DNA Breakdown Research Articles

Correlation of Apoptosis with Comet Formation Induced by Tea Polyphenols in Human Leukemia Cells

Induction of apoptosis is an important approach to cancer control. Apart from morphological changes in cells, apoptosis is characterized by fragmentation of nuclear DNA. The characteristic DNA ladder formation that is observed on gel electrophoresis does not reflect the DNA breakdown in individual cells; contributions from small subpopulations are usually overlooked. On the other hand, alkaline comet assay as measured by single cell gel electrophoresis accurately measures DNA fragmentation at a single cell level. The comet assay was originally developed as a cytogenetic test to measure the genotoxicity of various chemicals. However, the comet image generated by an apoptotic cell is different from that obtained with a cell treated for a short time with a genotoxic agent. In the present study using human leukemic cells, typical apoptotic features such as morphological characteristics, FACS analysis, caspase activation, and expression of apoptosis-related genes as induced by tea polyphenols have been found to correlate with the comet tail formation. It is apparent from the high degree of correlation observed between the comet tail moment and each parameter of apoptosis that the comet assay can accurately reflect the measure of DNA fragmentation and, hence, can be used to detect a cell undergoing apoptosis.

Neuronal death in primary retinal cultures is related to nitric oxide production, and is inhibited by erythropoietin in a glucose‐sensitive manner

The aim of this work was to investigate the interrelated effects of glucose, nitric oxide (NO) and erythropoietin on neuronal survival in retinal cultures, thereby exploring the mechanism of neuronal death in the diabetic retina. Rat retinal cells were cultured in low (5 mM) or high (15 mM) glucose concentrations. After 9 days, cell viability was assessed by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and NO production was determined by the Griess reaction. Immunohistochemistry was used to quantify GABA-labelled neurones and cells staining for DNA breakdown. High or low glucose concentrations had no effect on basal NO production or the survival of neurones in culture, but treatment with N-nitro-L-arginine methyl ester reduced extracellular levels of NO and increased neuronal survival at both concentrations of glucose. Erythropoietin decreased cell death and NO levels, but only in cultures grown in low concentrations of glucose. It is concluded that erythropoietin's neurotrophic function in the retina is attenuated at glucose concentrations similar to those which occur in diabetes.

HGF protects corneal epithelial cells from apoptosis by the PI-3K/Akt-1/Bad- but not the ERK1/2-mediated signaling pathway.

Cell survival is critical during corneal epithelial regeneration after injury, and growth factors could be fundamental in cytoprotection. The goal of this study was to investigate the involvement of the paracrine hepatocyte growth factor (HGF) in the prevention of corneal epithelial cell apoptosis and to identify signal transducers in this process. Apoptosis in human and rabbit corneal epithelial (HCE and RCE) cells was induced with a nutrient-deprived exhausted medium (ExM) or by treatment with staurosporine (20-100 ng/mL) or the calcium ionophore A23187 (0.5 microM). Apoptotic cells were identified by DNA fragmentation in agarose gels and by Hoechst staining. Active Akt-1 overexpression (Akt-1 pUSEamp cDNA) and small interfering RNA (siRNA) specific for Akt mRNA were used. Immunofluorescence, Western immunoblot analysis, and Akt kinase assays were also used. Staurosporine, ExM, and A23187 induced DNA fragmentation in HCE and RCE cells. HGF (20 ng/mL) in combination with the apoptotic agents greatly reduced DNA breakdown and the number of Hoechst-positive cells. In the presence of phosphatidylinositol-3 kinase (PI-3K) inhibitors (wortmannin and LY294002), HGF did not overcome apoptosis. However, PD98059, the ERK1/2 cascade pathway inhibitor, was ineffective in preventing HGF protection. HGF induced a sustained activation of Akt-1, and overexpression of active Akt-1 reduced apoptosis. HGF stimulated the downstream targets of Akt, glycogen synthase kinase (GSK-3), and Bad, a proapoptotic member of the Bcl-2 family, an effect that was blocked by PI-3K inhibitors but not by ERK1/2 inhibition. Suppressing the expression of Akt by Akt siRNA led to a decrease in the phosphorylation of Bad and GSK-3. Translocation of Bad to the mitochondria, a critical stage in apoptosis, was prevented by HGF when apoptosis was induced. Moreover, in epithelial cells overexpressing active Akt-1, Bad translocation was also prevented. HGF modulates multiple signaling cascades in corneal epithelial cells. The results demonstrated that HGF, in a paracrine fashion, protects cells from apoptosis through a PI-3K/Akt/Bad pathway but not through an ERK1/2 pathway. It was also demonstrated that GSK-3 is a target of PI-3K/Akt-1.

Individual expression of poliovirus 2Apro and 3Cpro induces activation of caspase-3 and PARP cleavage in HeLa cells

The expression of individual viral genes enables the study of their effects on cellular functions. Our group previously generated stable HeLa cell lines that efficiently express poliovirus proteases 2A (clone 2A7d) and 3C (clone 3C7) under the control of tetracycline [Virology 266 (2000a) 352; J. Virol. 74 (2000b) 2383]. Upon induction of these proteases, the cells undergo drastic morphological alterations and eventually die. The present paper characterizes, in detail, the cellular and molecular events that lead to cell death in these lines. Several signs of apoptosis were observed in both 2A7d- and 3C7-induced cells, such as nuclear fragmentation, DNA breakdown (as determined by TUNEL), and phosphatidylserine translocation. Protease 2A induces the cleavage of poly-ADP-ribose-polymerase (PARP). This is blocked by the caspase-3 inhibitor DEVD in both 2A7d-On and 3C7-On cells suggesting that this enzyme might account for PARP cleavage in both cell lines. The results indicate that both poliovirus proteases induce apoptosis by mechanisms involving caspase activation, although the kinetics of apoptosis differs.

In situ monitoring of cell death using Raman microspectroscopy

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.

Microdissection genotyping of archival fixative treated tissue for gaucher disease

The genetic diagnosis of Gaucher disease by molecular methods is complicated by the existence of a highly homologous transcribed pseudogene (96% identity) that is found in close proximity to the true gene on chromosome 1q21. In addition, the pseudogene sequence can mimic disease-causing mutations in the true gene. Selective polymerase chain reaction (PCR) amplification of the true gene can be accomplished in extracted DNA from fresh-frozen samples by designing oligonucleotide primers to hybridize to defined regions that are not present in the pseudogene. This standard molecular approach, which entails amplification of relatively long segments of intact DNA, is not feasible in archival, paraffin-embedded, solid-tissue specimens in which the negative effects of chemical fixation result in DNA strand scission and breakdown of nucleic acid. A novel approach, specifically created for use with archival, fixative-treated tissue specimens, was developed for detection and characterization of common mutations of Gaucher disease. Three separate robust PCR reactions were formulated, 2 for selective amplification of portions of only the true gene exons 2 and 9, with a third reaction targeting exon 10, wherein both the true and pseudogene were coamplified. In the latter, DNA sequencing was used to determine the presence of true and pseudogene allele content in addition to identification of base sequence alterations. This method, requiring a single, 4-μm-thick histologic section, was successfully applied to archival paraffin block tissue specimens that had been in storage for up to 75 years. It was capable of accurately genotyping common Gaucher disease mutations as well as discovering a novel mutation and genetic polymorphism. We recommend our approach when only fixative-treated tis sue is available for molecular genotyping.

Programmed cell death features in apple suspension cells under low oxygen culture

Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple celi death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

Programmed cell death features in apple suspension cells under low oxygen culture.

Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

Differential Efficacy of Caspase Inhibitors on Apoptosis Markers during Sepsis in Rats and Implication for Fractional Inhibition Requirements for Therapeutics

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

Activation of K+ channels: an essential pathway in programmed cell death.

Cell apoptosis and proliferation are two counterparts in sharing the responsibility for maintaining normal tissue homeostasis. In recent years, the process of the programmed cell death has gained much interest because of its influence on malignant cell growth and other pathological states. Apoptosis is characterized by a distinct series of morphological and biochemical changes that result in cell shrinkage, DNA breakdown, and, ultimately, phagocytic death. Diverse external and internal stimuli trigger apoptosis, and enhanced K+ efflux has been shown to be an essential mediator of not only early apoptotic cell shrinkage, but also of downstream caspase activation and DNA fragmentation. The goal of this review is to discuss the role(s) played by K+ transport or flux across the plasma membrane in the regulation of the apoptotic volume decrease and apoptosis. Attention has also been paid to the role of inner mitochondrial membrane ion transport in the regulation of mitochondrial permeability and apoptosis. We provide specific examples of how deregulation of the apoptotic process contributes to pulmonary arterial medial hypertrophy, a major pathological feature in patients with pulmonary arterial hypertension. Finally, we discuss the targeting of K+ channels as a potential therapeutic tool in modulating apoptosis to maintain the balance between cell proliferation and cell death that is essential to the normal development and function of an organism.

Detection of apoptosis in cartilage in situ and in isolated chondrocytes.

Apoptosis is a form of programmed cell death conceptually opposed to necrosis. In view of the inherent difficulty in accurately detecting apoptosis in chondrocytes, this chapter describes complementary techniques that may be used in combination. During apoptotic death, protein and DNA breakdown is accomplished by caspases (cysteine proteases) in a highly regulated manner. Activation of caspases occurs in the initiation and/or the execution phase of certain apoptotic programs and represents an early physiologic marker of apoptosis. Here we present an immunoblotting technique that allows the detection of caspase-3 processing in cultured human chondrocytes. Apoptosis leads to plasma membrane asymmetry and to externalization of phosphatidylserine residues, which are bound with high affinity by annexin V. In the early stages of apoptosis, cells typically have an intact cell membrane. Apoptotic cells will not stain positive with propidium iodide, whereas externalization of phosphatidylserine will be detected by annexin V. Terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) works on the principle that DNA strand breaks (single or double) that occur during apoptosis can be identified by labeling free 3'-hydroxyl termini. Labeled nucleotides are polymerized to these termini in a reaction catalyzed by TdT. The tissue can then be examined histologically for identification of TUNEL-positive cells in situ.

Expression and significance of cell immunohistochemical markers (HHF-35, CD-31, Bcl-2, P-53 and apopDETEC) in hypertrophic cardiomyopathy.

There are several hypotheses concerning the pathogenesis of hypertrophic cardiomyopathy (genetic, ischaemic, immune, inflammatory and apoptosis induction). We have studied three types of cardiomyopathy in order to observe the expression and assess the significance of different immunohistochemical markers (muscular actin, CD-31, proliferation cell nuclear antigen -PCNA-, Ki-67, and markers related with programmed cell death, bcl-2, p-53 and apopDETEC). We studied different microscopic (haematoxylin-eosin and Masson's thrichrome) and immunohistochemical parameters (streptavidin-biotin-peroxidase and "in situ" hybridisation) of forty cases: ten each of hypertensive hypertrophic cardiomyopathy, essential hypertrophic cardiomyopathy, hypertrophic cardiomyopathy in patients treated with chemotherapy and morphologically "normal" hearts. Our findings point to an absence of structural marker expression (actin and CD-31) in cases of hypoxic damage. The distribution and intensity of apoptosis markers, a seen by "in situ" hybridisation were irregular, and the rest of the markers studied showed negative results, with the exception of acridin orange (a marker of hypoxic damage). In our opinion, the above immunohistochemical markers, especially actin and CD-31, could be used for differentiating hypoxic lesions in these three types of cardiomyopathy. Moreover, it is difficult to know the significance of the apoptosis markers, because the autolysis process produces cross reactions with false positive results. We think that there is a need for new studies on DNA breakdown processes during the post-mortem interval. To avoid autolysis problems the post-mortem material needs to be as fresh as possible.

Inhibition of nitric oxide synthesis potentiates apoptosis in the rabbit corpus luteum

To determine if nitric oxide (NO) plays a role in corpus luteum (CL) physiology by affecting progesterone secretion or luteal apoptosis, an in-vitro pseudopregnant rabbit ovarian perfusion system was used to measure the effects of an inhibitor of NO synthesis, N G-nitro- l-arginine methyl ester ( l-NAME), on progesterone secretion and corpus luteal apoptosis as measured by internucleosomal DNA breakdown. Pseudopregnant rabbit ovaries perfused in vitro with l-NAME did not demonstrate any significant differences compared with control ovaries in progesterone secretion. However, apoptosis, as measured by internucleosomal breakdown, was significantly increased in l-NAME-perfused CL compared with controls. While NO does not appear to directly affect progesterone secretion, there does appear to be a role for NO in CL maintenance, or a role for inhibition of NO production in CL regression.

Bromoenol Lactone Promotes Cell Death by a Mechanism Involving Phosphatidate Phosphohydrolase-1 Rather than Calcium-independent Phospholipase A2

Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.

Persistence of DNA of Gaeumannomyces graminis var. tritici in soil as measured by a DNA-based assay

There are an increasing number of assays available for fungal plant pathogens based on DNA technology. We have developed such an assay for Gaeumannomyces graminis var. tritici ( Ggt) in soil, using slot-blot hybridisation. To ensure the validity of DNA-based soil assays for the fungus, it is important to determine the stability of Ggt DNA in soil. This study was undertaken to quantify the DNA degradation of dead Ggt in soil using a DNA-based assay. Mycelia were killed using various treatments, then DNA was extracted and estimated by a slot-blot hybridisation technique using the specific Ggt DNA probe, pG158. Mycelia were also killed using a fungicide (triadimefon) at a concentration of 150–250 μg ml −1. The amount of detectable DNA of Ggt, killed using triadimefon, declined by 82–93%. Inoculum in the form of diseased wheat roots, artificially inoculated ryegrass seed, particulate soil organic matter and whole soil was killed using heat-treatment. The amount of detectable DNA of Ggt declined markedly (90%) in both heat-treated roots and inoculated ryegrass seeds, and declined by 50% in both treated soil and soil organic matter. The rate of DNA degradation of Ggt in soil varied with the type of inoculum. The amount of detectable DNA of Ggt in dead mycelia declined by 99.8% after 4 days of incubation in soil. No DNA was detected after 8 days of incubation. In contrast, Ggt DNA in live mycelia declined by 70% after 8 days of incubation and declined to 10% of original DNA level after 32 days. In ground ryegrass seed inoculum, DNA in both killed and live Ggt declined by 50% after 8 days. In diseased roots, DNA from both live and killed Ggt did not appear to decline over 16 days. Estimates of the amount of Ggt in the soil using a DNA-based assay reflect both live and dead populations of the fungus. The rate of breakdown of DNA of the dead fungus is very high and the presence of dead fungi in roots probably a rare event so the DNA from dead fungus probably contributes little to the total DNA level.

A History of Commercial Plant Growth Regulators in Apple Production

DNA breakdown (Miller et al., 1955). Kinetin was significantly more active than adenine in the tobacco tissue culture bioassay. The use of the plant cell culture bioassay was a key to the eventual isolation of zeatin from corn by Letham (1963). ETHYLENE. The growth regulating properties of ethylene were first recognized by the Russian scientist Nejebulov in 1901 (Beyer et al., 1984). His experiments showed that illuminating gas could cause leaf abscission and epinasty. The fruit physiologist Crocker developed the Alaskan pea bioassay that used the triple response of shortening and thickening of the hypocotyl, agravitropic growth, and maintained hypocotyl hook (Reid and Howell, 1995) to assess ethylene levels. He was the first to suggest that ethylene was an endogenous plant hormone. However, few scientist at the time agreed with Crocker since it was difficult to visualize that a gas could act as an endogenous regulating substance. It was not until the 1960s when gas chromatography was used to quantify endogenous ethylene that the significance of ethylene was recognized and acknowledged as an endogenous hormone. ABSCISIC ACID.While researchers had long speculated that a hormone which affected abscission, dormancy, and stress reduction existed, it was not until the 1950s that work on the isolation and identification of this compound began. In 1953 Bennet-Clark and Kefford discovered an acid fraction of plant extracts that was a potent growth inhibitor. In 1955 Osborne reported on the diffusion of a senescence factor from older leaves that could speed the abscission of bean petioles. By 1963, Addicott s group studying cotton fruit abscission in California had identified and characterized abscisin II. At the same time, Wareing s group in Wales identified the dormin as a compound that promoted dormancy in woody plants. In 1968, these groups formally redesignated abscisin II and dormin as abscisic acid (Milborrow, 1984).

In vivo hormonal environment leads to differential susceptibility of the corpus luteum to apoptosis in vitro.

We evaluated the involvement of the in vivo hormonal environment on the ability of the rat corpus luteum (CL) to undergo apoptosis. Gel electrophoretic DNA fragmentation analysis revealed no apoptosis in CL isolated either the 2 last days of pregnancy (Days 21 and 22) or throughout the 4 days following parturition, suggesting that the number of cells undergoing apoptosis at the same time is not sufficient to allow for visualization of DNA breakdown. In contrast, CL incubated in serum-free medium underwent significant apoptosis, as evaluated by chromatin condensation and DNA fragmentation, regardless of their developmental stage in pregnancy. However, CL obtained on Day 7 of pregnancy and on Day 4 postpartum demonstrated higher sensitivity to apoptosis in vitro, but lactation reduced significantly the capacity of the CL to undergo apoptosis when maintained in culture. These data suggest that the exposure of the CL to different hormonal environments throughout pregnancy and after parturition is responsible for the differential susceptibility to apoptosis observed in vitro. We have previously shown that progesterone is a direct factor for survival of the CL. Prolactin stimulates luteal progesterone production; therefore, we examined whether prolactin prevents apoptosis in luteal cells independently of its stimulatory action on progesterone production. We used a luteal cell line (GG-CL) that expresses the prolactin receptor but does not produce progesterone. These cells undergo apoptosis under conditions of serum starvation, and addition of prolactin to the culture medium significantly reduced DNA fragmentation. These results indicate that the extent of luteal cell death induced by incubation of CL under serum-free conditions depends on the hormonal environment to which this endocrine gland is exposed in vivo. These results also indicate an important role for lactation in preventing apoptosis, which is further supported by the antiapoptotic activity of prolactin observed in luteal cells.

Apoptosis of human carcinoma cells in the presence of potential anti-cancer drugs: III. Treatment of Colo-205 and SKBR3 cells with: cis -platin, Tamoxifen, Melphalan, Betulinic acid, L-PDMP, L-PPMP, and GD3 ganglioside.

Breast cancer is the most common type of cancer, predominantly among women over 20, whereas colo-rectal cancer occurs in both men and women over the age of 50. Chemotherapy of both cancers affect rapidly growing normal as well as cancer cells. Cancer cells are non-apoptotic. Seven anti-cancer agents (cis -platin, Tamoxifen, Melphalan, Betulinic acid, D-PDMP, L-PPMP, and GD3) have been tested with human breast (SKBR3) and colon (Colo-205) carcinoma cells for their apoptotic effect and found to be positive by several assay systems. Colo-205 cells were obtained from ATCC, and the SKBR3 cells were a gift from the Cleveland Clinic. All of these six agents killed those two cell lines in a dose-dependent manner. In the early apoptotic stage (6 h), these cells showed only a flopping of phosphatidylserine on the outer lamella of the plasma membranes as evidenced by the binding of a novel fluorescent dye PSS-380. After 24 h of the treatment, those apoptotic cells showed damage of the plasma as well as the nuclear membrane as evidenced by binding of propidium iodide to the nuclear DNA. DNA laddering assay viewed further breakdown of DNA by 1% agarose gel electrophoresis analysis. It is concluded that during apoptosis the signaling by Mitochondrial Signaling Pathway (MSP) is stimulated by some of these agents. Caspase 3 was activated with the concomitant appearance of its p17 polypeptide as viewed by Westernblot analyses. Incorporation of radioactivity from [U-(14)C]-L-serine in total sphingolipid mixture was observed between 2 and 4 micromolar concentrations of most of the agents except ci s-platin. However, apoptosis in carcinoma cells in the presence of cis -platin is induced by a caspase 3 activation pathway without any increase in synthesis of ceramide.

N-acetylcysteine prevents MAA induced male germ cell apoptosis: role of glutathione and cytochrome c

Exposure to methoxyacetic acid (MAA), a major byproduct of the paint industry, causes testicular atrophy in multiple species. This study demonstrates DNA breakdown in rat germ cells after exposure to MAA in vivo within 12 h, leading to 40% germ cell death by 24 h. Within 4 h of treatment, cytochrome c is released from the mitochondria into the cytosol without the involvement of mitochondrial potential loss, reactive oxygen species generation or lipid peroxidation events. Peak activation of caspase-9 and caspase-3 is detectable post treatment at 4 and 8 h respectively. There is a decrease in germ cell glutathione levels within 2 h of MAA treatment. Replenishment of glutathione by pretreatment of the animals with the antioxidant N-acetylcysteine prior to MAA treatment could prevent the release of cytochrome c, DNA fragmentation and cell death.

Radiocontrast-induced renal tubular cell apoptosis: hypertonic versus oxidative stress.

Radiocontrast-induced nephropathy (RCIN) is a major complication of intravascular radiocontrast administration. Renal tubular cell apoptosis is a feature of RCIN, which is related to hypertonicity of contrast agents. Because a hyperosmolal extracellular environment induces oxidative stress via reactive oxygen species, we tested the hypothesis that antioxidants decrease hypertonicity-induced apoptosis of renal epithelial cells. We analyzed the effects of the antioxidants N-acetylcysteine (NAC) and taurine on hypertonicity-induced apoptosis of renal epithelial cells in vitro. Madin Darby Canine Kidney (MDCK) cells were incubated with the highly hyperosmolal, ionic radiocontrast agent diatrizoate (20% vol/vol, 6 hours) or with equally hyperosmolal (640 mOsm/kg) NaCl solutions. DNA fragmentation, which is a hallmark feature of apoptosis, was assessed quantitatively using flow cytometry after propidium iodide staining and qualitatively using agarose gel electrophoresis. Both diatrizoate and NaCl induced DNA fragmentation in MDCK cells. Taurine (10 mmol/L) reduced DNA degradation in both diatrizoate- [79.5 +/- 2.3% versus 72.2 +/- 3.0%; P = 0.0088] and NaCl- [49.5 +/- 4.0% versus 39.4 +/- 1.0%; P = 0.0271] treated cells. In contrast, NAC (10 mmol/L) failed to reduce the DNA breakdown in this model of hypertonicity-induced renal tubular cell apoptosis. The radiocontrast/hypertonicity-induced DNA fragmentation of MDCK cells is attenuated by taurine but not by NAC. Because both agents are antioxidants, the antioxidant property is not sufficient for the observed cytoprotective effect. Hence, the antiapoptotic effect of taurine has to be attributed to other, yet to be defined mechanisms. Our results suggest that pharmacological doses of taurine may be particularly protective against RCIN.