Wortmannin-enhanced X-ray-induced apoptosis of human T-cell leukemia MOLT-4 cells possibly through the JNK/SAPK pathway

We demonstrated that enhancement of X-ray-induced apoptosis/rapid cell death by wortmannin accompanied by increased activation of JNK/SAPK in human leukemia MOLT-4 cells. Rapid cell death/apoptosis was determined either by the dye exclusion test or by the appearance of Annexin V-positive cells and cleaved PARP fragments. Enhancement was observed only at higher concentrations of wortmannin, i.e. 1 microM or more. At these high concentrations, both DNA-PK and ATM were inhibited. X-ray-induced phosphorylation of Ser 15 of p53/TP53, accumulation of both p53/TP53 and p21/WAF1/CDKN1A, and phosphorylation of XRCC4 were all suppressed. The enhancement of apoptosis/rapid cell death by wortmannin was prevented by addition of caspase inhibitors, Z-VAD-FMK or Ac-DEVD-CHO, or by transfection and overexpression of mouse Bcl2, which is known as an anti-apoptosis protein. The requirement for a high concentration of wortmannin, i.e. 1 microM or more, indicates that inhibition of both DNA-PK and ATM was necessary for the enhanced apoptosis/rapid cell death. Phosphorylation of AKT/PKB was completely suppressed at a much lower concentration, i.e. 0.1 microM wortmannin, where no enhancement of X-ray-induced apoptosis/rapid cell death was observed. On the other hand, X-ray-induced phosphorylation of JNK and its kinase activity as well as apoptosis/rapid cell death were all significantly enhanced only at high concentrations of wortmannin, i.e. 1 microM or more. Furthermore, the extent of enhancement of both JNK phosphorylation and of apoptosis/rapid cell death by wortmannin was less in Rh1a cells, which are ceramide- and radiation-resistant variant cells compared to the parental MOLT-4 cells. Therefore, activation of the JNK pathway was considered important for the enhancement of X-ray-induced apoptosis/rapid cell death of MOLT-4 cells by wortmannin, because of the requirement for a higher concentration of wortmannin than that required for inhibition of AKT phosphorylation. The suppression of the AKT-dependent pathway by wortmannin may have some underlying role in activating the JNK pathway toward the enhancement of cell death in the current system.     

Rapid intracellular acidification and cell death by H2O2 and alloxan in pancreatic beta cells

Pancreatic beta-cell death induced by oxidative stress plays an important role in the pathogenesis of diabetes mellitus. We studied the relation between rapid intracellular acidification and cell death of pancreatic beta-cell line NIT-1 cells exposed to H2O2 or alloxan. Intracellular pH was measured by a pH-sensitive dye, and cell damage by double staining with Annexin-V and propidium iodide using flow cytometry. H2O2 and alloxan caused a rapid fall in intracellular pH and suppressed Na+/H+ exchanger activity in the NH4Cl prepulse method. H2O2 induced necrotic cell death, which shifted to apoptotic cell death when initial acidification was prevented by pH clamping to 7.4 using nigericin (unclamped cells vs clamped cells, necrosis 43.8 +/- 5.8% vs 21.1 +/- 10.6%, P < 0.05; apoptosis 8.0 +/- 1.9% vs 44.5 +/- 5.0%, P < 0.01). pH-clamped cells showed enhanced caspase 3 activity and proapoptotic Bax expression. On the other hand, NIT-1 cells were resistant to alloxan toxicity, but treatment with alloxan and nigericin strikingly enhanced the cytotoxicity. Antioxidants partly prevented cell death, although intracellular pH remained similarly acidic. The rapid intracellular acidification was not the cause of cell death but a significant determinant of the mode of death of H2O2 -treated beta cells, whereas no link between cell death and acidification was demonstrated in alloxan toxicity.     

Lack of cell death enhancement after irradiation with monochromatic synchrotron X rays at the K-shell edge of platinum incorporated in living SQ20B human cells as cis-diamminedichloroplatinum (II)

In this paper we describe the results of experiments using synchrotron radiation to trigger the Auger effect in living human cancer cells treated with a widely used chemotherapy drug: cis-diamminedichloroplatinum (II) (cisplatin). The experiments were carried out at the ID17 beamline of the European Synchrotron Radiation Facility, which produces a high-fluence monochromatic beam that is adjustable from 20 to 80 keV. Cisplatin was chosen as the carrier of platinum atoms in the cells because of its alkylating-like activity and the irradiation was done with monochromatic beams above and below the platinum K-shell edge (78.39 keV). Cell survival curves were comparable with those obtained for the same cells under conventional irradiation conditions. At a low dose of cisplatin (0.1 microM, 48 h), no difference was seen in survival when the cells were irradiated above and below the K-shell edge of platinum. Higher cisplatin concentrations were investigated to enhance the cellular platinum content. The results with 1 microM cisplatin for 12 h showed no difference when the cells were irradiated with beams above or below the platinum K-shell edge with the exception of the higher cell death resulting from drug toxicity. The intracellular content of platinum was significant, as measured macroscopically by inductively coupled plasma mass spectrometry. Its subcellular localization and particularly its presence in the cell nucleus were verified by microscopic synchrotron X-ray fluorescence. This was the first known attempt at K-shell edge photon activation of stable platinum in living cells with a platinum complex used for chemotherapy. Its evident toxicity in these cells leads us to put forth the hypothesis that cisplatin toxicity can mask the enhancement of cell death induced by the irradiation above the K-shell edge. However, K-shell edge photon activation of stable elements provides a powerful technique for the understanding of the biological effects of Auger processes. Further avenues of development are discussed.     

Triclabendazole protects yeast and mammalian cells from oxidative stress: identification of a potential neuroprotective compound

The Prestwick and NIH chemical libraries were screened for drugs that protect baker’s yeast from sugar-induced cell death (SICD). SICD is triggered when stationary-phase yeast cells are transferred from spent rich medium into water with 2% glucose and no other nutrients. The rapid, apoptotic cell death occurs because reactive oxygen species (ROS) accumulate. We found that triclabendazole, which is used to treat liver flukes in cattle and man, partially protects against SICD. Characterization of triclabendazole revealed that it also protects yeast cells from death induced by the Parkinson’s disease-related protein alpha-synuclein (α-syn), which is known to induce the accumulation of ROS.     

Rapid discrimination of cytK-1 and cytK-2 genes in Bacillus cereus strains by a novel duplex PCR system

Bacillus cereus is the causative agent of gastrointestinal diarrhoea. At least three known enterotoxins may be involved in this syndrome: nonhaemolytic (Nhe) enterotoxin, Hbl enterotoxin and cytotoxin K. Two different forms were recently described for cytotoxin K, encoded by cytK-1 and cytK-2 genes. The CytK-1 toxin appeared to carry a high toxicity, but there is currently no method available to rapidly detect and discriminate the B. cereus strains able to produce this CytK-1 form. In this study, a duplex PCR assay was developed and validated on 162 known cytotoxin-containing strains. This PCR method is the first molecular tool to provide rapid detection and discrimination of cytK-1- and cytK-2-carrying B. cereus strains.     

Huntingtin interacting protein 1 induces apoptosis via a novel caspase-dependent death effector domain

Huntington disease is a devastating neurodegenerative disease caused by the expansion of a polymorphic glutamine tract in huntingtin. The huntingtin interacting protein (HIP-1) was identified by its altered interaction with mutant huntingtin. However, the function of HIP-1 was not known. In this study, we identify HIP-1 as a proapoptotic protein. Overexpression of HIP-1 resulted in rapid caspase 3-dependent cell death. Bioinformatics analyses identified a novel domain in HIP-1 with homology to death effector domains (DEDs) present in proteins involved in apoptosis. Expression of the HIP-1 DED alone resulted in cell death indistinguishable from HIP-1, indicating that the DED is responsible for HIP-1 toxicity. Furthermore, substitution of a conserved hydrophobic phenylalanine residue within the HIP-1 DED at position 398 eliminated HIP-1 toxicity entirely. HIP-1 activity was found to be independent of the DED-containing caspase 8 but was significantly inhibited by the antiapoptotic protein Bcl-x(L), implicating the intrinsic pathway of apoptosis in HIP-1-induced cell death. Co-expression of a normal huntingtin fragment capable of binding HIP-1 significantly reduced cell death. Our data identify HIP-1 as a novel proapoptotic mediator and suggest that HIP-1 may be a molecular accomplice in the pathogenesis of Huntington disease.     

The differential effects of single or repeated restraint stress on kainic acid-induced neuronal death in the hippocampal CA3 region: the role of glucocorticoid and various signal molecules

The effect of stress mediators following the stress period and addition time is a controversial issue until now. Thus, we aim to clarify the differential effects of single restraint stress (SS) or repeated restraint stress (RS) on kainic acid (KA)-induced neuronal death especially as addressing not only the role of glucocorticoid (Gc) and its receptor but also the signal pathway leading to cAMP response element binding protein phosphorylation (pCREB) and its functional role during stress. In the present study, we found that although RS did not show any difference on serum Gc level and hippocampal Gc receptor level compared to SS, SS exacerbated KA-induced neuronal death in hippocampal CA3 region, but RS did not. Moreover, pre-treatment with RU 38486 (Gc receptor antagonist) abolished the effect of SS on KA-induced neuronal death without an effect on KA toxicity itself. Furthermore, RS aggravates KA-induced neuronal death when CREB phosphorylation was deprived by KN-93 (calcium/calmodulin-dependent protein kinase II inhibitor). However, other signal molecules inhibitors such as PD98059 (MEK1/2 inhibitor) and SP600125 (p-p38 inhibitor) have no effect on KA-induced neuronal death after RS although these signal molecule were increased during SS or RS. These findings suggest that pCREB expression via calcium/calmodulin-dependent protein kinase II phosphorylation during RS comprise one of the balancers against Gc induced by stress.     

Is There Adaptive Value to Reproductive Termination in Japanese Macaques? A Test of Maternal Investment Hypotheses

Evolutionary biologists often argue that menopause evolved in the human female as the result of selection for a postreproductive phase of life, during which increased maternal investment in existing progeny could lead to enhanced survivorship of descendents. Adaptive theories relating menopause to enhanced maternal investment are known as the mother (first-generation) and grandmother (second-generation-offspring) hypotheses. Although menopause—universal midlife termination of reproduction—has not been documented in primates other than humans, some researchers have argued that postreproductive alloprimates also have a positive impact on the survivorship of first and second generation progeny. We tested the maternal investment hypotheses in Japanese macaques by comparing the survivorship of offspring, final infants, and great-offspring of females that terminated reproduction before death with females that continued to reproduce until death. SURVIVAL analyses revealed no significant difference in the survivorship of descendents of postreproductive and reproductive females, though final infants of postreproductive females were 13% more likely to survive than final infants of females that reproduced until death were. We also explored possible differences between these two groups of females, other than survivorship of progeny. We found no difference in dominance rank, matrilineal affiliation, body weight, infant sex ratio, age at first birth, fecundity rate or lifetime reproductive success. However, postreproductive females are significantly longer-lived than reproductive females and as a result experienced more years of reproduction and produced more infants in total. Apart from final infants, offspring survival is marginally lower in postreproductive females. Since offspring survival is not significantly enhanced in postreproductive females, the greater number of infants produced did not translate into greater lifetime reproductive success. Our findings fail to support the maternal investment hypotheses and instead suggest that reproductive termination in this population of Japanese macaques is most closely associated with enhanced longevity and its repercussions.     

Neurotoxicity of 25-OH-Cholesterol on NGF-Differentiated PC12 Cells

PC12 cells induced to differentiate with nerve growth factor were used to study the neurotoxicity of 25-OH-cholesterol. This agent induced a dose- and time-dependent cell death in neuronal PC12 cells. Cells treated with this agent showed condensed nuclei, a morphology similar to that of cells dying of programmed cell death. However, agents known to prevent neuronal programmed cell death (cyclic AMP, KCl, aurintricarboxylic acid, and cycloheximide) failed to prevent the 25-OH-cholesterol-mediated cytotoxicity. On the other hand, cell death induced by 25-OH-cholesterol was prevented by treatment with vitamin E and methyl-beta-cyclodextrin. In contrast to observations made in other cell types, whole-cell patch clamp recording of neuronal PC12 cells revealed that treatment with 25-OH-cholesterol did not significantly alter calcium influx through voltage-dependent channels. These results provide the first characterization of the toxicity of cholesterol oxides toward neuronal PC12 cells, which should be useful in future studies on the interactions between cholesterol oxides and cells from the nervous system.     

Metabolic effects of TiO2 nanoparticles,a common component of sunscreens and cosmetics,on human keratinocytes

The long-term health risks of nanoparticles remain poorly understood, which is a serious concern given their prevalence in the environment from increased industrial and domestic use. The extent to which such compounds contribute to cellular toxicity is unclear, and although it is known that induction of oxidative stress pathways is associated with this process, the proteins and the metabolic pathways involved with nanoparticle-mediated oxidative stress and toxicity are largely unknown. To investigate this problem further, the effect of TiO₂ on the HaCaT human keratinocyte cell line was examined. The data show that although TiO₂ does not affect cell cycle phase distribution, nor cell death, these nanoparticles have a considerable and rapid effect on mitochondrial function. Metabolic analysis was performed to identify 268 metabolites of the specific pathways involved and 85 biochemical metabolites were found to be significantly altered, many of which are known to be associated with the cellular stress response. Importantly, the uptake of nanoparticles into the cultured cells was restricted to phagosomes, TiO₂ nanoparticles did not enter into the nucleus or any other cytoplasmic organelle. No other morphological changes were detected after 24-h exposure consistent with a specific role of mitochondria in this response.     

Vanadium and diabetes.

Vanadium is an ultratrace element, widely distributed in nature, yet with no presently known specific physiological function in mammals. The apparent role of vanadium in regulation of intracellular signaling, as a cofactor of enzymes essential in energy metabolism, and as a possible therapeutic agent in diabetes is of increasing interest as more and more research reports present evidence of vanadium's potentially unique biological function. In this mini-review, the author summarizes current knowledge of the bioinorganic chemistry of vanadium, the basic features of diabetes mellitus and its metabolic sequelae, and the in vitro and in vivo effects of both inorganic and organically-chelated vanadium compounds. Results of clinical trials to date, as well as kinetic studies of tissue uptake are covered. Examples of ways to enhance the positive effects of vanadium as an oral therapeutic adjunct in diabetic control, while minimizing potential toxicity, are compared with regard to desirable features and possible drawbacks.     

Effects of dichloroacetate and glyoxylate on low density lipoprotein uptake and on growth of cultured fibroblasts

The effects of dichloroacetate, a known hypocholesterolemic agent, were studied in cultured growing and confluent human fibroblast cells. Microscopic examination showed no visible adverse effects of dichloroacetate on confluent cells during exposure to concentrations as high as 5 mM for 96 hr. Higher concentrations resulted in cell death after varying periods of incubation. There were no viable cells after 24 hr of exposure to 100 mM dichloroacetate. In contrast, much lower concentrations proved lethal to growing cells; cell growth, as determined by cell numbers at specified times after splitting, was suppressed by 1 mM dichloroacetate and 5 mM concentrations resulted in cell death. Similar effects were noted with glyoxylate. The hypocholesterolemic effect of dichloroacetate is probably not due to any effect on the low density lipoprotein pathway, since concentrations of up to 1 mM dichloroacetate did not affect the cellular binding and uptake of 125I-labeled low density lipoprotein. It is concluded that growing and rapidly metabolizing cells are much more sensitive to the toxic effects of dichloroacetate and glyoxylate than confluent cells.     

The clc element of Pseudomonas sp. strain B13, a genomic island with various catabolic properties

Pseudomonas sp. strain B13 is a bacterium known to degrade chloroaromatic compounds. The properties to use 3- and 4-chlorocatechol are determined by a self-transferable DNA element, the clc element, which normally resides at two locations in the cell's chromosome. Here we report the complete nucleotide sequence of the clc element, demonstrating the unique catabolic properties while showing its relatedness to genomic islands and integrative and conjugative elements rather than to other known catabolic plasmids. As far as catabolic functions, the clc element harbored, in addition to the genes for chlorocatechol degradation, a complete functional operon for 2-aminophenol degradation and genes for a putative aromatic compound transport protein and for a multicomponent aromatic ring dioxygenase similar to anthranilate hydroxylase. The genes for catabolic functions were inducible under various conditions, suggesting a network of catabolic pathway induction. For about half of the open reading frames (ORFs) on the clc element, no clear functional prediction could be given, although some indications were found for functions that were similar to plasmid conjugation. The region in which these ORFs were situated displayed a high overall conservation of nucleotide sequence and gene order to genomic regions in other recently completed bacterial genomes or to other genomic islands. Most notably, except for two discrete regions, the clc element was almost 100% identical over the whole length to a chromosomal region in Burkholderia xenovorans LB400. This indicates the dynamic evolution of this type of element and the continued transition between elements with a more pathogenic character and those with catabolic properties.     

Tetrazoles as anticancer agents: A review on synthetic strategies,mechanism of action and SAR studies

Cancer is a leading cause of death worldwide. Even after the availability of numerous drugs and treatments in the market, scientists and researchers are focusing on new therapies because of their resistance and toxicity issues. The newly synthesized drug candidates are able to demonstrate in vitro activity but are unable to reach clinical trials due to their rapid metabolism and low bioavailability. Therefore there is an imperative requisite to expand novel anticancer negotiators with tremendous activity as well as in vivo efficacy. Tetrazole is a promising pharmacophore which is metabolically more stable and acts as a bioisosteric analogue for many functional groups. Tetrazole fragment is often castoff with other pharmacophores in the expansion of novel anticancer drugs. This is the first systematic review that emphasizes on contemporary strategies used for the inclusion of tetrazole moiety, mechanistic targets along with comprehensive structural activity relationship studies to provide perspective into the rational design of high-efficiency tetrazole-based anticancer drug candidates.     

Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells

Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper­ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver.     

Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 μM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 μM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na⁺/K⁺-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway. © 1996 John Wiley & Sons, Inc.     

Terminal repeats of the Drosophila transposable element copia: nucleotide sequence and genomic organization

We have determined the nucleotide sequence of the terminal regions of two members of the copia sequence family of D. melanogaster. The first 276 bp at one end of a copia element are repeated in direct orientation at its other end. The direct repeats on a single copia element are identical to each other, but they differ by two nucleotide substitutions between the two elements which were examined; this suggests that during transposition only one direct repeat of the parent element is used as a template for both direct repeats of the transposed element. Each direct repeat itself contain a 17 bp imperfectly matched inveted terminal repetition. The ends of copia show significant sequence homology both to the yeast Ty1 element and to the integrated provirus of avian spleen necrosis virus, two other eucaryotic elements known to insert at many different chromosomal locations. Analysis of the genomic organization of the direct repeat sequence demonstrates that it seldom, if ever, occurs unlinked to an entire copia element.     

Gain in toxic function of stefin B EPM1 mutants aggregates: Correlation between cell death,aggregate number/size and oxidative stress

EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death.We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.     

Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans

The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in agriculture. Despite successful and extensive use of these toxins in transgenic crops, little is known about toxicity and resistance pathways in target insects since these organisms are not ideal for molecular genetic studies. To address this limitation and to investigate the potential use of these toxins to control parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with toxin effects in insects. We have screened for and isolated 10 recessive mutants that resist the toxin's effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more components are required for Bt toxicity than previously known. We find that a second, unrelated nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold promise as nematicides.