Early Phase Of Apoptosis Research Articles

Serum M30 levels: A potential biomarker of severe liver disease in nonalcoholic fatty liver disease and normal aminotransferase levels #

We thank Dr. Yilmaz et al. for comments on our article.1 The results of the pilot study Yilmaz and colleagues conducted in patients with nonalcoholic fatty liver disease (NAFLD) and normal alanine aminotransferase (ALT) levels confirm our finding that the severity of insulin resistance is an independent predictor of liver damage in these patients, and suggest that serum levels of M30, a neoepitope of cytokeratin 18 (CK-18) released during the early phases of apoptosis, may prove useful to discriminate patients with nonalcoholic steatohepatitis (NASH) from those with simple steatosis. There is growing interest in evaluating apoptosis markers as potential predictors of liver damage in NAFLD, and preliminary studies in patients with increased ALT levels have provided encouraging results.2 Recent data showed that cytokeratin CK-18 (M65 antigen) and caspase-cleaved CK-18 (M30 antigen), two soluble forms of extracellular cytokeratin 18, have both a good sensitivity and specificity, with M65 having a higher predictive value to diagnose NASH.3 Larger prospective cooperative studies are needed to test the performance of these new apoptosis markers in discriminating patients with NASH and potentially progressive liver disease from those with benign disease, even before severe fibrosis ensues, and to compare these tests with other noninvasive risk scores.4 Based on the present results, and the preliminary data reported by Yilmaz, we think that future research should evaluate the combined role of genetic, apoptotic, and serological markers of insulin resistance in the noninvasive diagnosis of NASH including patients with or without increased ALT. Anna Ludovica Fracanzani*, Luca Valenti*, Silvia Fargion*, * Dipartimento Medicina Interna, Centro Studio Ecura Malattie Meta Boliche, Fondazione IRCCS Ospedale Maggiore Policlinico, Università di Milano, Milan, Italy.

Geldanamycin-induced Lyn dissociation from aberrant Hsp90-stabilized cytosolic complex is an early event in apoptotic mechanisms in B-chronic lymphocytic leukemia

AbstractLyn, a tyrosine kinase belonging to the Src family, plays a key role as a switch molecule that couples the B-cell receptor to downstream signaling. In B-CLL cells, Lyn is overexpressed, anomalously present in the cytosol, and displays a high constitutive activity, compared with normal B lymphocytes. The aim of this work was to gain insights into the molecular mechanisms underlying these aberrant properties of Lyn, which have already been demonstrated to be related to defective apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Herein, Lyn is described to be in an active conformation as integral component of an aberrant cytosolic 600-kDa multiprotein complex in B-CLL cells, associated with several proteins, such as Hsp90 through its catalytic domain, and HS1 and SHP-1L through its SH3 domain. In particular, Hsp90 appears tightly bound to cytosolic Lyn (CL), thus stabilizing the aberrant complex and converting individual transient interactions into stable ones. We also demonstrate that treatment of B-CLL cells with geldanamycin, an Hsp90 inhibitor already reported to induce cell death, is capable of dissociating the CL complex in the early phases of apoptosis and thus inactivating CL itself. These data identify the CL complex as a potential target for therapy in B-CLL.

Influence of 4-aminopyridine on human ovarian luteinized granulosa cell proliferation, production, and apoptosis through inhibiting voltage-gated K+ channel

To study the influence of 4-aminopyridine (4-AP) on proliferation, production, and apoptosis through inhibiting voltage-gated K(+) channel (Kv) in ovarian luteinized granulosa cells. Ovarian luteinized granulosa cells were recovered from 25 women with regular menses who underwent in vitro fertilization programme. The cultured granulosa cells were divided into 4 groups:blank group, 4-AP treated group, human chorionic gonadotropin (hCG)-induced group and hCG + 4-AP co-treated group. The final concentrations of hCG and 4-AP were 1250 U/L and 5 nmol/L respectively. The progesterone production was detected by the chemoluminescence method. The expression of Kv mRNA on human ovarian luteinized granulosa cell was detected by RT-PCR. The influence on the early apoptosis of granulosa cells by 4-AP was observed by flow cytometry. Cellular caspase-3 activities were observed with colorimetric method and the inhibition of the cell proliferation was studied using methyl thiazolyl tetrazolium (MTT) method. (1) Kv mRNA was expressed in granulosa cell. (2) The progesterone production of the blank group, 4-AP treated group, hCG-induced group and hCG + 4-AP co-treated group were (547 +/- 64), (206 +/- 32), (1991 +/- 172) and (763 +/- 79) nmol/L, respectively after 24 hours culture. Exposure of the granulosa cells to 4-AP reduced the production of progesterone in blank and hCG-induced granulosa cells. (3) The flow cytometry analysis and the cellular caspase-3 A(405) showed that 4-AP increased the percentage of early phase apoptosis (P < 0.01): 4-AP treated group vs blank group [(40 +/- 5)% and 0.049 +/- 0.009] vs [(17 +/- 4)% and 0.029 +/- 0.008], hCG + 4-AP co-treated group vs hCG-induced group [(25 +/- 4)% and 0.039 +/- 0.008] vs [(15 +/- 3)% and 0.022 +/- 0.007]. (4) 24 hours after treated with 4-AP and hCG, the inhibitory rate of cultured granulosa cells of 4-AP treated group was higher than the blank group (19.7% vs 0), and that of hCG + 4-AP co-treated group was obviously higher than hCG-induced group (34.6% vs 0, P < 0.01). The voltage-gated K(+) channels expressed by ovarian luteinized granulosa cell play an important role in cell proliferation, production, and apoptosis. 4-AP may inhibit differentiation of progesterone in granulosa cells through the inhibition of proliferation and induction of apoptosis.

The effect of combination treatment with docosahexaenoic acid and 5-fluorouracil on the mRNA expression of apoptosis-related genes, including the novel gene BCL2L12, in gastric cancer cells

Docosahexaenoic acid (DHA) plays an important role in suppressing the growth of cancer. In this paper, the synergetic anticancer effect of combination DHA with 5-fluorouracil (5-FU) was investigated in gastric carcinoma cells. We found that DHA inhibited the growth of cultured SGC7901 cells at different concentrations in a dose- and time-dependent manner. Furthermore, the growth-inhibition activities of increasing concentration of 5-FU were markedly enhanced when different doses of 5-FU were administered in the combination with dose as low as 40 microg/ml of DHA. The early phase of apoptosis was increased in DHA- and 5-FU-treated cells. In the case of apoptotic genes expression in the combination-treated cells, BAX mRNA expression increased, whereas FAS, BCL-2, BCL2L12, and CASPASE-9 mRNA expression decreased. These results suggest that DHA strongly enhances the anticancer effect of 5-FU. Moreover, the application of both compounds on gastric cancer cells provides a new potential approach for cancer therapy.

BioPEGylation of Polyhydroxyalkanoates: Influence on Properties and Satellite-Stem Cell Cycle

The addition of poly(ethylene glycol), PEG, to bioprocessing systems producing polyhydroxyalkanoates (PHAs), has been reported as a means of their molecular weight control and can also support bioPEGylation, resulting in hybrids with amphiphillic properties. However, the study of such natural-synthetic hybrids of PHA-b-PEG is still in its infancy. In this study, we report the influence of bioPEGylation of polyhydroxyoctanoate (PHO) on its physiochemical, material, and biological properties. Consistent with previous studies, bioPEGylation with diethylene glycol (DEG) showed a significant reduction in PHA molecular weight (57%). In comparison to solvent cast films of PHO, PHO-b-DEG films possessed a noticeable X-ray diffraction peak at 9.82 degrees and increased Young's modulus of 11 Gpa (83%). Potential biocompatibility was investigated by measuring the early phase of apoptosis in myoblastic satellite-stem cells (C2C12). Comparative analysis of cell proliferation and progression in the presence of the mcl-PHA and its hybrid showed that the latter induced significant cell cycle progression: the first time a biomaterial has been shown to do so. Microtopographies of the film surfaces demonstrated that these differences were not due to changes in surface morphology; both polymers possessed average surface rugosities of 1.4 +/- 0.2 microm. However, a slight decrease in surface hydrophobicity (3.5 +/- 0.9 degrees) due to the hydrophilic DEG may have exerted an influence. The results support the further study of bioPEGylated PHAs as potential biomaterials in the field of tissue engineering.

Leptin prevents apoptosis of trophoblastic cells by activation of MAPK pathway

Leptin (Ob), the peripheral signal produced by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. Recently, we have demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work we aimed to study the signal transduction pathways that mediate the trophic effect of leptin in placenta, by using the human placenta choriocarcinoma JEG-3 cell line, as well as trophoblastic cells from human placenta. We have assayed the early phase of apoptosis, triggered by serum deprivation, by using Annexin V–propidium iodide (PI) labeling and flow cytometric analysis, as well as the late phase of apoptosis by studying the activation of caspase-3. We have studied the major signalling pathways known to be triggered by the leptin receptor, and we have investigated the relative importance of these pathways in the effect of leptin by using pharmacological inhibitors. We have found that leptin stimulates Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway by promoting JAK-2 and STAT-3 tyrosine phosphorylation. We have also demonstrated the activation of mitogen-activated protein kinase (MAPK) pathway by studying phosphorylation of extracellular-signal regulated kinase (Erk) kinase (MEK) and Erk1/2. PI3K pathway is also triggered by leptin stimulation as assessed by the study of protein kinase B (PKB) phosphorylation. These signaling pathways were confirmed in trophoblastic cells obtained from placenta of healthy donors. The effect of leptin on JEG-3 survival was completely reversed by blocking Erk1/2 activation employing the MEK inhibitor PD98059, whereas it was not affected by PI3K inhibition using wortmannin. These data suggest that the leptin antiapoptotic effect in placenta is mediated by the MAPK pathway.

Leptin promotes cell survival and activates Jurkat T lymphocytes by stimulation of mitogen-activated protein kinase

Leptin (Ob) is a non-glycosylated peptide hormone that regulates energy homeostasis centrally, but also has systemic effects including the regulation of the immune function. We have reported previously that leptin activates human peripheral blood lymphocytes co-stimulated with phytohaemagglutinin (PHA) (4 microg/ml), which prevented the employment of pharmacological inhibitors of signalling pathways. In the present study, we used Jurkat T cells that responded to leptin with minimal PHA co-stimulation (0.25 microg/ml). The long isoform of leptin receptor is expressed on Jurkat T cells and upon leptin stimulation, the expression of early activation marker CD69 increases in a dose-dependent manner (0.1-10 nM). We have also found that leptin activates receptor-associated kinases of the Janus family-signal transucers and activators of transcription (JAK-STAT), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K) signalling pathways. Moreover, we sought to study the possible effect of leptin on cell survival and apoptosis of Jurkat T cells by culture in serum-free conditions. We have assayed the early phases of apoptosis by flow cytometric detection of fluorescein isothiocyanate (FITC)-labelled annexin V simultaneously with dye exclusion of propidium iodide (PI). As well, we have assayed the activation level of caspase-3 by inmunoblot with a specific antibody that recognizes active caspase-3. We have found that leptin inhibits the apoptotic process dose-dependently. By using pharmacological inhibitors, we have found that the stimulatory and anti-apoptotic effects of leptin in Jurkat T cells are dependent on MAPK activation, rather than the PI3K pathway, providing new data regarding the mechanism of action of leptin in T cells, which may be useful to understand more clearly the association between nutritional status and the immune function.

Interferon-α-induced apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent and -independent manner

IFN-alpha regulates tumor cell growth at least through induction of apoptosis. We have recently demonstrated that IFN-alpha causes apoptosis through upregulation of TNF-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma and U266 myeloma cells. However, other cell lines such as Ramos and RPMI 8226 underwent apoptosis without any apparent involvement of TRAIL following IFN-alpha stimulation. In this study, we examined whether the IFN-alpha-induced upregulation of TRAIL is essential for the induction of apoptosis. IFN-alpha-induced early phase (48 h) of loss of DeltaPsim was substantially prevented in Daudi B lymphoma cells overexpressing the dominant-negative form of Fas-associated death domain (dnFADD) compared with vector control, whereas a late phase (72 h) of DeltaPsim was comparable to the control. The IFN-alpha-induced early phase of apoptosis was also reduced in the dnFADD-expressing cells, while the late phase of apoptosis was unaffected. IFN-alpha-induced upregulation of TRAIL protein in the dnFADD-expressing Daudi or U266 cells was comparable to their control cells, suggesting that FADD is not involved in the IFN-alpha-induced upregulation of TRAIL. Moreover, the early phase of mitochondrial depolarization was severely prevented by the presence of fusion protein of TRAIL receptor 1 and Fc portion of immunoglobulin (TRAIL-R1:Fc) and TRAIL-R2:Fc. Together, IFN-alpha induces apoptosis in a TRAIL-dependent or -independent manner, depending on the course of the apoptotic process.

Transport and metabolism of l-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis

Having confirmed that externally added l-lactate can enter cerebellar granule cells, we investigated whether and how l-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions.(1)l-lactate enters mitochondria, perhaps via an l-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an l-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis.(2)In both normal and apoptotic cell homogenates, externally added l-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored l-lactate uptake by mitochondria and occurred with a hyperbolic dependence on l-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of l-lactate. The rate of the process depended on l-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular l-lactate/pyruvate shuttle, whose activity was limited by the putative l-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier.(3)l-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative l-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.

Anti tumor immunity of cyclophosphamide(CY) mediated by homeostatic proliferation of memory type T cells and reduction of regulatory T cells. (48.29)

Abstract In addition to its cytotoxic effect, CY has been known to have a certain type of immune modulation effects. In this study, we investigated the modulation effect of high dose CY on anti-tumor immunity of tumor bearing mouse under a tumor-mediated immune suppression status. In murine melanoma tumor model, a single injection of CY(200mg/kg) to tumor bearing mouse delayed the tumor progression and increased the mean survival days by 2fold. The phenotypic analysis by FACS revealed CY caused significant increase of both percentage and absolute number of CD4+and CD8+ T cells in draining LNs on 10 days post injection (DPI). Of note, the proportion of CD4+CD25+Foxp3+ T cell was significantly decreased whereas that of CD4+CD44hi and CD8+CD44hiT cell was increased. Caspase-3 staining of CD44hiT cells on 3 DPI indicated the increase was preceded by the early-phase apoptosis. In vitro functional analysis by ELISPOT revealed the number of IFN-γproducing cells stimulated by ConA was increased to the level comparable to tumor non-bearing mouse. We could not find any significant changes in the percentage of monocyte and NK cells with CY treatment. In conclusion, the enhancement of anti-tumor immunity by CY may be due to the increase of the ratio of CD44himemory type T cells to regulatory T cells, which would be induced by early phase depletion followed by preferential homeostatic proliferation of memory type T cells over regulatory T cells.

Clearance deficiency and systemic lupus erythematosus (SLE)

Systemic lupus erythematosus (SLE) is a fairly heterogeneous autoimmune disease. Impaired clearance functions for dying cells may explain accumulation of nuclear autoantigens in various tissues of SLE patients. Our data show that in a subgroup of patients with SLE, apoptotic cells accumulated in the germinal centres of the lymph nodes. Apoptotic material was attached to the surfaces of follicular dendritic cells. Furthermore, we found an accumulation of apoptotic cells in the skin of patients with cutaneous lupus after UV exposure. Granulocytes and monocytes in whole blood of SLE patients showed a reduced uptake of albumin- and polyglobin-coated beads. Furthermore, we analysed sera from SLE patients in migration assays and observed that the attraction signals for macrophages were reduced by sera of approximately 25% of the SLE patients. Analyses of high-affinity DNA binding IgG autoantibodies of SLE patients revealed that those antibodies had gained their DNA reactivity in a germinal centre reaction. We suggest a stepwise maturation from a non-anti-DNA reactive B cell to an anti-dsDNA autoreactive B cell. We conclude that impaired clearance in early phases of apoptosis leads to a secondary necrotic status of the cells. Danger signals are released; modified autoantigens are accessible, favouring an autoimmune reaction.

Enhanced T-cell immunity induced by dendritic cells with phagocytosis of heat shock protein 70 gene-transfected tumor cells in early phase of apoptosis

The dual role of heat shock protein 70 (HSP70), as antigenic peptide chaperone and danger signal, makes it especially important in dendritic cell (DC)-based vaccination. In this study, we investigated the impacts of apoptotic transgenic MCA/HSP tumor cells expressing HSP70 on DC maturation, T-cell stimulation and vaccine efficacy. We found that DCs with phagocytosis of MCA/HSP in early phase of apoptosis expressed more pMHC I complexes, stimulated stronger cytotoxic T lymphocyte (CTL) responses (40% specific killing at an E:T cell ratio of 50) and induced immune protection in 90% of mice against MCA tumor cell challenge, compared with 25% specific CTL killing activity and 60% immune protection seen in mice immunized with DC with phagocytosis of MCA/HSP in late phase of apoptosis (P<0.05). Similar results were confirmed in another EG7 tumor model also expressing HSP70. Taken together, our data demonstrate that HSP70 on apoptotic tumor cells stimulate DC maturation, and DC with phagocytosis of apoptotic tumor cells expressing HSP70 in early phase of apoptosis more efficiently induced tumor-specific CTL responses and immunity than DCs with phagocytosis of apoptotic tumor cells in late phase of apoptosis. These results may have an important impact in designing DC-based antitumor vaccines.

55 LIVE PLASMA MEMBRANE ANALYSIS OF EARLY APOPTOTIC CELL DEATH IN PORCINE ADULT DERMAL FIBROBLASTS PRIOR TO SOMATIC CELL CLONING

The aim of our study was to determine the in vitro developmental capability of porcine nuclear-transferred (NT) embryos reconstructed with adult dermal fibroblast cells, which had been analyzed for apoptosis by live plasma membrane fluorescent labelling. Frozen/thawed fibroblasts, which had been in vitro cultured to confluency, were used for analysis. To detect the early apoptotic changes in the plasma membrane involving the externalization of phosphatidylserine molecules and the subsequent loss of lipid composition asymmetry, the fibroblasts were tagged using a conjugate of annexinV with enhanced green fluorescent protein (eGFP). In the somatic cell cloning procedure, enucleated in vitro-matured oocytes were reconstituted with non-apoptotic dermal fibroblast cell nuclei. Afterwards, NT-derived oocytes were stimulated with a combination of electrical and chemical activation. Simultaneous fusion and electrical activation were induced by application of two successive DC pulses of 1.2 kV cm–1 for 60 �s. A two-step chemical activation procedure was initiated after a 1.5–2 h delay. The cybrids were exposed to 15 µm calcium ionomycin for 5 to 7 min and then incubated in the culture medium supplemented with 10 µg mL–1 cycloheximide for 3 h. Reconstructed embryos were in vitro cultured in NCSU-23 medium for 6–7 days. Fluorescence analysis of the adult dermal fibroblast cells revealed that a relatively high proportion of donor cells exhibited proapoptotic changes in the plasma membrane. The percentage of late apoptotic cells with advanced morphological changes did not exceed 30%. Moreover, an extremely low rate (ranging from 0 to 2%) of early apoptotic cells, with a morphologically normal, i.e., smooth (non-corrugated) and intact (non-blebbing), plasmolemma but which emitted the green eGFP-derived chemiluminescence, was detected. A total of 219 enucleated oocytes were subjected to reconstruction and 185/219 (84.5%) were successfully fused with non-apoptotic nuclear donor cells. Out of 185 cultured NT embryos, 108 (58.4%) cleaved. The frequencies of cloned embryos, that reached the morula and blastocyst stages, were 84/185 (45.4%) and 26/185 (14.0%), respectively. In conclusion, annexin V-eGFP is a sensitive method able to detect the early phases of apoptosis in cultured adult dermal fibroblast cells, because it identified that very small proportion of morphologically normal cells (without shrinkage of the plasmolemma) that also emitted the annexin V-eGFP-derived biochemiluminescence. Nonetheless, the probability of their random erroneous selection for somatic cell cloning appears to be extremely low. It was also found that the preimplantation developmental potential of NT embryos originating from non-apoptotic adult dermal fibroblast cells is relatively high. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

Topoisomerase II and tubulin inhibitors both induce the formation of apoptotic topoisomerase I cleavage complexes

Topoisomerase I (Top1) is a ubiquitous enzyme that removes DNA supercoiling generated during transcription and replication. Top1 can be trapped on DNA as cleavage complexes by the anticancer drugs referred to as Top1 inhibitors as well as by alterations of the DNA structure. We reported recently that Top1 cleavage complexes (Top1cc) are trapped during apoptosis induced by arsenic trioxide and staurosporine. In the present study, we generalize the occurrence of apoptotic Top1cc in response to anticancer drugs, which by themselves do not directly interact with Top1: the topoisomerase II inhibitors etoposide, doxorubicin, and amsacrine, and the tubulin inhibitors vinblastine and Taxol. In all cases, the Top1cc form in the early phase of apoptosis and persist throughout the apoptotic process. Their formation is prevented by the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone and the antioxidant N-acetyl-L-cysteine. We propose that the trapping of Top1cc is a general process of programmed cell death, which is caused by alterations of the DNA structure (oxidized bases and strand breaks) induced by caspases and reactive oxygen species.

Inhibitor of double stranded RNA-dependent protein kinase protects against cell damage induced by ER stress

Endoplasmic reticulum (ER)-stress is known to induce neuronal cell death and to play roles in neurodegenerative diseases. Phosphorylation of double stranded RNA-dependent protein kinase (PKR) has been demonstrated in brain tissues in patients with Alzheimer's, Parkinson's, and Huntington's diseases. Here, we examined the effect of a PKR inhibitor (an imidazolo–oxindole derivative that acts as an ATP-binding site-directed inhibitor of PKR) on the neuronal cell death induced by ER-stress in cultured human neuroblastoma cells (SH-SY5Y). Cell damage was induced by tunicamycin (an ER-stress inducer), and cell viability was measured by Hoechst 33342 and YO-PRO-1 double staining and by the resazurin-reduction test (to evaluate metabolic activity). Treatment with tunicamycin at 2 μg/ml for 24 h induced apoptotic cell death accompanied by nuclear condensation and/or fragmentation, and these cells were positive for YO-PRO-1 (early-phase apoptosis and necrosis indicator). Treatment with the PKR inhibitor at 0.1 or 0.3 μM led to a decrease in the number of apoptotic cells induced by tunicamycin. In the resazurin-reduction test, the PKR inhibitor (at 0.1 and 0.3 μM) concentration-dependently inhibited the tunicamycin-induced decrease in metabolic activity. On the other hand, treatment with the PKR inhibitor alone (at 0.3 μM) had no effect on cell morphology or viability (versus in normal control cells). These results indicate that inhibition of PKR activation may be neuroprotective against ER stress-induced cell damage.

Induction of apoptosis by YTX in myoblast cell lines via mitochondrial signalling transduction pathway

Yessotoxin (YTX) can induce apoptotic events in myoblast L6 and BC3H1 cell lines from rat and mouse, respectively. The present study indicates that apoptosis induced by YTX in these cell lines can occur through activation of the mitochondrial pathway indicating an intracellular response. Terminal events during mitochondrial-mediated apoptosis involve perturbations to mitochondria resulting in loss of mitochondrial membrane potential (Δ Ψ m), permeability transition pore (PTP) opening and the release of proapoptotic factors cytochrome c, smac/DIABLO into the cytosol. Results from western blotting, electron and fluorescent microscopy of YTX-treated myoblast cells provided experimental data for evaluation of cytochrome c, smac/DIABLO release and caspase-9 activation. Loss of mitochondrial membrane potential and swelling of mitochondria indicated an active role of mitochondria during the early phase of apoptosis in L6 and BC3H1 cells after YTX exposure. These observations show that YTX targets mitochondria and involve activation of a cascade of events through mitochondrial regulation.

Algal Toxins as Guidance to Identify Phosphoproteins with Key Roles inApoptotic Cell Death

The protein phosphatase inhibiting toxins microcystin and nodularin act rapidly to induce apoptotic cell death. Their inhibitory effect on protein phosphatases 1 and 2A can be utilized as tools to understand the phosphorylation-dependent regulatory mechanism underlying the early stage of apoptosis. The incubation of freshly isolated hepatocytes with these toxins results in a rapid hyperphosphorylation of cellular proteins before any morphological signs of apoptosis appears [Fladmark, K. E., Brustugun, O. T., Hovland, R., Boe, R., Gjertsen, B. T., Zhivotovsky, B. and Doskeland, S. O. (1999) Cell Death Differ. 6, 1099-108]. Proteins subjected to phosphorylation in this early phase of apoptosis may play key roles in this cellular process and become valuable targets for drug development. The ultra-rapid apoptosis-induction by microcystin and nodularin provides a unique amount of synchronized apoptotic cells with "large" amounts of mainly serine/threonine phosphorylated proteins. This ultra-rapid toxin-induced up-concentration of phosphorylated proteins reduces the material needed as well as simplifies our effort in order to obtain enough phosphoproteins for mass spectrometric identification and characterization. We will here give an overview of our strategy for identification of low-abundance phosphoproteins involved in algal toxin-induced apoptosis and most likely also in a general apoptotic pathway.

High‐resolution magic‐angle‐spinning 1H NMR spectroscopy reveals different responses in choline‐containing metabolites upon gene therapy‐induced programmed cell death in rat brain glioma

Changes in the concentrations of choline-containing metabolites (CCM) have been implicated in both cell proliferation and death processes. In this study, high-resolution magic-angle-spinning (HRMAS) 1H NMR spectroscopy was used to study metabolite changes in the CCM chemical shift region in rat glioma ex vivo during apoptosis induced by thymidine kinase-ganciclovir gene therapy. Cell density and apoptotic activity in the tumours were quantified by histological methods. HRMAS 1H NMR was able to resolve peaks from choline (Cho), glycerophosphocholine (GPC), phosphocholine (PC), taurine (Tau) and myo-inositol (myo-Ins), all of which contribute to the in vivo 1H NMR peak centred at 3.23 ppm. The early phase of apoptosis (treatment day 4), with a approximately 2.8-fold increase in the number of apoptotic nuclei (at constant cell density of 1.8 +/- 0.1 x 10(5) cells/mm3) was associated with increases in resonance intensity from GPC and PC, while Cho and Tau remained unchanged. Later stage apoptosis, accompanied by synchronous cell death (cell density declined to 0.7 +/- 0.02 x 10(5) cells/mm3), resulted in a significant decline in Tau relative to untreated tumours, while the contents of CCMs and myo-Ins detectable by 1H HRMAS were unchanged. These observations demonstrate that, while the in vivo 1H NMR peak at 3.23 ppm is indicative of cellular processes involved in apoptosis, the biochemical changes monitored by this resonance involve a number of different and chemically distinct metabolites.

Involvement of hydroperoxide in mitochondria in the induction of apoptosis by the eicosapentaenoic acid

Eicosapentaenoic acid (EPA) induced apoptosis of rat basophilic leukemia cells (RBL2H3 cells), whereas 100 μM linoleic acid (LA) had no significant effect. Cytochrome c was released at 4 h. Apoptosis was detected at 6 h after exposure to EPA and docosahexaenoic acid (DHA), and preceded the activation of caspase-3. Liberation of apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus were observed at 4 h. A broad-specificity caspase inhibitor, z-VAD-fmk, failed to suppress the apoptosis, suggesting that EPA induced caspase-independent apoptosis. On other hand, a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that blocks AIF translocation to the nucleus suppressed EPA-induced apoptosis. The level of hydroperoxide in the cells and mitochondria increased at the early phase of apoptosis within 2 h. On the contrary, elevation of hydroperoxide in mitochondria was not observed after treatment with LA. The EPA-induced apoptosis was abolished by prevention of the hydroperoxide elevation in mitochondria via overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). Neither cytochrome c nor AIF were released from mitochondria in the mitochondrial PHGPx-overexpressing cells. EPA also induced apoptosis in HeLa cells, but not in L929 or RAW264.7 cells. Enhancement of the hydroperoxide level in mitochondria was found in the EPA-sensitive HeLa cells after treatment with EPA, whereas no such enhancement was observed in the apoptosis-resistant L929 and RAW264.7 cells. These results suggest that the generation of hydroperoxide in mitochondria induced by EPA is associated with AIF release from mitochondria and the induction of apoptosis.

Absence of cardiotoxicity of the experimental cytotoxic drug cyclopentenyl cytosine (CPEC) in rats

The experimental anticancer drug cyclopentenyl cytosine (CPEC) was associated with cardiotoxicity in a phase I study. The aim of the present study was twofold; first we investigated whether the observed effects could be reproduced in in-vitro and in-vivo rat models. Second, we intended to investigate the underlying mechanism of the possible cardiotoxicity of CPEC. Effects on frequency and contractility were studied on the isolated atria of 18 male Wistar rats. Atria were incubated with 0.1 mmol L(-1) (n = 6) or 1 mmol L(-1) (n = 6) CPEC for 1.5 h and compared with control atria (incubation with buffer solution, n = 6). The cardiac apoptosis-inducing potential was studied in-vivo on 66 rats by 99mTc-Annexin V scintigraphy, followed by postmortem determination of radioactivity in tissues, histological confirmation with the TUNEL assay (late-phase apoptosis), and immunohistochemical staining for cleaved caspase 3 and cytochrome C (early-phase apoptosis). Serum levels of the necrotic cardiomyopathy marker troponin T were also determined. No effect on heart frequency was found in the isolated atria after CPEC treatment. A trend towards a decrease of contraction force was observed. However, the differences were not statistically significant. 99mTc-Annexin V scintigraphy showed no increase in cardiac uptake ratio upon CPEC treatment in the in-vivo rat model, which was confirmed by determination of radioactivity in heart versus blood ratios. At each section a few individual isolated late apoptotic cells (< 5) could be identified by the TUNEL assay in the highest CPEC dose group (90 mg kg(-1)) but not in controls or in rats treated with 60 mg kg(-1) CPEC. Staining for the early apoptosis markers cleaved caspase 3 and cytochrome C did not reveal any significant differences between treated and control rats. Cardiac troponin T levels were not increased after CPEC treatment. CPEC does not affect heart frequency or contraction force in our cardiotoxicity models. Moreover, we did not find an indication of CPEC-induced apoptosis in heart tissue.