Heat shock at the germinal vesicle breakdown stage induces apoptosis in surrounding cumulus cells and reduces maturation rates of porcine oocytes in vitro

The objectives were to determine the effects of cumulus cells (CC) on porcine oocyte maturation in vitro (IVM) after heat shock (HS). Treated oocytes were cultured at 39 degrees C for 20h, followed by HS treatment (42 degrees C for 1h), and then matured in vitro for 23h. The CC were removed before maturation (H1), after HS (H2), or after maturation (H3). Control oocytes were continuously cultured under the same conditions and CC were similarly removed before maturation (C1), after 21h of IVM (C2), and after maturation (C3). Maturation rates were affected by HS (P<0.01) and by an interaction between HS and CC (P<0.01). A significant decrease in maturation rate only occurred when CC were not removed from cumulus oocyte complexes during IVM after HS (H3, 39.2+/-5.7% versus C3, 78.2+/-8.2%, P<0.01). Mature oocytes in all treatment groups were electrically activated and cultured for 8 d in NCSU23. Blastocyst rates in group H1 (7.2+/-3.5%) and C1 (6.3+/-3.1%) were lower than in other groups (H2, 21.4+/-4.4%, C2, 20.5+/-7.0%, H3, 23.1+/-2.0%, C3, 24.3+/-3.1%, P<0.05). Damaged DNA was detected in CC by a comet assay at 0h after HS (60.8+/-12.5% compared with 9.2+/-2.2% in control, P<0.05); in HS groups, both DNA damage (comet assay, 74.9+/-6.3% compared with 10.0+/-2.1% in control) and apoptosis (TUNEL assay, 21.6+/-1.6% compared with 5.6+/-0.6% in control) in CC were increased (P<0.05) at 44h of maturation. In conclusion, heat shock (42 degrees C for 1h) during IVM induced DNA damage and apoptosis of porcine CC; furthermore, apoptotic CC may contribute to maturation failure of oocytes in vitro.     

Survival and proliferation of cells expressing caspase-uncleavable Poly(ADP-ribose) polymerase in response to death-inducing DNA damage by an alkylating agent

To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (-/-) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-alpha, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, approximately 25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (-/-) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.     

Possible Involvement of Poly(ADP-Ribosyl) Polymerase in Triggering Stress-Induced Apoptosis

U937 human myeloid leukemia cells respond to mild treatment with hydrogen peroxide and hyperthermia by undergoing apoptosis, an active mode of cell suicide. Higher concentrations of hydrogen peroxide, or longer incubation at the hyperthermic temperature, change the mode of cell death from apoptosis to the passive necrosis. Stress treatments cause a severe drop in the intracellular NAD concentration. 3-Aminobenzamide (3-ABA), a specific inhibitor of poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme which is activated by breaks in DNA to catabolize intracellular NAD, is capable of relieving such a drop. This suggests that breaks in DNA have been induced by both oxidative stress and heat shock, thereby activating PARP. Upon stress, NAD concentration has a first initial sharp drop; then, for mild stress treatments, it recovers, just when apoptosis begins to be detectable (8 h of recovery). At 20 h, when the apoptotic ladder-like pattern of DNA is visible, NAD concentration has dropped again, probably because of a second PARP activation due to the extensive DNA degradation that accompanies apoptosis. The presence of 3-ABA, concomitantly with the preservation of the intracellular NAD content, reduces the extent of apoptosis upon oxidative stress and strongly enhances cell survival, thus suggesting a role for PARP in triggering stress-induced apoptosis. All apoptotic U937 cells have a reduced NAD content, independently of the inducing agent; however, upon treatments which do not cause immediate DNA breaks, the drop in NAD concentration occurs only after the apoptotic ladder is detectable and can be ascribed to the activation of PARP by the free ends of DNA formed during the endonucleolitic degradation. Moreover, in these instances the inhibition of PARP, although effective in blocking the drop in NAD concentration, has no effect on apoptosis, thus being only circumstantial.     

Heat Stress Induces Apoptosis through a Ca2+-Mediated Mitochondrial Apoptotic Pathway in Human Umbilical Vein Endothelial Cells

Background

Heat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined.

Methods

The percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O₂ ⁻, H₂O₂, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry.

Results

In this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca²⁺, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca²⁺ into the cytoplasm after heat stress.

Conclusion

Our findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca²⁺, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process.     

Depletion of intracellular NAD(+) and ATP levels during ricin-induced apoptosis through the specific ribosomal inactivation results in the cytolysis of U937 cells

Our previous studies demonstrated that ricin induces the apoptotic death of U937 cells as evidenced by DNA fragmentation, nuclear morphological changes, and increases in caspase-like activities. In this study, we have found that intracellular NAD(+) and ATP levels decrease in ricin-treated U937 cells and that this decrease is followed by the ricin-mediated protein synthesis inhibition. The PARP inhibitor, 3-aminobenzamide (3-ABA), prevents the depletion in NAD(+) and ATP levels and concomitantly protects U937 cells from the lysis that follows ricin treatment. Hence, the protective action of 3-ABA is due to the inhibition of PARP and does not result from its other pharmacological side effects. Moreover, the enzymatic activity of PARP gradually increases and reaches a maximum level after ricin exposure for 3 h, whereas no significant change in activity was observed in untreated cells. However, 3-ABA has no effect on ricin-mediated DNA fragmentation. In addition, immunoblot analysis revealed that significant PARP cleavage occurred more than 12 h after ricin addition, while DNA fragmentation reached a maximum level within 6 h of incubation. Thus, in the case of ricin-induced apoptosis, it appears that PARP cleavage is not an early apoptotic event associated with the onset of apoptosis. Our results suggest that multiple apoptotic signaling pathways may be triggered by ricin-treatment. Probably, the pathway leading to cell lysis via PARP activation and NAD(+) depletion is independent of the pathway leading to DNA fragmentation in which caspases may be profoundly involved. Other protein synthesis inhibitors, including diphtheria toxin and cycloheximide, were less effective in terms of inducing DNA fragmentation and cytolysis, even at concentrations that cause significant inhibition of protein synthesis. Thus, a specific ricin action mechanism through which ribosomes are inactivated may be responsible for the apoptotic events induced by ricin.     

Generation of an oxidative stress precedes caspase activation during 7beta-hydroxycholesterol-induced apoptosis in U937 cells

The oxysterol 7beta-hydroxycholesterol (7beta-OH) has been shown to induce apoptosis in a number of cell lines. Though not fully elucidated, the mechanism through which this oxysterol induces cell death is thought to involve the generation of an oxidative stress leading to perturbation of the mitochondrion and release of cytochrome c into the cytosol. Cytochrome c together with Apaf-1 causes activation of the initiator caspase, caspase-9, which in turn activates caspase-3 ultimately leading to the degradation of poly(ADP-ribose) polymerase (PARP). The objective of the present study was to investigate the signalling pathway in 7beta-OH-induced apoptosis in U937 cells, a human monocytic blood cell line known to undergo apoptosis upon treatment with 7beta-OH, over a time course of 48 h. Apoptosis was evident after 24 h incubation. Glutathione levels were decreased after 6 h and this was coupled with an increase in SOD activity. Through western blot analysis we examined expression of caspase-3, -8, and -9 and cleavage of the caspase-3 substrate PARP. The sequence proceeded with activation of caspase-9 after 9 h, caspase-3 at the 12 h timepoint, and cleavage of PARP after 24 h treatment with 7beta-OH. Caspase-8 did not appear to play a major role in this particular apoptotic pathway.     

Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon

Apoptosis is a form of cell death that can function to eliminate cells damaged by environmental stress. One stress that can compromise embryonic development is elevated temperature (i.e., heat shock). For the current studies, we hypothesized that heat shock induces apoptosis in bovine embryos in a developmentally regulated manner. Studies were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis is developmentally regulated, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. As determined by TUNEL reaction, exposure of bovine embryos > or =16 cells on Day 5 after insemination to 41 or 42 degrees C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41 degrees C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41 degrees C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40 degrees C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41 degrees C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.     

Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.     

In vitro thermal stress induces apoptosis and reduces development of porcine parthenotes

The precise physiological causes that result in reduced development of oocytes after heat shock (HS) are not clear. In this study, apoptosis, heat shock protein70 (hsp70), and in vitro development of porcine oocytes were evaluated after HS. Porcine cumulus-oocyte complexes (COCs) were subjected to in vitro maturation for 42 h. The matured oocytes were then heated at 41.5 degrees C for 0 h (control, C0h), 1 h (HS1h), 2 h (HS2h), or 4 h (HS4h). An additional group of oocytes was cultured for 4 h without HS (control, C4h). In Experiment 1, expression of hsp70 was detected by Western-blotting and no difference between controls and HS groups was observed. In Experiment 2, apoptosis of matured oocytes after HS was examined by Annexin V-FITC and TUNEL. No significant TUNEL-positive signals were detected in the heated oocytes compared to the controls, but the intensity of Annexin V-FITC labeling among different groups increased with length of HS and in vitro culture (P<0.05). Oocytes were parthenogenetically activated by an electric pulse plus 6-DMAP (Experiment 3). Mean (+/-S.E.M.) embryonic development in HS2h (cleavage: 42+/-29%; blastocyst: 11+/-10%) and HS4h (cleavage: 36+/-28%; blastocyst: 11+/-8%) were decreased when compared to those in C0h (cleavage: 63+/-12%; blastocyst: 24+/-14%) and C4h (cleavage: 66+/-8%; blastocyst: 21+/-11%). Numbers of blastocysts with TUNEL-positive signals were similar among groups, but the signals increased before the eight-cell stage in HS groups (P<0.05). In conclusion, developmental competence of matured pig oocytes was compromised after heat shock, but it was not closely associated with the expression of oocyte hsp70. However, there may be a link between apoptosis and developmental competence of porcine oocytes.     

SMT-A07, a 3-(Indol-2-yl) indazole derivative, induces apoptosis of leukemia cells in vitro

N-(2-(1H-indazol-3-yl)-1H-pyrrolo[3,2-b]pyridin-5-yl)-4-chloro-N-methylbenzamide (SMT-A07) is a novel 3-(Indol-2-yl) indazole derivative. The anticancer activities in vitro and the cell apoptosis-induction abilities of SMT-A07 on human leukemia HL60 and NB4 cell lines were investigated in this study. The results of MTT assay showed SMT-A07 was a potential and highly efficient antitumor compound with IC₅₀ values ranging from 0.09 to 1.19 μM in five leukemia cell lines. SMT-A07 treatment for 24 h caused the increment of apoptosis rate from 6.88 to 49.72% in HL60 cells and from 8.72 to 56.28% in NB4 cells by flow cytometry analysis. Agarose gel electrophoresis showed DNA fragmentation that appeared after cells were exposed to SMT-A07. After SMT-A07 incubation, DAPI staining revealed the presence of DNA fragmentation, and perinuclear apoptotic body. SMT-A07 also resulted in a loss of ΔΨm in both HL60 and NB4 cells by JC-1 staining. Moreover, apoptosis-related proteins were examined by western blotting to explore the mechanism of its cytotoxicity. SMT-A07 exposure caused down-regulation and cleavage of procaspase-8, procaspase-3, Bid, PARP and up-regulation of cleaved caspase-8, cleaved caspase-3, PARP (Cleaved Fragment). In addition, the presence of pan-caspase inhibitor BOC-D-FMK prevented cells from caspase-3 activation, PARP cleavage, and subsequent apoptosis. Our study demonstrates that SMT-A07 displays an apparent antitumor activity with extensive anti-leukemia spectrum, and SMT-A07 can induce the apoptosis of HL60 and NB4 cells activation of the caspase cascade, which deserves further development.     

Mechanisms involved in the induction of apoptosis by T-2 and HT-2 toxins in HL-60 human promyelocytic leukemia cells

T-2 and HT-2 toxins belong to a group of mycotoxins that are widely encountered as natural contaminants known to elicit toxic responses in hematopoietic cells. In the present study, HL-60 cells were used to characterize the apoptotic effects of T-2 and a major metabolite, HT-2, and to examine the mechanisms involved. Apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation, by flow cytometric analysis, and by DNA gel electrophoresis. T-2 and HT-2 induced concentration-dependent apoptosis after 24 h in HL-60 cells, starting at concentrations of 3.1 and 6.25 ng/ml respectively. An increased number of apoptotic cells could be observed 4–6 h after exposure to 12.5 ng/ml of toxin. Little cytotoxicity (plasma membrane damage) was observed even after exposure to concentrations of toxins (25–50 ng/ml) inducing apoptosis in 60–100% of the cells. The apoptotic process was almost completely blocked in the presence of the general caspase inhibitor zVAD.fmk. In contrast, no or only minor effects were observed with the more specific caspase inhibitors DEVD.CHO, IETD.fmk, and DEVD.fmk. As judged by Western blotting, the levels of several procaspases (-3, -7, -8, -9, but not -12) were reduced 3–6 h after exposure to toxin. Substantial increases in the presumed active form(s) of caspase-8 and -9 were observed. Furthermore, poly(ADP-ribose) polymerase (PARP) was already markedly cleaved 3 h after toxin treatment, indicative of active caspase-3 and -7. No or only minor changes in Bcl-2, Bcl-XL and Bax levels were observed. BAPTA-AM and ZnCl₂ blocked the degradation of procaspases, the fragmentation of PARP, and the induction of apoptosis. In summary, both T-2 and HT-2 induced apoptosis, with T-2 being somewhat more potent than HT-2. The divalent calcium concentration, [Ca²⁺], appears to be involved in the activation of several caspases, resulting in DNA fragmentation, chromosomal condensation, and nuclear fragmentation.     

Induction of apoptosis in normal cultured rat hepatocytes and in Hep3B, a human hepatoma cell line

The in vitro occurrence of apoptosis in hepatic cells has not been well characterized because it depends on apoptosis inducing-agents and culture conditions. Furthermore, for a given hepatic cell and the same agent, discrepant results have been reported depending on the technique used to evaluate the proportion of apoptotic cells. In this study, we compared the effects of several apoptosis-inducing agents – transforming growth factor β₁ (TGF-β₁), retinoic acid (RA), okadaic acid (OA), and cycloheximide (CY) – on two types of hepatic cells, the human hepatoma cell line Hep3B and normal rat hepatocytes, maintained either plated for 24 to 48 h or in suspension for 20 h. Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI staining. DNA fragmentation was investigated biochemically by agarose gel electrophoresis and poly(ADP-ribose) polymerase (PARP) cleavage was studied by western blot. Apoptotic cells were quantified either by counting cells on UV microscopy after DAPI staining or by flow cytometry. Nuclear changes, the ladder pattern on DNA electrophoresis and PARP cleavage were observed in plated cells, hepatoma cells and normal rat hepatocytes, with all inducers but especially with OA. Semiquantification confirmed that OA was a strong inducer in plated cells under the present conditions, since about 14% and 30% of Hep3B cells (with DAPI staining and flow cytometry, respectively) were apoptotic after 48 h treatment, while, with the other inducers, apoptosis was weaker and discrepancies were also observed between the two counting methods (TGF-β₁; 4% and 12%; RA, 7% and 12%; CY, 4% and 16%, with DAPI staining and flow cytometry, respectively). OA induced a moderate apoptosis in cultured hepatocytes (13% with DAPI staining), while TGF-β₁, RA and CY were found to be weakly apoptotic (respectively 4% for the first two and 6% for the last ) after 48 h. In contrast, in suspension cells, apoptosis was observed neither in Hep3B cells nor in normal hepatocytes, whatever the apoptotic inducer and whatever the techniques used to detect apoptosis. In conclusion, our results show that induction of apoptosis in hepatic cells depends not only on the apoptosis-inducing agent but also on the culture conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Apoptosis in sulfur mustard treated A549 cell cultures

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.     

Involvement of caspase-3 in apoptosis induced by Viscum album var. coloratum agglutinin in HL-60 cells

A cytotoxic lectin (Viscum album L. coloratum agglutinin, VCA) from Korean mistletoe was isolated by affinity chromatography on Sepharose 4B immobilized with asialofetuin. In HL-60 cells, addition of VCA resulted in a dose- and time-dependent growth suppression, morphological changes of apoptotic nuclei, and DNA fragmentation characteristics of apoptosis. To investigate how caspase-3 activation during VCA-induced apoptosis induces cleavages of PARP, the expression of PARP and the pattern of caspase-3 activation in HL-60 cells were investigated. The native and processed PARP forms typically seen in apoptotic cells were observed, and a decrease in expression of the 32-kDa form of caspase-3 in a dose-dependent manner was observed. The VCA-induced apoptosis was significantly inhibited by a caspase-3 specific inhibitor, z-DEVD-FMK, and the PARP processing and caspase-3 activation were also inhibited by the inhibitor. A possible involvement of cell cycle arrest in VCA-induced apoptosis was investigated by flow cytometry and the results suggested that the apoptotic effect of VCA is not involved in the induction of cell cycle arrest.     

Farnesylthiosalicylic acid induces caspase activation and apoptosis in glioblastoma cells

Primary glioblastomas (GBMs) commonly overexpress the oncogene epidermal growth factor receptor (EGFR), which leads to increased Ras activity. FTA, a novel Ras inhibitor, produced both time- and dose-dependent caspase-mediated apoptosis in GBM cell lines. EGFR-mediated increase in 3H-thymidine uptake was inhibited by FTA. FACS analysis was performed to determine the percent of apoptotic cells. The sub-Go population of GBM cells was increased from 4.5 to 13.8% (control) to over 45-53.6% in FTA-treated cells within 24 h. Furthermore, FTA also increased the activities of both caspase-3 and -9, and PARP cleavage. Treatment of GBMs with FTA before or after EGF addition to the cultures blocked phosphorylation of Akt and mitogen-activated protein kinases (MAPK). FTA also significantly reduced the amount of EGF-induced Ras-GTP as reflected by a decrease in the level of Ras bound to Raf-RBD-GST. This study demonstrates that inhibition of Ras methylation may provide a therapeutic target for the treatment of GBMs overexpressing EGFR.     

The effects of cysteine addition during in vitro maturation on the developmental competence, ROS, GSH and apoptosis level of bovine oocytes exposed to heat stress

In the present study, we investigated the effects of various concentrations of cysteine (0.0, 0.6, 1.2 and 1.8 mM) added to the maturation medium on nuclear maturation and subsequent embryonic development of bovine oocytes exposed to heat stress (HS: set at 39.5 °C for 5 h, 40.0 °C for 5 h, 40.5 °C for 6 h, and 40.0 °C for 4 h versus 38.5 °C for 20 h as the control group). This regime mimicked the circadian rhythm of the vaginal temperature of lactating dairy cows during the summer season in southwestern Japan. Moreover, we also evaluated the oocyte's reactive oxygen species (ROS) and glutathione (GSH) levels and the apoptosis levels of the oocytes and cumulus cells in the presence or absence of 1.2 mM cysteine. As a result, HS in the without-cysteine group significantly suppressed (p < 0.05) both the nuclear maturation rate up to the metaphase (M)II stage and the blastocyst formation rate compared with that of the control group. In addition, this group showed significantly higher (p < 0.05) ROS levels and significantly lower (p < 0.05) GSH levels than those of the control group. Moreover, the level of TdT-mediated dUTP nick end labelling (TUNEL)-positive cumulus cells in the HS without-cysteine group was significantly higher (p < 0.05) than that of the control group. However, the addition of 1.2 mM cysteine to the maturation medium restored not only the nuclear maturation, blastocyst formation rates and GSH contents, but also increased the ROS and TUNEL-positive levels of the cumulus cells, but not oocytes, to that of the control group. These results indicate that the addition of 1.2 mM cysteine during in vitro maturation (IVM) may alleviate the influence of heat stress for oocyte developmental competence by increasing GSH content and inhibiting the production of oocyte ROS followed by apoptosis of cumulus cells.