Morphological Evidence Of Apoptosis Research Articles

A novel cell death pathway that is partially caspase dependent, but morphologically non‐apoptotic, elicited by proteasomal inhibition of rat sympathetic neurons

Proteasomal dysfunction has been linked to neurodegeneration. Pharmacological proteasomal inhibitors may have pro-survival or pro-death effects in neuronal cells. We have previously found that application of such agents to mouse sympathetic neurons leads to activation of the intrinsic apoptotic pathway. We show here that in rat sympathetic neurons proteasomal inhibition leads to a form of death that is morphologically non-apoptotic, with features of autophagy. The intrinsic apoptotic pathway is activated in a delayed fashion compared with mouse neurons, and is in part responsible for death, as evidenced by the partial protective effects of bcl-xL and the general caspase inhibitor Boc-aspartyl-fluoromethylketone. Death is accompanied by induction of Bim and caspase activation, but caspase 3 activation is lacking; 3-methyl-adenine inhibits macroautophagy, but has a relatively small pro-survival effect. We conclude that a complex array of pro- and anti-apoptotic effects elicited by proteasomal inhibition in rat sympathetic neurons leads to partial engagement of the intrinsic apoptotic pathway and a morphologically non-apoptotic, autophagic form of death. The species difference with mouse neurons is underscored by the fact that proteasomal inhibitors are protective against apoptosis elicited by nerve growth factor deprivation in rat, but not mouse, sympathetic neurons. The type of death described herein may be relevant to neurodegenerative diseases, where morphological evidence for apoptosis has been scant.

A Molecular Signature of Gastric Metaplasia Arising in Response to Acute Parietal Cell Loss

Loss of gastric parietal cells is a critical precursor to gastric metaplasia and neoplasia. However, the origin of metaplasia remains obscure. Acute parietal cell loss in gastrin-deficient mice treated with DMP-777 leads to the rapid emergence of spasmolytic polypeptide/trefoil factor family 2 (TFF2)-expressing metaplasia (SPEM) from the bases of fundic glands. We now sought to characterize more definitively the pathway for emergence of SPEM. Emerging SPEM lineages in gastrin-deficient mice treated with DMP-777 were examined for immunolocalization of TFF2, intrinsic factor, and Mist1, and morphologically with electron microscopy. Emerging SPEM was isolated with laser-capture microdissection and RNA was analyzed using gene microarrays. Immunohistochemistry in mouse and human samples was used to confirm up-regulated transcripts. DMP-777-induced SPEM was immunoreactive for TFF2 and the differentiated chief cell markers, Mist1 and intrinsic factor, suggesting that SPEM derived from transdifferentiation of chief cells. Microarray analysis of microdissected SPEM lineages induced by DMP-777 showed up-regulation of transcripts associated with G1/S cell-cycle transition including minichromosome maintenance deficient proteins, as well as a number of secreted factors, including human epididymis 4 (HE4). HE4, which was absent in the normal stomach, was expressed in SPEM of human and mouse and in intestinal metaplasia and gastric cancer in human beings. Although traditionally metaplasia was thought to originate from normal mucosal progenitor cells, these studies indicate that SPEM evolves through either transdifferentiation of chief cells or activation of a basal cryptic progenitor. In addition, induction of metaplasia elicits the expression of secreted factors, such as HE4, relevant to gastric preneoplasia.

Small interfering RNA-mediated silencing of mitochondrial NADP +-dependent isocitrate dehydrogenase enhances the sensitivity of HeLa cells toward tumor necrosis factor-α and anticancer drugs

Tumor necrosis factor-α (TNF-α) and several anticancer drugs induce the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP +-dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In the present report, we show that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by TNF-α and anticancer drugs. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of anticancer agent-induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by TNF-α and anticancer drugs and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.

Early release of arachidonic acid prevents an otherwise immediate formation of toxic levels of peroxynitrite in astrocytes stimulated with lipopolysaccharide/interferon‐γ

Addition of bacterial lipopolysaccharides (LPS) and interferon-gamma (IFN-gamma) to rat astrocytes in primary culture promotes an early release of arachidonic acid (ARA) associated with an immediate inhibition of neuronal nitric oxide synthase (nNOS). Preventing the release of constitutive nitric oxide (NO) is indeed critical for activation of the nuclear factor kappa B, and for the expression of inducible nitric oxide synthase responsible for the formation of large amounts of NO. LPS/IFN-gamma also promotes an early release of superoxide, via activation of NADPH oxidase, but the generation of peroxynitrite (ONOO-) is prevented by the different timing of superoxide (minutes) and NO (hours) formation. Upstream inhibition of the ARA-dependent nNOS inhibitory signaling, however, caused the parallel release of superoxide and constitutive NO, thereby leading to formation of ONOO- levels triggering loss of ATP and mitochondrial membrane potential followed by the mitochondrial release of cytochrome c, activation of caspase 3 and morphological evidence of apoptosis. Nanomolar levels of exogenous ARA prevented all these events via inhibition of early ONOO- formation. Thus, the ARA-dependent nNOS inhibition observed in astrocytes exposed to pro-inflammatory stimuli, as LPS/IFN-gamma, is critical for both the expression of nuclear factor kappa B-dependent genes and for survival.

Cathepsin D Deficiency Induces Persistent Neurodegeneration in the Absence of Bax-Dependent Apoptosis

Neuronal ceroid lipofuscinosces/Batten disease (NCL) is a devastating group of neurodegenerative diseases caused by genetic disruptions in lysosomal function. Cathepsin D (CD) is a major lysosomal protease, and mutations in CD that render it enzymatically defective have been reported recently in subsets of NCL patients. The targeted deletion of CD in mice results in extensive neuropathology, including biochemical and morphological evidence of apoptosis and autophagic stress (aberrant autophagosome accumulation), effects that are similar to those observed in NCL. To determine the contribution of Bax-dependent apoptosis in this mouse model of NCL, combined Bax- and CD-deficient mice were generated. Morphological analysis of CD-deficient mouse brains indicated large numbers of pyknotic neurons and neurons with marked cytoplasmic swellings containing undigested lipofuscin. Cell death and apoptosis were evidenced by increases in terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactivity and activation of caspase-3, respectively. DeOlmos silver-positive neurons were abundant in CD-deficient brain and correlated with neuron loss, as indicated by significant decreases in NeuN (neuronal nuclear antigen)-positive neurons. Lysosome dysfunction and autophagic stress were apparent in CD-deficient brain as indicated by the accumulation of autofluorescent storage material and by increased levels of LC3-II (light chain 3-II, a selective autophagosome marker), respectively. Bax deletion significantly inhibited caspase-3 activation and hippocampal TUNEL reactivity but did not prevent the majority of CD deficiency-induced neuropathology, including the persistence of pyknotic neurons, elevated cortical TUNEL reactivity, lysosome dysfunction and autophagic stress, neurodegeneration, and neuron loss. Together, these results suggest that CD deficiency-induced neuropathology does not require Bax-dependent apoptosis and highlights the importance of caspase-independent neuron death and neurodegeneration resulting from the genetic disruption of lysosome function.

Mortality of wild swans associated with naturally infection with highly pathogenic H5N1 avian influenza virus in Iran

In the February 2006 in two wetlands in northern Iran, the mortality among wild swans was observed. Paralysis was the most prominent feature of the disease. Histologically, diffused necrosis of acinar cells in pancreas, degeneration and necrosis of some neurons in central nervous system (CNS), sever necrotic and hemorrhagic enteritis, foci of haemorrahge and myocardial cell necrosis in the heart, mild to moderate multifocal hepatocytic necrosis, limited focal necrosis in the kidney and testis and morphological evidence of apoptosis in the spleen were observed. Immunohistochemically, influenza virus antigen were detected more often in the acinar cells of pancreas, some neuron and glial cells in CNS, renal tubule of kidney and hepatocytes of the liver. Moreover, antigen was seen in the meisner plexus of the intestine and testis, but seldom in the lung. The RT-PCR tests showed the presence of H5, N1 and NP genes of influenza virus in trachea, lung, liver, kidney, spleen, and cerebellum. Deduced amino acid sequence at cleavage site was PQGERRRKKR G which is typical for highly pathogen avian influenza viruses. The phylogenetic analysis of HA(heamagglutinin) protein showed a very close similarity of Z/101(H5N1) virus with HA proteins of H5 influenza viruses isolates from cat, wild duck, chicken, ostrich and turkey in various geographical regions. Our data confirmed that these new emerging viruses are systemically able to infect wild swans. The high HA protein sequence similarity among H5N1 viruses from various geographical areas of the word shows that the wild aquatic birds are the major cause of worldwide spreading of H5N1 viruses.

Berberine induces apoptosis through a mitochondrial/caspase pathway in human promonocytic U937 cells

Berberine, an isoquinoline plant alkaloid, is known to generate a wide variety of biochemical and pharmacological effects. To elucidate the molecular mechanism of berberine-induced antiproliferative activities, the human promonocytic U937 cells were used. Berberine exhibited dose-dependent antiproliferative effects. Morphological evidence of apoptosis, including apoptotic DNA fragmentation, were observed in cells treated with 75 μg ml −1 of berberine for 24 h. Flow cytometry analysis revealed that berberine had no effect on cell cycle profile of U937 cells, however, sub-G 0 fraction (apoptotic cell population) was detected. The percentage of sub-G 0 fraction of cells treated with 75 μg ml −1 of berberine was 25.3 ± 1.6%. Berberine induces significant changes in mitochondrial membrane potential of U937 cells. The highest tested concentration of berberine decreased the mitochondrial membrane potential to 15.8 ± 2.4% of control. Additionally, berberine-treated cells had an elevated level of ROS production. Activation of caspase-9 and caspase-3 was also detected, with no caspase-8 activation observed. Taken together, the results clearly demonstrate that berberine induces apoptosis of U937 cells through the mitochondrial/caspase-dependent pathway.

Muscle Coenzyme Q10 Level in Statin-Related Myopathy

Statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) reduce the level of cholesterol by inhibiting the synthesis of mevalonate, an intermediary in the cholesterol biosynthetic pathway. Use of statin drugs has been associated with a variety of skeletal muscle-related complaints. Coenzyme Q10 (CoQ10), a component of the mitochondrial respiratory chain, is also synthesized from mevalonate, and decreased muscle CoQ10 concentration may have a role in the pathogenesis of statin drug-related myopathy. To measure the CoQ10 concentration and respiratory chain enzyme activities in muscle biopsy specimens from 18 patients with statin drug-related myopathy and to look for evidence of apoptosis using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) assay. An open-labeled study of CoQ10 concentration in muscle from patients with increased serum creatine kinase concentrations while receiving standard statin drug therapy. Neuromuscular centers at 2 academic tertiary care hospitals. Muscle structure was essentially normal in 14 patients and showed evidence of mitochondrial dysfunction and nonspecific myopathic changes in 2 patients each. Muscle CoQ10 concentration was not statistically different between patients and control subjects, but it was more than 2 SDs below the normal mean in 3 patients and more than 1 SD below normal in 7 patients. There was no TUNEL positivity in any patients. These data suggest that statin drug-related myopathy is associated with a mild decrease in muscle CoQ10 concentration, which does not cause histochemical or biochemical evidence of mitochondrial myopathy or morphologic evidence of apoptosis in most patients.

Erythropoietin attenuates renal injury in experimental acute renal failure ischaemic/reperfusion model

Erythropoietin (EPO), originally identified for its critical role in promoting erythrocyte survival and differentiation, has been shown to exert multiple paracrine/autocrine functions. Protective effects of EPO have been demonstrated in various tissues and experimental models of ischaemia-induced injury. In the present study, we investigated the effect of EPO on an in vivo rat model of renal ischaemia/reperfusion (I/R) injury and the possible mechanisms implicated in the EPO-mediated anti-apoptotic action. Male Wistar rats, subjected to renal ischaemia for 45 min, were administered either saline or EPO (500 U/kg, i.p.) 20 min prior to I/R. A sham-operated group served as the control. At 48 h of reperfusion, the renal dysfunction and injury was assessed by measurement of serum biochemical markers (urea, creatinine) and histological grading. Apoptosis was assessed by the TUNEL method and morphological criteria. Expression of Bax and NF-kappaB (p65) was also evaluated. High levels of serum urea and creatinine were identified at 48 h after ischaemia. The EPO-treated group had significantly lower serum and creatinine levels. Semi-quantitative assessment of the histological lesions showed that rats subjected to I/R developed marked structural damage, whereas significantly less tubular damage was observed in the EPO-treated group. I/R caused an increase in TUNEL-positive cells that was accompanied by morphological evidence of apoptosis. In the EPO-treated rats only a few scattered TUNEL-positive cells were observed. Up-regulation of Bax in the tubular epithelial cells and increased expression of NF-kappaB was observed in the I/R-treated rats, while diminished expression of Bax and positive immunostaining of NF-kappaB was observed in the EPO-treated rats. Administration of EPO as a single dose before the onset of ischaemia produced a significant reduction in tubular injury, which was accompanied by a marked amelioration of renal functional impairment. The cytoprotective action of EPO against I/R injury seems to be associated with its anti-apoptotic action. Moreover, transcription factor NF-kappaB is likely to play a pivotal role in the pathophysiology of I/R renal injury and might have a key role in EPO-mediated protective effects.

Citrinin induces apoptosis in HL-60 cells via activation of the mitochondrial pathway

The mycotoxin citrinin (CTN), a frequent natural contaminants of certain food and feeds, is known to be cytotoxic and genotoxic to various mammalian cells. To investigate the death mode of cells exposed to CTN, human promyelocytic leukemia (HL-60) cells were chosen to identify the apoptotic process induced by CTN. Morphological evidence of apoptosis, including nuclei fragmentation and DNA laddering formation, was clearly observed 24 h after exposure to CTN. Flow cytometry analysis revealed that apoptotic cells in the hypodiploid region dramatically increased in cultures treated with CTN at concentrations above 50 μM. Results of Western blotting showed that CTN induced the formation of processed caspase-3, -6, -7, -9, but not caspase-8, in a dose-dependent manner; CTN also induced a time-dependent increase in caspase-3 catalytic activity. The apoptosis triggered by CTN in HL-60 was accompanied by the cytochrome c release from mitochondria to cytoplasm. The presence of antioxidants in cultures did not effectively suppress CTN-induced cytotoxicity and caspase-3 activity. These findings suggest that CTN induces apoptosis in HL-60 cells by stimulating cytochrome c release followed by activation of multiple caspases, but oxidative stress may not play a role in the apoptotic process.

Morphological Evidence of Apoptosis in Hepatocytes of Rats (Rattus norvegicus) Exposed to Arabian Incense

Morphological Evidence of Apoptosis in Hepatocytes of Rats (Rattus norvegicus) Exposed to Arabian Incense

Calcineurin Triggers Reactive/Inflammatory Processes in Astrocytes and Is Upregulated in Aging and Alzheimer's Models

Astrocyte reactivity (i.e., activation) and associated neuroinflammation are increasingly thought to contribute to neurodegenerative disease. However, the mechanisms that trigger astrocyte activation are poorly understood. Here, we studied the Ca2+-dependent phosphatase calcineurin, which regulates inflammatory signaling pathways in immune cells, for a role in astrogliosis and brain neuroinflammation. Adenoviral transfer of activated calcineurin to primary rat hippocampal cultures resulted in pronounced thickening of astrocyte somata and processes compared with uninfected or virus control cultures, closely mimicking the activated hypertrophic phenotype. This effect was blocked by the calcineurin inhibitor cyclosporin A. Parallel microarray studies, validated by extensive statistical analyses, showed that calcineurin overexpression also induced genes and cellular pathways representing most major markers associated with astrocyte activation and recapitulated numerous changes in gene expression found previously in the hippocampus of normally aging rats or in Alzheimer's disease (AD). No genomic or morphologic evidence of apoptosis or damage to neurons was seen, indicating that the calcineurin effect was mediated by direct actions on astrocytes. Moreover, immunocytochemical studies of the hippocampus/neocortex in normal aging and AD model mice revealed intense calcineurin immunostaining that was highly selective for activated astrocytes. Together, these studies show that calcineurin overexpression is sufficient to trigger essentially the full genomic and phenotypic profiles associated with astrocyte activation and that hypertrophic astrocytes in aging and AD models exhibit dramatic upregulation of calcineurin. Thus, the data identify calcineurin upregulation in astrocytes as a novel candidate for an intracellular trigger of astrogliosis, particularly in aging and AD brain.

The effect of α-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. α-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of γ-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP++NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.

Oncolytic herpes simplex virus mutants are more efficacious than wild‐type adenovirus Type 5 for the treatment of high‐risk neuroblastomas in preclinical models

High-risk neuroblastoma (Nb) is incurable using current treatment regimens in the majority of patients. Oncolytic virotherapy is a novel approach being tested for several types of adult cancers. To compare the susceptibility of Nb tumor models to oncolytic adenovirus and HSV mutants and delineate the mechanisms of resistance or sensitivity. Human Nb cell lines were used to determine susceptibility to adenovirus type 5 wild-type and HSV1 mutant (NV1066) infection, adenovirus receptor expression, support of NV1066 replication, and induction of apoptosis. Human xenograft tumors in immunodeficient mice were evaluated for histological effects and tumor response to intratumoral injection of an oncolytic HSV mutant. All eight Nb cell lines tested in culture were relatively resistant to infection with wild type and attenuated adenoviruses. Cells expressed the cocksackie-adenovirus attachment receptor (CAR) but had low or absent expression of the internalization receptors (alphavbeta3, alphavbeta5 integrins). In contrast, all cells were uniformly sensitive to infection with the attenuated HSV mutant, NV1066. Productive virus replication and induction of apoptosis were observed in HSV-infected cells. CHLA-20 and LAN-5 xenograft tumors injected with a single dose of NV1066 showed a significant antitumor response, and the animals had a prolonged survival post infection in comparison to the PBS-treated control group. HSV injected tumors showed extensive areas of necrosis and morphologic evidence of apoptosis. Nb tumor models are resistant to adenovirus mediated oncolysis but highly sensitive to HSV mediated oncolysis. Further studies of HSV virotherapy as a novel treatment for Nb are warranted.

Ischemia Leads to Apoptosis—and Necrosis‐like Neuron Death in the Ischemic Rat Hippocampus

Morphological evidence of apoptosis in transient forebrain ischemia is controversial. We therefore investigated the time sequence of apoptosis-related antigens by immunohistochemistry and correlated it with emerging nuclear patterns of cell death in a model of transient forebrain ischemia in CA1 pyramidal cells of the rat hippocampus. The earliest ischemic changes were found on day 2 and 3, reflected by an upregulation of phospho-c-Jun in a proportion of morphologically intact CA1 neurons, which matched the number of neurons that succumbed to ischemia at later time points. At day 3 and later 3 ischemic cell death morphologies became apparent: pyknosis, apoptosis-like cell death and necrosis-like cell death, which were confirmed by electron microscopy. Activated caspase-3 was present in the vast majority of cells with apoptosis-like morphology as well as in a small subset of cells undergoing necrosis; its expression peaked on days 3 to 4. Silver staining for nucleoli, which are a substrate for caspase-3, revealed a profound loss of nucleoli in cells with apoptosis-like morphology, whereas cells with necrosis-like morphology showed intact nucleoli. Overall, cells with apoptosis-like morphology and/or caspase-3 expression represented a minor fraction (<10%) of ischemic neurons, while the vast majority followed a necrosis-like pathway. Our studies suggest that CA1 pyramidal cell death following transient forebrain ischemia may be initiated through c-Jun N-terminal kinase (JNK) pathway activation, which then either follows an apoptosis-like cell death pathway or leads to secondary necrosis.

Animal model of chondrocyte apoptosis in the epiphyseal cartilage of the neonatal bone

AbstractApoptosis is considered to be the mechanism responsible for the death of chondrocytes during endochondral bone formation. It is also claimed that apoptosis of the chondrocytes is age related and that the apoptotic index increases with age. However, a detailed analysis of the apoptotic activity of the neonatal epiphyseal cartilage is lacking. A model that evaluates apoptosis in the femoral rat epiphyseal cartilage both quantitatively and qualitatively is reported. Apoptotic incidence in the epiphyseal cartilage reached a maximum at age 6 days, but the age in our study did not significantly affect the percentile rate of apoptotic chondrocytes (P > 0.05, Kruskal-Wallis test). Apoptosis in the zone of hypertrophic cartilage played the most important role in the growth plate’s homeostasis. Morphologic evidence of apoptosis was necessary in addition to positive nick end labeling of cells. Electron microscopy studies revealed atypical modes of programmed death of the growth plate chondrocytes in addition to the classical apoptotic mode.

The pathobiology of moderate diffuse traumatic brain injury as identified using a new experimental model of injury in rats

Experimental models of traumatic brain injury have been developed to replicate selected aspects of human head injury, such as contusion, concussion, and/or diffuse axonal injury. Although diffuse axonal injury is a major feature of clinical head injury, relatively few experimental models of diffuse traumatic brain injury (TBI) have been developed, particularly in smaller animals such as rodents. Here, we describe the pathophysiological consequences of moderate diffuse TBI in rats generated by a newly developed, highly controlled, and reproducible model. This model of TBI caused brain edema beginning 20 min after injury and peaking at 24 h post-trauma, as shown by wet weight/dry weight ratios and diffusion-weighted magnetic resonance imaging. Increased permeability of the blood–brain barrier was present up to 4 h post-injury as evaluated using Evans blue dye. Phosphorus magnetic resonance spectroscopy showed significant declines in brain-free magnesium concentration and reduced cytosolic phosphorylation potential at 4 h post-injury. Diffuse axonal damage was demonstrated using manganese-enhanced magnetic resonance imaging, and intracerebral injection of a fluorescent vital dye (Fluoro-Ruby) at 24-h and 7-day post-injury. Morphological evidence of apoptosis and caspase-3 activation were also found in the cerebral hemisphere and brainstem at 24 h after trauma. These results show that this model is capable of reproducing major biochemical and neurological changes of diffuse clinical TBI.

PAP Modulations in Daudi Cells and Molt-3 Cells Treated with Etoposide are Mutually Associated with Morphological Evidence of Apoptosis

Daudi (B-cell line) and Molt-3 (T-cell line) cells provide a model for the study of apoptosis, the induction of which is often accompanied by concominant modulations of proteins involved in mRNA maturation. One of these proteins is poly(A) polymerase (PAP), which is responsible for mRNA cleavage and polyadenylation. A number of recent reports also suggest involvement of mRNA maturation and stability in the induction of specific pathways of cell apoptosis. In this study we identified PAP activity levels and isoform modulations in two different cell lines (Daudi and Molt-3) and related them to DNA fragmentation (a hallmark of apoptosis) and cell cycle phase specificity in terms of the temporal sequence of events and the time that elapsed between administration of the apoptosis inducer (the widely used anticancer drug etoposide) and the observed effects. Treatment of both cell lines with 20 microg/mL etoposide induced apoptosis after four hours in Molt-3 cells and only after 24 hours in Daudi cells, as revealed by two independent methods. In Daudi cells the PAP activity levels and isoforms were downregulated prior to deltapsim reduction, DNA fragmentation and the morphological changes of the nucleus, whereas in Molt-3 cells no PAP activity and isoform modulations were observed prior to the early hallmarks of apoptosis.

CYTOTOXICITY INDUCED BY NIVALENOL, DEOXYNIVALENOL, AND FUMONISIN B1 IN THE SF-9 INSECT CELL LINE

The toxicity of the mycotoxins nivalenol (NIV), deoxynivalenol (DON), and fumonisin B1 (FB1) was studied in the lepidopteran Spodoptera frugiperda (SF-9) cells, by the trypan blue dye-exclusion and 3-(4,5-dimethylthiozole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) tests, uptake analyses of cytotoxicity, and cell metabolism, respectively. Deoxyribonucleic acid analysis by flow cytometry was used to identify apoptosis and cell cycle distribution. After 48 h of exposure, the MTT and trypan blue dye-exclusion tests indicated that NIV was significantly more toxic than DON, and both were significantly more toxic than FB1. The IC50 (mycotoxin concentration resulting in 50% inhibition of proliferation) values for NIV and DON were 4.5 and 41 microM, and the CC50 (mycotoxin concentration that caused 50% cytotoxicity) values were 9.5 and 45 microM, respectively. At the highest concentration of FB1 (100 microM), there was 80% viability. With the same incubation time, cell cycle distribution showed an arrest of cells in the G0/G1 phase in the presence of NIV (up to 0.3 microM), DON (up to 3 microM), and FB1 (up to 10 microM). Morphological evidence of apoptosis was related to the toxicity of the substances in that the more toxic NIV induced late apoptosis, whereas DON and FB1 produced less-severe morphological changes characteristic of early apoptosis. This study suggests that NIV is more toxic than DON, which in turn is more toxic than FB1. These mycotoxins can modify the normal progression of the cell cycle and induce an apoptotic process.

PAP modulations in Daudi cells and Molt-3 cells treated with etoposide are mutually associated with morphological evidence of apoptosis

Daudi (B-cell line) and Molt-3 (T-cell line) cells provide a model for the study of apoptosis, the induction of which is often accompanied by concominant modulations of proteins involved in mRNA maturation. One of these proteins is poly(A) polymerase (PAP), which is responsible for mRNA cleavage and polyadenylation. A number of recent reports also suggest involvement of mRNA maturation and stability in the induction of specific pathways of cell apoptosis. In this study we identified PAP activity levels and isoform modulations in two different cell lines (Daudi and Molt-3) and related them to DNA fragmentation (a hallmark of apoptosis) and cell cycle phase specificity in terms of the temporal sequence of events and the time that elapsed between administration of the apoptosis inducer (the widely used anticancer drug etoposide) and the observed effects. Treatment of both cell lines with 20 microg/mL etoposide induced apoptosis after four hours in Molt-3 cells and only after 24 hours in Daudi cells, as revealed by two independent methods. In Daudi cells the PAP activity levels and isoforms were downregulated prior to deltapsim reduction, DNA fragmentation and the morphological changes of the nucleus, whereas in Molt-3 cells no PAP activity and isoform modulations were observed prior to the early hallmarks of apoptosis.