Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid derived from the modification of lysine by spermidine. Two genes, TIF51A and TIF51B, encode eIF5A in the yeast Saccharomyces cerevisiae. In an effort to understand the structure-function relationship of eIF5A, we have generated yeast mutants by introducing plasmid-borne tif51A into a double null strain where both TIF51A and TIF51B have been disrupted. One of the mutants, tsL102A strain (tif51A L102A tif51aDelta tif51bDelta) exhibits a strong temperature-sensitive growth phenotype. At the restrictive temperature, tsL102A strain also exhibits a cell shape change, a lack of volume change in response to temperature increase and becomes more sensitive to ethanol, a hallmark of defects in the PKC/WSC cell wall integrity pathway. In addition, a striking change in actin dynamics and a complete cell cycle arrest at G1 phase occur in tsL102A cells at restrictive temperature. The temperature-sensitivity of tsL102A strain is due to a rapid loss of mutant eIF5A with the half-life reduced from 6 h at permissive temperature to 20 min at restrictive temperature. Phenylmethyl sulfonylfluoride (PMSF), an irreversible inhibitor of serine protease, inhibited the degradation of mutant eIF5A and suppressed the temperature-sensitive growth arrest. Sorbitol, an osmotic stabilizer that complement defects in PKC/WSC pathways, stabilizes the mutant eIF5A and suppresses all the observed temperature-sensitive phenotypes.

An aqueous extract of Fagonia cretica induces DNA damage, cell cycle arrest and apoptosis in breast cancer cells via FOXO3a and p53 expression

Background - Plants have proved to be an important source of anti-cancer drugs. Here we have investigated the cytotoxic action of an aqueous extract of Fagonia cretica, used widely as a herbal tea-based treatment for breast cancer. Methodology/Principal Findings - Using flow cytometric analysis of cells labeled with cyclin A, annexin V and propidium iodide, we describe a time and dose-dependent arrest of the cell cycle in G0/G1 phase of the cell cycle and apoptosis following extract treatment in MCF-7 (WT-p53) and MDA-MB-231 (mutant-p53) human breast cancer cell lines with a markedly reduced effect on primary human mammary epithelial cells. Analysis of p53 protein expression and of its downstream transcription targets, p21 and BAX, revealed a p53 associated growth arrest within 5 hours of extract treatment and apoptosis within 24 hours. DNA double strand breaks measured as ?-H2AX were detected early in both MCF-7 and MDA-MB-231 cells. However, loss of cell viability was only partly due to a p53-driven response; as MDA-MB-231 and p53-knockdown MCF-7 cells both underwent cell cycle arrest and death following extract treatment. p53-independent growth arrest and cytotoxicity following DNA damage has been previously ascribed to FOXO3a expression. Here, in MCF-7 and MDA-MB-231 cells, FOXO3a expression was increased significantly within 3 hours of extract treatment and FOXO3 siRNA reduced the extract-induced loss of cell viability in both cell lines. Conclusions/Significance - Our results demonstrate for the first time that an aqueous extract of Fagonia cretica can induce cell cycle arrest and apoptosis via p53-dependent and independent mechanisms, with activation of the DNA damage response. We also show that FOXO3a is required for activity in the absence of p53. Our findings indicate that Fagonia cretica aqueous extract contains potential anti-cancer agents acting either singly or in combination against breast cancer cell proliferation via DNA damage-induced FOXO3a and p53 expression.

Heat shock proteins in leukaemia cell differentiation and cell death

When HL60 cells were induced to differentiate to granulocyte-like cells with the agents N-methylformamide and tunicamycin an concentrations marginally below those which were cytotoxic, there was a decrease in the synthesis of the glucose- regulated proteins which preceded the expression of markers of a differentiated phenotype. There was a transient increase in the amount of hsp70 after 36 hours in NMF treated cells but in differentiated cells negligible amounts were detected. Inducers which were known to modulate hsp70 such as azetadine carboxylic acid did not induce differentiation suggesting early changes in the endoplasmic reticulum may be involved in the commitment to terminal differentiation of HL60 cells. These changes in group synthesis were not observed when K562 human chronic myelogenous leukemia cells were induced to differentiate to erythroid-like cells but there was a comparable increase in amounts of hsp70. When cells were treated with concentrations of drugs which brought about a loss in cell viability there was an early increase in the amount of hsp70 protein in the absence of any increase in synthesis. HL60 cells were treated with NMF (225mM), Adriamycin (1μM), or CB3717 (5μM) and there was an increase in the amounts of hsp70, in the absence of any new synthesis, which preceded any loss of membrane integrity and any significant changes in cell cycle but was concomitant with a later loss in viability of > 50% and a loss in proliferative potential. The amounts of hsp70 in the cell after treatment with any of the drugs was comparable to that obtained after a heat shock. Following a heat shock hsp70 was translocated from the cytoplasm to the nucleus, but treatment with toxic concentrations of drug caused hsp70 to remain localised in the cytoplasm. Changes in hsp70 turn-over was observed after a heat shock compared to NMF-treated cells. Morphological studies suggested that cells that had been treated with NMF and CB3717 were undergoing necrosis whereas the Adriamycin cells showed characteristics that were indicative of apoptosis. The data supports the hypothesis that an increase in amounts of hsp70 is an early marker of cell death.

Efficient purification of chromatin architectural proteins:histones, HMGB proteins and FKBP3 (FKBP25) immunophilin

A two-step process of high ionic strength lysis of chicken erythrocyte cell nuclei followed by cation-exchange chromatography has separated at very high yield all the histone and HMGB (high-mobility group B) nuclear proteins, except the less-soluble histone tetramers. Surprisingly high yields of the nuclear immunophilin FKBP3 (FKBP25) and Hsp70 (heat-shock protein 70) co-fractionate with HMGB1 and HMGB3. Furthermore, these proteins can be separated by anion-exchange chromatography. The purified nuclear proteins retain their native, post-translational modification (PTM) marks, including those associated with chromatin-fibre remodelling. These marks are intimately associated with the control of the cell cycle. The methods herein are therefore of value for targeting these and other nuclear proteins for future proteomic studies in healthy and diseased cells. This journal is © 2012 The Royal Society of Chemistry.

Ascorbic acid induces an oxidative stress in CCRF cells activating the transcription factors AP-1 and NFRB

We have previously tested the effects of high dose AA supplements on human volunteers in terms of reducing DNA damage, as a possible mechanism of the vitamin’s proposed protective effect against cancer and detected a transient, pro-oxidant effect at high doses (500 mg/day). Herein, we present evidence of a pro-oxidant effect of the vitamin when added to CCRF cells at extracellular concentrations which mimic those present in human serum in vivo (50–150AM). The activation of the transcription factor AP-1 was optimal at 100 AM AA following 3h exposure at 37jC. A minimum dose of 50 AM of AA activated NFnB but there appeared to be no dose-dependent effect. Increases of 2–3 fold were observed for both transcription factors when cells were exposed to 100 AM AA for 3h, comparing well with the pro-oxidant effect of H2O2 at similar concentrations. In parallel experiments the activation of AP-1 (binding to DNA) was potentiated when cells were pre-incubated with AA prior to exposure with H2O2. Cycloheximide pretreatment (10 Ag/ml for 15min) caused a 50% inhibition of AP-1 binding to DNA suggesting that it was due to a combination of increasing the binding of pre-existing Fos and Jun and an increase in their de novo synthesis. Cellular localisation was confirmed by immunocytochemistry using antibodies specific for c-Fos and c-Jun proteins. These results suggest that extracellular AA can elicit an intracellular stress response resulting in the activation of the oxidative stress-responsive transcription factors AP-1 and NFnB. These transcription factors are involved in the induction of genes associated with an oxidative stress response, cell cycle arrest and DNA repair confirmed by our cDNA microarray analysis (Affymetrix). This may explain the abilty for AA to appear to inhibit 8-oxodG, yet simultaneously generate another oxidative stress biomarker, 8-oxo-dA. These results suggest a completely novel DNA repair action for AA. Whether this action is relevant to our in vivo findings will be the subject of our future research.

Raf kinase inhibitor protein RKIP enhances signaling by glycogen synthase kinase-3β

Raf kinase inhibitory protein (RKIP) is a physiologic inhibitor of c-RAF kinase and nuclear factor ?B signaling that represses tumor invasion and metastasis. Glycogen synthase kinase-3ß (GSK3ß) suppresses tumor progression by downregulating multiple oncogenic pathways including Wnt signaling and cyclin D1 activation. Here, we show that RKIP binds GSK3 proteins and maintains GSK3ß protein levels and its active form. Depletion of RKIP augments oxidative stress-mediated activation of the p38 mitogen activated protein kinase, which, in turn, inactivates GSK3ß by phosphorylating it at the inhibitory T390 residue. This pathway de-represses GSK3ß inhibition of oncogenic substrates causing stabilization of cyclin D, which induces cell-cycle progression and ß-catenin, SNAIL, and SLUG, which promote epithelial to mesenchymal transition. RKIP levels in human colorectal cancer positively correlate with GSK3ß expression. These findings reveal the RKIP/GSK3 axis as both a potential therapeutic target and a prognosis-based predictor of cancer progression.

The role of cytoskeletal proteins in the mechanism of insulin release

This thesis is concerned with the role of /3-cell cytoskeletal proteins in the mechanism of insulin release from islets of experimental animals, the Aston obese diabetic hyperglycaemic (ob/ob) mouse and their lean littermates and the cultural insulin secreting /?-cell lines, HIT-TT5 and RINm5F. Investigations were carried out into the glucose induced insulin response of the lean and obese mouse islets and HIT-TI5 cells and the D-glyceraldehyde response of RINm5F cells using a static incubation system. Colchicine was found to inhibit insulin release from both lean and obese mouse islets more significantly than cultured TTT-TI5 and RINm5F cells. (Colchicine pre-treatment also inhibited the second phase of insulin release from perifused lean mouse islets and HIT-TI5 cells). Cytocha-lasin B, used to investigate the role of the microfilamentous system in the mechanism of insulin release enhanced insulin release from both lean and obese mouse islets to a significantly greater degree than that from cultured HIT-TI5 and RINm5F cells. Pre-treatment of isolated lean and obese mouse islets and cultured /?-cells with a combination of colchicine and cytochalasin B significantly reduced the insulin response of the HIT-TI5 and RINm5F cells compared with the control values suggesting that intact microtubules are more important for the sustained release of insulin than the microfilamentous system. However, the response was not so clearly defined with the lean and obese mouse islets. Tubulin was separated from the extracts of lean mouse islets and the HIT-TI5 and RINm5F cells and actin was separated from all of the cell types including the obese mouse islets by SDS- polyacrylamide electrophoresis. A tubulin radioimmunoassay and a colchicine binding assay were developed to measure the tubulin content of lean and obese mouse islets, and the shift between the proportions of tubulin dimers and polymerized tubulin under stimulatory and non-stimulatory conditions. The assay methods developed were not prone to be accurate, sensitive and precise but gave some indication of the shift from unpolymerised to polymerised tubulin during glucose stimulated insulin release. These studies show that microtubules do play a fundamental role in the mechanism of insulin release from both islets and cultured HIT-TI5 and RINm5F cells.

The importance of Na(plus)-K(plus)-Cl- cotransport in nitrogen mustard induced cell death

The incubation of murine leukaemic L1210 cells in vitro for 4 hours (hr) with 10uM nitrogen mustard (HN2), a bifunctional alkylating agent, inhibited the influx of the potassium congener, 88rubidium+ ( 86Rb+) by the selective inhibition of the Na+-K+-CI- cotransporter. The aim of this project was to investigate the importance of this lesion in HN2-induced cytotoxicity. 86Rb+ uptake in human erythrocytes was inhibited by high concentrations of HN2 (2mM) and occurred in two phases.In the first hour both the Na+/K+ ATPase pump and the Na+-K+-CI- cotransporter were equally inhibited but after 2 hrs exposure to 2mM HN2, the Na+ -K+ -CI- cotransporter was significantly more inhibited than the Na+/K+ ATPase pump. In contrast, both potassium transport systems were equally inhibited in L1210 cells incubated for 10 minutes with 1mM HN2. The selective inhibition of the Na+-K+-CI- cotransporter, after a 3 hrs exposure to 10uM HN2, was not absolved by coincubation with 5ug/ml cycloheximide (CHX), an inhibitor of protein synthesis. Incubation of L1210 cells with concentrations of diuretics which completely inhibited Na+-K+-CI- cotransport did not enhance the cytotoxicity of either HN2 or its monofunctional analogue 2-chloroethyldimethylamine (Me-HN1). The incubation of L1210 cells with a twice strength Rosewell Park Memorial Institute 1640 media did not enhance the toxicity of HN2. An L1210 cell line (L1210FR) was prepared which was able to grow in toxic concentrations of furosemide and exhibited a similiar sensitivity to HN2 as parental L1210 cells. Treatment of L1210 cells with 10uM HN2 resulted in a decrease in cell volume which was concurrent with the inhibition of the Na+-K+-CI- cotransporter. This was not observed in L1210 cells treated with either 1 or O.SuM HN2. Thus, possible differences in the cell death, in terms of necrosis and apoptosis, induced by the different concentrations of HN2 was investigated. The cell cycle of L1210 cells appeared to be blocked non-specifically by 10uM HN2 and in S and G2/M by either 1 or 0.5uM HN2. There were no significant changes in the cytosolic calcium concentrations of L1210 cells for up to 48 hrs after exposure to the three concentrations of HN2. No protection against th_ toxic effects of HN2 was observed in L1210 cells incubated with 5ug/ml CHX for up to 6 hrs. Incubation for 12 or 18 hrs with a non-toxic concentration (5mM) of L-Azetidine-2- carboxylic acid (ACA) enhanced the toxicity of low concentrations (<0.5uM) of HN2.

Characterization of tissue transglutaminase 2 in macrophage function

Apoptosis is a highly regulated process that removes damaged or unwanted cells in vivo and defective clearance of apoptotic cells by macrophages has significant immunological implications. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in cell proliferation, differentiation, carcinogenesis, programmed death, and aging. TG2 as a guanosine triphosphate (GTP)-binding or GTP- hydrolyzing protein for mediating signal transduction and as a cell cycle regulator emphasized the importance of this enzyme in aging process. The ubiquitous presence of TG2 compared to the other organ-specific TGases has attracted special attention as a cellular aging device. TG2 activity and expression are known to increase in aging humans suggesting possible involvement in several age-related processes such as decrease in vascular compliance and increased stiffening of conduit arteries, cataract formation, Alzheimer's disease and senescent epidermal keratinocytes. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function. THP-1 cell derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors resulted in significant inhibition of interaction. Macrophage cell surface TG2 and, in particular, its cell surface cross linking activity was found to be crucial in apoptotic cell clearance. Syndecan-4 association with TG2 implies possible cooperation of these proteins and knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance.

The effects of the fungicides fenhexamid and myclobutanil on SH-SY5Y and U-251 MG human cell lines

Mixtures of pesticides in foodstuffs and the environment are ubiquitous in the developed world and although agents are usually exhaustively tested individually, the toxicological implications of pesticide mixtures are underreported. In this study, the effects of two fungicides, fenhexamid and myclobutanil were investigated individually and in combination on two human cell lines, SH-SY5Y neuronal cells and U-251 MG glial cells. After 48. h of incubation with increasing concentrations of pesticides ranging from 1 to 1000. μM, gene expression profiles were studied in addition to toxicity end points, including cell viability, mitochondrial depolarisation as well as cellular glutathione maintenance. There were no significant differences between the susceptibility of the two cell lines in terms of cell viability assessment or mitochondrial membrane potential, when agents were administered either individually or in combination. By contrast, in the presence of the fungicides, the SH-SY5Y cells showed significantly greater susceptibility to oxidative stress in terms of total thiol depletion in comparison with the astrocytic cells. Treatment with the two pesticides led to significant changes in the cell lines' expression of several genes which regulate cell cycle control and growth (RB1, TIMP1) as well as responses to DNA attrition (ATM and CDA25A) and control of apoptosis (FAS). There was no evidence in this study that the combination of fenhexamid and myclobutanil was significantly more toxic than individual exposure, although gene expression changes suggested there may be differences in the sub-lethal response of both cell lines to both individual and combined exposure.

Anticancer effects of Curcuma C20-dialdehyde against colon and cervical cancer cell lines

Background: Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. Materials and Methods: Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. Results: Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 μg/ml, 58.4±5.20 μg/ml and 72.0±0.03 μg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. Conclusions: Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.