The action of three antiseptics/disinfectants against enveloped and non-enveloped viruses

Studies were carried out to investigate the protective effects of pyruvate, a key glycolytic intermediate and alpha-keto-monocarboxylate, against oxidative stress-induced apoptosis. Oxidative stress was induced by treating mouse thymocytes with 25 microM hydrogen peroxide for 15 min at 37 degrees C under 5% CO2 in air. Pre- and post-treatment of cells with 10 mM pyruvate inhibited morphological changes, internucleosomal DNA fragmentation, and translocation of phosphatidylserine to the plasma membrane surface, which are characteristic features of apoptosis. L-lactate (10 mM) and acetate (10 mM) were ineffective in inhibiting apoptosis and appeared to be toxic to the cells under similar conditions. The results suggest that pyruvate has therapeutic potential for use in the treatment of oxidative stress-induced disorders associated with increased apoptosis.     

Frequency-shaped amplification changes the neural representation of speech with noise-induced hearing loss

Apoptosis is a mode of active cell death. We have examined whether 2-chloroethylethyl sulfide (CEES), a sulfur vesicating agent, triggers apoptosis as a cytotoxic mechanism. Incubation of thymocytes with CEES, resulted in an induction of apoptotic features of cell death. Treatment of cells with 100 microM CEES for 5 h increased DNA fragmentation to approximately 40% of control. The fragmentation of DNA was visualized by agarose gel electrophoresis. It showed ladder pattern of DNA fragmentation, which indicates internucleosomal cleavage of DNA. Further evidence of apoptosis was observed in morphological changes of nuclei by using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The percentage of TUNEL positive cells was dependent upon CEES concentrations. CEES induced the classical morphological features of apoptosis in nucleus. These features were accompanied by condensation of chromatin, which arranged in sharply declined clumps and fragmentation of nucleus. To study requirement for synthesis of new protein in CEES-induced apoptosis, we studied the effect of cycloheximide for apoptotic activity. This protein synthesis inhibitor did not suppress the CEES-induced apoptotic activity. Taken together, these results suggest that CEES-induced apoptosis as a cytotoxicmechanism and this process occurs independent of synthesis of new protein.     

Oxidative stress induces DNA fragmentation and caspase activation via the c-Jun NH2-terminal kinase pathway in H9c2 cardiac muscle cells

The aim of this study was to test the hypothesis that oxidative stress induces apoptosis in the H9c2 cardiac muscle cell line, and that signaling via mitogen-activated protein kinase (MAPK) pathways is involved. Three forms of oxidative stress were utilized: the superoxide generator menadione; hydrogen peroxide; or simulated ischemia followed by reperfusion. Relatively low concentrations of menadione (10 micrometer) or H2O2 (250 micrometer) caused maximal DNA fragmentation and caspase activation, both markers for apoptotic cell death, and preferential activation of the c-Jun NH 2-terminal kinase (JNK) and p38 MAPK pathways. In contrast, higher concentrations of menadione or H 2O2 caused less DNA fragmentation, more necrotic cell death and preferential activation of the extracellular signal-regulated kinase (ERK) pathway. Simulated ischemia alone did not induce DNA fragmentation or caspase activation and activated only the p38 MAPK pathway. However, ischemia plus reperfusion resulted in DNA fragmentation, caspase activation, necrotic cell death and activation of all three MAPK pathways. Selective inhibition of the ERK or p38 MAPK pathways (by PD98059 or SB-203580, respectively) had no effect on the extent of oxidative stress-induced DNA fragmentation or caspase activation. In contrast, inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of DNA fragmentation and caspase activation induced by oxidative stress. In conclusion, these data suggest that the JNK pathway plays an important role in signaling oxidative stress-induced apoptosis of H9c2 cardiac muscle cells.     

Oxidative DNA damage and apoptosis induced by benzene metabolites

Benzene is a widely recognized human carcinogen. The mechanism of DNA damage induced by major benzene metabolites 1,4-benzoquinone (1,4-BQ) and hydroquinone (1,4-HQ) was investigated in relation to apoptosis and carcinogenesis. Pulsed-field gel electrophoresis showed that cellular DNA strand breakage was induced by benzene metabolites. Internucleosomal DNA fragmentation and morphological changes of apoptotic cells were observed at higher concentrations of benzene metabolites. Flow cytometry showed an increase of peroxides in cultured cells treated with benzene metabolites. 1,4-BQ induced these changes at a much lower concentration than 1,4-HQ. Damage to DNA fragments obtained from the c-Ha-ras-1 proto-oncogene was investigated by a DNA sequencing technique. 1,4-BQ + NADH and 1,4-HQ induced piperidine-labile sites frequently at thymine residues in the presence of Cu(II). Catalase and bathocuproine inhibited DNA damage, suggesting that H2O2 reacts with Cu(I) to produce active species causing DNA damage. Electron spin resonance studies showed that semiquinone radical was produced by NADH-mediated reduction of 1,4-BQ and autoxidation of 1,4-HQ, suggesting that benzene metabolites produce O2- and H2O2 via the formation of semiquinone radical. These results suggest that these benzene metabolites cause DNA damage through H2O2 generation in cells, preceding internucleosomal DNA fragmentation leading to apoptosis. The fates of the cells to apoptosis or mutation might be dependent on the intensity of DNA damage and the ability to repair DNA.     

Role of oxygen radicals generated by NADPH oxidase in apoptosis induced in human leukemia cells

We have used a human leukemia cell line that, after homologous recombination knockout of the gp91-phox subunit of the phagocyte respiratory-burst oxidase cytochrome b-558, mimics chronic granulomatous disease (X-CGD) to study the role of oxygen radicals in apoptosis. Camptothecin (CPT), a topoisomerase I inhibitor, induced significantly more apoptosis in PLB-985 cells than in X-CGD cells. Sensitivity to CPT was enhanced after neutrophilic differentiation, but was lost after monocytic differentiation. No difference between the two cell lines was observed after treatment with other apoptosis inducers, including etoposide, ultraviolet radiation, ionizing radiation, hydrogen peroxide, or 7-hydroxystaurosporine. After granulocytic differentiation of both cell lines, CPT still induced apoptosis, suggesting independence from replication in fully differentiated and growth-arrested cells. Pyrrolidine dithiocarbamate (an antioxidant inhibitor of NF-kappaB) and catalase partially inhibited CPT-induced DNA fragmentation in granulocytic-differentiated PLB-985 cells, but had no effect in X-CGD cells. Flow cytometry analysis revealed that reactive oxygen intermediates were generated in CPT-treated PLB-985 cells. These data indicate that oxygen radicals generated by NADPH oxidase may contribute directly or indirectly to CPT-induced apoptosis in human leukemia and in neutrophilic-differentiated cells.     

Light-induced apoptosis: differential timing in the retina and pigment epithelium

Apoptosis is a genetically regulated form of cell death. Individual cells show condensed nuclear chromatin and cytoplasm, and biochemical analysis reveals fragmentation of the DNA. Ensuing cellular components, apoptotic bodies, are removed by macrophages or neighboring cells. Genes involved in the regulation of apoptosis as well as stimuli and signal transduction systems, are only beginning to be understood in the retina. Therefore, we developed a new in vivo model system for the investigation of events leading to apoptosis in the retina and the pigment epithelium. We induced apoptosis in retinal photoreceptors and the pigment epithelium of albino rats by exposure to 3000 lux of diffuse, cool white fluorescent light for short time periods of up to 120 minutes. Animals were killed at different time intervals during and after light exposure. The eyes were enucleated and the lower central retina was processed for light- and electron microscopy. DNA fragmentation was analysed in situ by TdT-mediated dUTP nick-end labeling (TUNEL) or by gel electrophoresis of total retinal DNA. We observed that the timing of apoptosis in the photoreceptors and pigment epithelium was remarkably different, the pigment epithelium showing a distinct delay of several hours before the onset of apoptosis. In photoreceptors, apoptosis was induced within 90 minutes of light exposure, with the morphological appearance of apoptosis preceding the fragmentation of DNA. In the pigment epithelium, the morphological appearance of apoptosis and DNA fragmentation were coincident. Different regulative mechanisms may lead to apoptotic cell death in the retinal photoreceptors and pigment epithelium. This in vivo model system will allow measurement of dose-responses, a potential spectral dependence and the molecular background of apoptotic mechanisms in the retina.     

Distinct steps in DNA fragmentation pathway during camptothecin-induced apoptosis involved caspase-, benzyloxycarbonyl- and N-tosyl-L-phenylalanylchloromethyl ketone-sensitive activities

Monocytic-like leukemia U-937 cells rapidly undergo morphological changes and DNA fragmentation that is typical of apoptosis following treatment with DNA topoisomerase I inhibitor [20-S-camptothecin lactone (CPT)]. The tripeptide derivative benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone blocks Asp-Glu-Val-Asp-ase (DEVDase) activity and prevents the occurrence of high molecular weight and oligonucleosome-sized DNA fragments associated with apoptosis in CPT-treated cells. In contrast, N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) does not prevent DEVDase activity and high molecular weight DNA fragmentation but completely abrogates the appearance of oligonucleosome-sized DNA fragmentation. These results suggest that caspase 3-like activities are involved with high molecular weight DNA fragmentation pathway, whereas TPCK-sensitive activities are involved in oligonucleosome-sized DNA fragmentation pathway in CPT-treated cells. Electron micrographs reveal that caspase inhibition by benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone also abrogates the typical morphological changes associated with apoptosis, whereas TPCK does not delay these morphological changes that are typical of apoptosis. Caspase inhibition slows passage of the cells through G2 and causes a transient accumulation of these cells at the G0/G1 phase of the cell cycle following CPT treatment. In a cell-free system, when purified nuclei are incubated with apoptotic cytosolic extracts obtained from CPT-treated U-937 cells, TPCK causes a similar effect in abrogating the oligonucleosome-sized DNA fragmentation but does not affect DEVDase activity. Addition of either benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group or acetyl-Asp-Glu-Val-Asp-aldehyde completely inhibits DEVDase activity in these extracts. However, acetyl-Asp-Glu-Val-Asp-aldehyde does not affect the occurrence of oligonucleosome-sized DNA fragmentation in the cell-free system, whereas the benzyloxycarbonyl derivatives benzyloxycarbonyl-Val-Ala-Asp-free carboxyl group, benzyloxycarbonyl-Val-Ala-free hydroxyl group, benzyloxycarbonyl-Val-free hydroxyl group, and benzyloxycarbonyl hydrazide abolish it markedly. Taken together, these observations show the pivotal role of DEVDase activity in triggering the apoptotic process and high molecular weight DNA fragmentation, whereas TPCK- and benzyloxycarbonyl-sensitive activities are involved in the oligonucleosome-sized DNA fragmentation pathway induced by CPT.     

Evidence against a direct role for the induction of c-jun expression in the mediation of drug-induced apoptosis in human acute leukemia cells

Previous reports have demonstrated that a variety of anticancer drugs, e.g., 1-beta-D-arabinofuranosylcytosine (ara-C), mitoxantrone, etoposide, camptothecin, and cisplatin, induce the expression of c-jun oncogene in leukemic cells prior to producing internucleosomal DNA fragmentation and the morphological features of apoptosis. This has led to the impression that the induction of c-jun expression may be directly involved in the molecular signaling of the final common pathway of programmed cell death or apoptosis. In the present study, we examined the role of c-jun expression in three different settings of anticancer drug-induced apoptosis in human leukemic cells. First, exposure of human myeloid leukemia HL-60 cells to high-dose ara-C for 4 h produced internucleosomal DNA fragmentation preceded by c-jun induction. However, pretreatment of HL-60 cells with staurosporine, a protein kinase C inhibitor, repressed c-jun yet enhanced DNA fragmentation and apoptosis due to ara-C. Second, in human pre-B leukemia 697/BCL-2 cells which are transfected with the cDNA of the bcl-2 oncogene and overexpress p26BCL-2, although ara-C or mitoxantrone treatment caused greater c-jun induction than in the 697/neo cells, significantly reduced endonucleolytic DNA fragmentation and apoptosis was observed in 697/BCL-2 cells. Finally, taxol-induced internucleosomal DNA fragmentation and morphological features of apoptosis in HL-60 cells were not associated with the induction of c-jun expression. These lines of evidence indicate that the induction of c-jun expression may not have a direct role in the molecular signaling of anticancer drug-induced apoptosis, and that the anticancer drug-induced apoptosis can occur by a mechanism that does not involve the induction of c-jun expression.     

7-Hydroxystaurosporine (UCN-01) induces apoptosis in human colon carcinoma and leukemia cells independently of p53

7-hydroxystaurosporine (UCN-01) is a more selective protein kinase C inhibitor than staurosporine. UCN-01 exhibits antitumor activity in experimental tumor models and is presently in clinical trials. Our study reveals that human myeloblastic leukemia HL60 and K562 and colon carcinoma HT29 cells undergo internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis after UCN-01 treatment. These three cell lines lack functional p53, and K562 and HT29 cells are usually resistant to apoptosis. DNA fragmentation in HT29 and K562 cells occurred after 1 day of treatment while it took less than 4 h in HL60 cells. Cycloheximide prevented UCN-01-induced DNA fragmentation in HT-29 cells, but not in HL60 and K562 cells, suggesting that macromolecular synthesis is selectively required for apoptotic DNA fragmentation in HT29 cells. UCN-01-induced DNA fragmentation was preceded by activation of cyclin B1/cdc2 kinase. Further studies in HL60 cells showed that UCN-01-induced apoptosis was associated with degradation of CPP32, PARP, and lamin B and that the inhibitor of caspases (ICE/CED-3 cysteine proteases), Z-VAD-FMK, and the serine protease inhibitor, DCI, protected HL60 cells from UCN-01-induced DNA fragmentation. However, only DCI and TPCK, but not Z-VAD-FMK, inhibited DNA fragmentation in the HL60 cell-free system, suggesting that serine protease(s) may play a role in the execution phase of apoptosis in HL60 cells treated with UCN-01. Z-VAD-FMK and DCI also inhibited apoptosis in HT29 cells. These data demonstrate that the protein kinase C inhibitor and antitumor agent, UCN-01 is a potent apoptosis inducer in cell lines that are usually resistant to apoptosis and lack p53 and that caspases and probably serine proteases are activated during UCN-01-induced apoptosis.     

Hydrogen peroxide-induced apoptosis mediated by p53 protein in glial cells

It is now generally accepted that massive neuronal death due to oxidative stress is a regular feature of brains in neurodegenerative diseases. However, much less attention has been given to the death of glial cells. In this study, we examined p53-sensitive apoptosis of cells by using human glioblastoma A172 cells and p53-deficient mouse astrocytes. In human A172 cells, hydrogen peroxide (H2O2) caused cell death in a time- and concentration-dependent manner, accompanied by nucleosomal DNA fragmentation and chromatin condensation. After treatment with H2O2, p53 protein was highly expressed and protein levels of Bak, p21WAF1/CIP1 and GADD45 were also enhanced. However, the protein levels of Bcl-2 and Bax did not change. On the other hand, primary cultured astrocytes from p53-deficient mouse brain grew faster than wild-type and heterozygous astrocytes. In addition, p53-deficient astrocytes were more resistant to H2O2-induced apoptosis than wild-type and heterozygous astrocytes. These results suggest that glial proliferation and the repair of damaged DNA may be regulated by p53-induced p21WAF1/CIP1 and GADD45, and that glial apoptosis caused by oxidative stress may be mediated by p53-induced Bak.     

Resistance to nitric oxide-mediated apoptosis in HL-60 variant cells is associated with increased activities of Cu,Zn-superoxide dismutase and catalase

Nitric oxide (NO) released from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO or NOC-18) induces apoptosis in human leukemia HL-60 cells. In this study, we isolated a HL-60 variant cell line, HL-NR6, that is resistant to DETA/NO toxicity as assessed by DNA fragmentation, morphology, and colony forming ability. The variant cells also showed resistance to reactive oxygen species (ROS) such as superoxide and hydrogen peroxide as well as NO donors, but not to anti-tumor drugs. We found that HL-NR6 cells when compared with HL-60 cells possessed twice the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase, but no change in Mn-SOD nor in glutathione peroxidase. Immunoblotting confirmed the high levels of both enzymes in the variant cell. We also observed that ROS generation following DETA/NO exposure was substantially higher in HL-60 cells than in HL-NR6 cells, using the 2',7'-dichlorofluorescein fluorometric method. Moreover, the SOD mimetic Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin and exogenous catalase effectively attenuated DETA/NO-elicited DNA fragmentation in HL-60 cells. Taken together, these data suggested that the NO resistance in HL-NR6 cells is associated with the increased Cu,Zn-SOD/catalase and that NO-mediated apoptosis in HL-60 cells is correlated with the generation of ROS and derived molecules like peroxynitrite.     

Differentiation and apoptosis of murine neuroblastoma cells N1E115

The mode and the kinetics of differentiation and death of murine N1E115 neuroblastoma cells induced by dimethyl sulfoxide and other nonspecific factors in vitro were investigated. After morphological differentiation neuroblastoma cells die by apoptosis which is indicated by characteristic morphological features and by internucleosomal DNA fragmentation. Durations of both differentiation and apoptosis are dependent on the nature of stimuli used. Protein synthesis inhibitor cycloheximide does not prevent differentiation and apoptosis of neuroblastoma cells induced by dimethyl sulfoxide and even accelerates both processes. The relationship between cell death and differentiation is discussed.     

Apoptotic effects of different drugs on cultured retinoblastoma Y79 cells

This paper deals with the apoptotic effect exerted in human retinoblastoma Y79 cells by a number of compounds. A remarkable effect was observed after treatment with DNA-damaging agents, such as camptothecin, etoposide, cisplatin and carboplatin; camptothecin was found to be the most efficacious. Treatment with these compounds induced the appearance of morphological features of apoptosis in the cells together with the distinct fragmentation of DNA, as shown by agarose gel electrophoresis. These effects were also accompanied by a remarkable increase in the level of p53. Many other compounds, which are not DNA-damaging agents, induced the morphological features of apoptosis but none of them were capable of increasing the level of p53. Among these compounds, Taxol, suramin and sodium butyrate also stimulated the oligonucleosomal fragmentation of DNA, while C2-ceramide, a cell-permeable analogue of ceramide, and vitamin D3 were not effective in the induction of DNA laddering in Y79 cells. Apoptosis was dependent on macromolecular synthesis with all the compounds tested.     

Induction of apoptosis in leukemia U937 cells by 5'-deoxy-5'-methylthioadenosine, a potent inhibitor of protein carboxylmethyltransferase

We found dramatic changes in leukemia U937 cells treated with 5'-deoxy-5'-methylthioadenosine (MTA), a potent inhibitor of protein carboxylmethyltransferase (protein methylase II). Initiation of cell death was observed by 1 day after MTA treatment, and it was induced in a dose- and time-dependent manner. However, cell viability measured by trypan blue exclusion was not consistent with the actual percentage of cell death. These results indirectly indicated that the type of cell death is apoptosis rather than necrosis. Nuclear fragmentation and DNA condensation of MTA-treated U937 cells were analyzed by both fluorescent and electron microscopy. MTA-treated cells first began to arrest in the M phase of the cell cycle, and they then exhibited a mitotic-like nuclear fragmentation process with partially membraneless chromatin. Furthermore, agarose gel electrophoresis of DNA extracted from cells treated with MTA showed DNA laddering with production of fragments of approximately 200 bp multiples. These studies indicated that cell death induced by MTA has the characteristics of apoptosis, although nuclear fragmentation is atypical. It seems likely that the process of apoptosis in U937 cells induced by MTA correlates with incomplete assembly of the nuclear envelope, since MTA itself could inhibit the carboxylmethylation of nuclear lamin B and delayed incorporation of lamin B into the nuclear envelope.     

Involvement of both caspase-like proteases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and pseudomonas toxin

We investigated the involvement of caspases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and Pseudomonas toxin in U937 cells. We found that caspase-3- and caspase-6-like activities, but not caspase-1-like activity, increased during toxin-induced apoptosis. Z-D-CH2-DCB, a caspase-like inhibitor, completely inhibited the generation of caspase-3- and caspase-6-like activities and blocked all features of apoptosis induced by toxins: nuclear morphological changes, DNA fragmentation, and cytotoxicity. However, three caspase-specific inhibitors, Ac-YVAD-CHO, Ac-DEVD-CHO, and Ac-VEID-CHO, had no effect, even though Ac-DEVD-CHO and Ac-VEID-CHO inhibited the increased caspase-3- and caspase-6-like activity, respectively. These results suggest that the generation of caspase-3- and caspase-6-like activities is redundant, and other caspases distinct from caspase-3 and -6 may be important in toxin-induced apoptosis. Furthermore, serine protease inhibitor, 3,4-dichloroisocoumarine (DCI), abolished the apoptotic cell death and DNA fragmentation caused by toxins, without affecting the increased caspase-3- and caspase-6-like activities. Our results suggest that multiple proteases with different preferences for apoptotic substrates participate in toxin-induced apoptotic death of U937 cells.     

Development of technologies aiding large-tissue engineering

Apoptosis associated oligonucleosomal fragmentation of DNA can result from the activation of endonucleases that exhibit different pH optima and are either sensitive or insensitive to divalent cations. DNA fragmentation due to activation of cation sensitive endonucleases occurs in the absence of a change in intracellular pH whereas intracellular acidification is a feature of apoptosis characterized by activation of cation insensitive acidic endonuclease. We have reported earlier that somatostatin (SST) induced DNA fragmentation and apoptosis is signaled in a receptor subtype selective manner uniquely via human somatostatin receptor subtype 3 (hSSTR3). In the present study we investigated the pH dependence and cation sensitivity of endonuclease induced in hSSTR3 expressing CHO-K1 cells by the SST agonist octreotide (OCT) and its effect on intracellular pH. We show that OCT induced apoptosis is associated with selective stimulation of a divalent cation insensitive acidic endonuclease. The intracellular pH of of cells undergoing OCT induced apoptosis was 0.9 pH units lower than that of control cells. The effect of OCT on endonuclease and pH was inhibited by orthovanadate as well as by pretreatment with pertussis toxin, suggesting that hSSTR3 initiated cytotoxic signaling is protein tyrosine phosphatase mediated and is G protein dependent. These findings suggest that intracellular acidification and activation of acidic endonuclease mediate wild type p53 associated apoptosis signaled by hormones acting via G protein coupled receptors.     

Effects of modulators of protein kinases on taxol-induced apoptosis of human leukemic cells possessing disparate levels of p26BCL-2 protein

Taxol-induced polymerization of tubulin into stable microtubules and cell cycle metaphase arrest have been demonstrated to result in internucleosomal DNA fragmentation and morphological features of apoptosis in human leukemia cells. Recent studies have also shown that Taxol-induced apoptosis, but not Taxol-induced microtubular bundling or mitotic arrest, is significantly inhibited in cells that overexpress the bcl-2 gene product p26BCL-2. In the present studies we examined the effects of several modulators of activities of protein kinases on Taxol-induced DNA fragmentation and apoptosis in human pre-B leukemia 697 cells transfected with the cDNA of the bcl-2 gene and expressing high intracellular levels of p26BCL-2 (697/BCL-2 cells). Treatment with 0.1-1.0 microM MTaxol for 24 h produced prolonged mitotic arrest of control 697/neo cells, which had been transfected with the neomycin resistance gene. This resulted in apoptosis-associated large DNA fragments ranging between 5 and 200 kb and internucleosomal DNA fragmentation. Cotreatment with the phorbol ester phorbol dibutyrate (PdBU) significantly reduced Taxol-induced internucleosomal and large DNA fragmentation and inhibited apoptosis of 697/neo cells. In contrast, a combined exposure to Taxol and staurosporine (ST; 5 or 50 ng/ml), a potent inhibitor of protein kinase C and other kinases, significantly increased DNA fragmentation and apoptosis of 697/neo cells. Additionally, in 697/BCL-2 cells, ST partially overcame the suppressive effects of high p26BCL-2 levels on Taxol-induced apoptosis. Cotreatment with the tyrosine kinase inhibitor Genistein (30 microM) markedly inhibited Taxol-induced DNA fragmentation and apoptosis of 697/neo cells. However, it is noteworthy that the modulations of Taxol-induced DNA fragmentation and apoptosis by PdBU, ST, and Genistein occurred without significant effects on Taxol-mediated mitotic arrest of 697/neo cells. These agents also did not affect intracellular p26BCL-2 levels in 697/neo or 697/BCL-2 cells. These findings indicate that Taxol-induced apoptosis can be modulated by agents that affect the activities of protein kinases, and these effects are not mediated by modulations of Taxol-induced mitotic arrest or by alterations of intracellular p26BCL-2 levels.     

Etoposide-induced PC12 cell death: apoptotic morphology without oligonucleosomal DNA fragmentation or dependency upon de novo protein synthesis

Etoposide, a topoisomerase II inhibitor used in cancer therapy, has been shown to induce apoptosis in vitro in a variety of cell types. In the present study, we have characterized the effects of etoposide on undifferentiated rat pheochromocytoma PC12 cells. Etoposide killed PC12 cells in a time- and concentration-dependent manner. 20-24 h incubation with 10 micrograms/ml etoposide induced 25-50% cell death. Hoechst 33258 staining revealed apoptotic morphology in dying cells. No evidence was found of either oligonucleosomal DNA fragmentation, as shown by agarose gel electrophoresis, or endonuclease involvement, as shown by the inability of aurintricarboxylic acid to prevent cell death. Cycloheximide and actinomycin-D were unable to prevent etoposide cytotoxicity indicating that the process is not dependent upon de novo protein or mRNA synthesis. NGF (5 ng/ml) prevented etoposide-induced PC12 cell death. These results offer an example of how the morphological features of apoptosis are not necessarily associated with oligonucleosomal DNA fragmentation or with de novo macromolecule synthesis.     

Biochemical evidence for autocrine/paracrine regulation of apoptosis in cultured uterine epithelial cells during mouse embryo implantation in vitro

During embryo implantation, apoptosis is observed morphologically at the implantation site of endometrium. The objectives of this study were to demonstrate biochemical evidence of apoptosis and quantitative assessment of DNA fragmentation in uterine epithelial cells using a mouse implantation model, and to investigate the autocrine/paracrine regulation of apoptosis in uterine epithelial cells during blastocyst outgrowth. Blastocysts from day 4 pregnant mice were cultured on uterine epithelial cells for 96 h. Uterine epithelial cells dislodged by trophoblasts in endometrium-trophoblast unit demonstrated morphological features of apoptosis by Acridine Orange staining. Electrophoresis demonstrated DNA ladder and DNA fragmentation by enzyme-linked immunosorbent assay markedly increased after 48 h period of incubation. Apoptosis increased in an exponential way in accordance with trophoblast outgrowth. In addition, DNA fragmentation was shown in the epithelial cells by adding embryo-conditioned medium (CM) and the effect of embryo CM on apoptosis was significantly inhibited by anti-transforming growth factor (TGF)-beta antibody. Delayed outgrowth was observed after 48 h of incubation in the blastocysts cultured with anti-TGF-beta antibody. These results suggest there is autocrine/paracrine regulation of apoptosis in uterine epithelial cells at mouse embryo implantation and that TGF-beta might play an important role in the occurrence of apoptosis in the endometrium-trophoblast unit.