression in low malignant potential ovarian cancer (LMP) where p53 mutations rarely occur. Elevated levels of Necdin expression were observed in LMP when compared to aggressive serous ovarian cancers. We propose that in some contexts, the constitutive expression of Necdin could contribute to cancer promotion by delaying appropriate p53 responses and potentially promote genomic instability.Age-Dependent Targeting of Protein Phosphatase 1 to Ca<sup>2+</sup>/Calmodulin-Dependent Protein Kinase II by Spinophilin in Mouse StriatumAnthony J. Baucum IIStefan StrackRoger J. Colbran10.1371/journal.pone.00315542012-02-13T14:00:00Z2012-02-13T14:00:00Z<p>by Anthony J. Baucum II, Stefan Strack, Roger J. Colbran</p>
Mechanisms underlying age-dependent changes of dendritic spines on striatal medium spiny neurons are poorly understood. Spinophilin is an F-actin- and protein phosphatase 1 (PP1)-binding protein that targets PP1 to multiple downstream effectors to modulate dendritic spine morphology and function. We found that calcium/calmodulin-dependent protein kinase II (CaMKII) directly and indirectly associates with N- and C-terminal domains of spinophilin, but F-actin can displace CaMKII from the N-terminal domain. Spinophilin co-localizes PP1 with CaMKII on the F-actin cytoskeleton in heterologous cells, and spinophilin co-localizes with synaptic CaMKII in neuronal cultures. Thr286 autophosphorylation enhances the binding of CaMKII to spinophilin <i>in vitro</i> and <i>in vivo</i>. Although there is no change in total levels of Thr286 autophosphorylation, maturation from postnatal day 21 into adulthood robustly enhances the levels of CaMKII that co-immunoprecipitate with spinophilin from mouse striatal extracts. Moreover, N- and C-terminal domain fragments of spinophilin bind more CaMKII from adult vs. postnatal day 21 striatal lysates. Total levels of other proteins that interact with C-terminal domains of spinophilin decrease during maturation, perhaps reducing competition for CaMKII binding to the C-terminal domain. In contrast, total levels of α-internexin and binding of α-internexin to the spinophilin N-terminal domain increases with maturation, perhaps bridging an indirect interaction with CaMKII. Moreover, there is an increase in the levels of myosin Va, α-internexin, spinophilin, and PP1 in striatal CaMKII immune complexes isolated from adult and aged mice compared to those from postnatal day 21. These changes in spinophilin/CaMKII interactomes may contribute to changes in striatal dendritic spine density, morphology, and function during normal postnatal maturation and aging.Dynamic Epitope Expression from Static Cytometry Data: Principles and ReproducibilityJames W. JacobbergerJayant AvvaSree N. SreenathMichael C. WeisTammy Stefan10.1371/journal.pone.00308702012-02-08T14:00:00Z2012-02-08T14:00:00Z<p>by James W. Jacobberger, Jayant Avva, Sree N. Sreenath, Michael C. Weis, Tammy Stefan</p>
Background <p>An imprecise quantitative sense for the oscillating levels of proteins and their modifications, interactions, and translocations as a function of the cell cycle is fundamentally important for a cartoon/narrative understanding for how the cell cycle works. Mathematical modeling of the same cartoon/narrative models would be greatly enhanced by an open-ended methodology providing precise quantification of many proteins and their modifications, etc. Here we present methodology that fulfills these features.</p> Methodology <p>Multiparametric flow cytometry was performed on Molt4 cells to measure cyclins A2 and B1, phospho-S10-histone H3, DNA content, and light scatter (cell size). The resulting 5 dimensional data were analyzed as a series of bivariate plots to isolate the data as segments of an N-dimensional “worm” through the data space. Sequential, unidirectional regions of the data were used to assemble expression profiles for each parameter as a function of cell frequency.</p> Results <p>Analysis of synthesized data in which the true values where known validated the approach. Triplicate experiments demonstrated exceptional reproducibility. Comparison of three triplicate experiments stained by two methods (single cyclin or dual cyclin measurements with common DNA and phospho-histone H3 measurements) supported the feasibility of combining an unlimited number of epitopes through this methodology. The sequential degradations of cyclin A2 followed by cyclin B1 followed by de-phosphorylation of histone H3 were precisely mapped. Finally, a two phase expression rate during interphase for each cyclin was robustly identified.</p> Conclusions <p>Very precise, correlated expression profiles for important cell cycle regulating and regulated proteins and their modifications can be produced, limited only by the number of available high-quality antibodies. These profiles can be assembled into large information libraries for calibration and validation of mathematical models.</p>Creatine Protects against Excitoxicity in an In Vitro Model of NeurodegenerationJust GeniusJohanna GeigerAndreas BenderHans-Jürgen MöllerThomas KlopstockDan Rujescu10.1371/journal.pone.00305542012-02-08T14:00:00Z2012-02-08T14:00:00Z<p>by Just Genius, Johanna Geiger, Andreas Bender, Hans-Jürgen Möller, Thomas Klopstock, Dan Rujescu</p>
Creatine has been shown to be neuroprotective in aging, neurodegenerative conditions and brain injury. As a common molecular background, oxidative stress and disturbed cellular energy homeostasis are key aspects in these conditions. Moreover, in a recent report we could demonstrate a life-enhancing and health-promoting potential of creatine in rodents, mainly due to its neuroprotective action. In order to investigate the underlying pharmacology mediating these mainly neuroprotective properties of creatine, cultured primary embryonal hippocampal and cortical cells were challenged with glutamate or H<sub>2</sub>O<sub>2</sub>. In good agreement with our <i>in vivo</i> data, creatine mediated a direct effect on the bioenergetic balance, leading to an enhanced cellular energy charge, thereby acting as a neuroprotectant. Moreover, creatine effectively antagonized the H<sub>2</sub>O<sub>2</sub>-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant. Additionally, creatine mediated a direct inhibitory action on the NMDA receptor-mediated calcium response, which initiates the excitotoxic cascade. Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible. In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity.Inhibition of Akt Activity and Calcium Channel Function Coordinately Drive Cell-Cell Fusion in the BeWO Choriocarcinoma Placental Cell LineManu VatishLydia TesfaDimitris GrammatopoulosEijiro YamadaClaire C. BastieJeffrey E. Pessin10.1371/journal.pone.00293532012-01-19T14:00:00Z2012-01-19T14:00:00Z<p>by Manu Vatish, Lydia Tesfa, Dimitris Grammatopoulos, Eijiro Yamada, Claire C. Bastie, Jeffrey E. Pessin</p>
To establish a simple and quantitative live cell fusion assay for placental syncytialization, we generated stable GFP and dsRed expressing fusogenic BeWo cell lines. Fluorescent Activated Cell Sorting was shown to provide a quantitative determination of forskolin (cAMP-mediated) fusion in a time and concentration dependent manner consistent with the increased secretion of beta human chorionic gonadotrophin (β-HCG) and appearance of multi-nucleated cells. Analyses of the fusion process demonstrated that in addition to increased cAMP levels, simultaneous reduction of intracellular calcium and inhibition of Type 1 phosphatidylinositol 3 kinase (PI3K)/Akt signaling also resulted in cell fusion. Although individual blockade of calcium channel function or PI3K/Akt signaling was without effect, the combination with forskolin resulted in a potentiation of cell fusion. These data demonstrate syncytialization is a complex process that depends upon the regulation of distinct signaling inputs that function in concert with each other.