Protection conferred by Bcl-2 expression involves reduced oxidative stress and increased glutathione production during hypothermia-induced apoptosis in AK-5 tumor cells

Hypothermia is known to retard mammalian cell growth, however, BC-8 cells, which have originated from AK-5 tumor after single cell cloning, were triggered into apoptotic pathway when grown at 30 degrees C. Cell death process showed typical apoptotic features like DNA fragmentation, cytochrome c release, etc. Introduction of Bcl-2 gene in BC-8 cells inhibited hypothermia-induced apoptotic process, which is ascribed to reduced ROS generation and higher glutathione production. Thus, Bcl-2 seems to control the apoptotic induction process at the level of redox regulation, in addition to its known effects at the mitochondrial dysregulation. These observations suggest that tumors, which are low in Bcl-2 expression, are sensitive to hypothermic shock and make hypothermia an interesting inducer of apoptosis in tumor cells.     

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC-8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC-8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC-8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6-8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.     

Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line.

Stress response is a universal phenomenon. However, a rat histiocytic cell line, BC-8, showed no heat shock response and failed to synthesize heat shock protein 70 (hsp70) upon heat shock at 42 degrees C for 30 min. BC-8 is a clone of AK-5, a rat macrophage tumor line that is adapted to grow in culture and has the same chromosome number and tumorigenic potential as AK-5. An increase in either the incubation temperature or time or both to BC-8 cells leads to loss of cell viability. In addition, heat shock conditions activated apoptotic cell death in these cells as observed by cell fragmentation, formation of nuclear comets, apoptotic bodies, DNA fragmentation and activation of ICE-like cysteine proteases. Results presented here demonstrate that BC-8 cells cannot mount a typical heat shock response unlike all other eukaryotic cells and that in the absence of induction of hsps upon stress, these cells undergo apoptosis at 42 degrees C.     

A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma

Increasing evidence provides support for oxidative stress to be closely linked to apoptosis. Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. Though heat shock is a universal phenomenon, BC-8, a macrophage-like cell line failed to mount a typical heat shock response. In the absence of heat shock proteins and functional p53, BC-8 cells undergo apoptosis through CD95 signaling. In the present study, we have investigated the role of ROS in the regulation of apoptosis in these cells. We show that cells transfected with hsp70 and functional p53 are resistant to heat-induced apoptosis through inhibition of CD95 expression and ROS induction. Furthermore, apoptosis in BC-8 cells resulted in two bursts of ROS generation, one correlated with heat stress and intracellular depletion of GSH and the other with Bax overexpression and cytochrome c release. Antioxidants could not protect these cells from heat-induced apoptosis and the death pathway seems to be dependent on initial signaling cascade subsequently altering the intracellular redox. Hence, our data suggest that ROS generation in BC-8 cells upon heat shock is facultative but not obligatory for apoptosis.     

N-t-Butyl hydroxylamine regulates heat shock-induced apoptosis in U937 cells

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Therefore, compounds that scavenge reactive oxygen species may regulate heat shock-induced cell death. Recently, it has been shown that the decomposition product of the spin-trapping agent alpha-phenyl-N-t-butylnitrone, N-t-butyl hydroxylamine (NtBHA), mimics alpha-phenyl-N-t-butylnitrone and is much more potent in delaying reactive oxygen species-associated senescence. We investigated the protective role of NtBHA against heat shock-induced apoptosis in U937 cells. Upon exposure to heat shock, there was a distinct difference between the untreated cells and the cells pre-treated with 0.1 mM NtBHA for 2 h in regard to apoptotic parameters, cellular redox status, and mitochondrial function. Upon exposure to heat shock, NtBHA pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax, and down-regulation of Bcl-2 compared to untreated cells. This study indicates that NtBHA may play an important role in regulating the apoptosis induced by heat shock, presumably through scavenging of reactive oxygen species.     

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.     

Heat shock and ceramide have different apoptotic pathways in radiation induced fibrosarcoma (RIF) cells

Heat shock induces various cellular responses including inhibition of protein synthesis, production of heat shock proteins (HSPs) and induction of thermotolerance. The molecular mechanisms of the processes have not been well understood. It has been proposed that ceramide formation during heat shock mediates heat shock induced apoptosis. We examined whether C2-ceramide mimicked the cellular response to heat shock in RIF-1 cells and their thermotolerant derivative TR-RIF-1 cells. Discernible effects between heat shock and C2-ceramide treatments were observed in cellular changes such as total protein synthesis, HSP synthesis, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activity and PARP cleavage. Heat shock immediately inhibited cellular protein synthesis, which was recovered by synthesizing HSPs first and then whole proteins later. Heat shock also activated SAPK/JNK and increased PARP cleavage in dose-dependent manner. Thermotolerant TR-RIF-1 cells responded to heat shock more insensitively than RIF-1 cells. On the other hand, C2-ceramide treatment did not accompany any changes induced by heat shock. No discernible differences between RIF-1 and TR-RIF-1 cells were observed by C2-ceramide treatment. We tried to figure out how C2-ceramide interacts with cellular membrane and found that exogenous C2-ceramide was incorporated into the outer monolayer and flipped into the inner monolayer of human erythrocytes in ATP-dependent manner. However, the rate of C2-ceramide incorporation was similar in control and thermotolerant cells. In summary, thermotolerant cells are resistant to heat shock induced apoptotic signaling but not resistant, rather sensitive to membrane disturbing C2-ceramide mediated apoptosis. These results suggest that heat shock and ceramide have different signal transduction pathways.     

Oxalomalate, a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase, regulates heat shock-induced apoptosis

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. The protective role of ICDH against heat shock-induced apoptosis in U937 cells was investigated in the control and the cells pre-treated with oxalomalate, a competitive inhibitor of ICDH. Upon exposure to heat shock, there was a distinct difference between the control cells and the cells pre-treated with 3mM oxalomalate for 3h in regard to apoptotic parameters, cellular redox status, and mitochondrial function. The oxalomalate pre-treated cells showed significant enhancement of apoptotic features such as activation of caspase-3, up-regulation of Bax, and down-regulation of Bcl-2 compared to the control cells upon exposure to heat shock. This study indicates that ICDH may play an important role in regulating the apoptosis induced by heat shock presumably through maintaining the cellular redox status.     

Silencing of mitochondrial NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances heat shock-induced apoptosis

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDPm) produces NADPH, an essential reducing equivalent for the antioxidant system. In this report, we demonstrate that silencing of IDPm expression in HeLa cells greatly enhances apoptosis induced by heat shock. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly decreased activity of IDPm, enhancing the susceptibility of heat shock-induced apoptosis reflected by morphological evidence of apoptosis, DNA fragmentation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. These results indicate that IDPm may play an important role in regulating the apoptosis induced by heat shock and the sensitizing effect of IDPm siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer therapy.     

In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis

Activation of 'initiator' (or 'apical') caspases-2, -8 or -9 (refs 1-3) is crucial for induction of apoptosis. These caspases function to activate executioner caspapses that, in turn, orchestrate apoptotic cell death. Here, we show that a cell-permeable, biotinylated pan-caspase inhibitor (bVAD-fmk) both inhibited and 'trapped' the apical caspase activated when apoptosis was triggered. As expected, only caspase-8 was trapped in response to ligation of death receptors, whereas only caspase-9 was trapped in response to a variety of other apoptosis-inducing agents. Caspase-2 was exclusively activated in heat shock-induced apoptosis. This activation of caspase-2 was also observed in cells protected from heat-shock-induced apoptosis by Bcl-2 or Bcl-xL. Reduced sensitivity to heat-shock-induced death was observed in caspase-2(-/-) cells. Furthermore, cells lacking the adapter molecule RAIDD failed to activate caspase-2 after heat shock treatment and showed resistance to apoptosis in this setting. This approach unambiguously identifies the apical caspase activated in response to apoptotic stimuli, and establishes caspase-2 as a proximal mediator of heat shock-induced apoptosis.     

Heat shock suppresses the permeability transition in rat liver mitochondria

Heat shock proteins inhibit apoptotic and necrotic cell death in various cell types. However, the specific mechanism underlying protection by heat shock proteins remains unclear. To test the hypothesis that heat shock proteins inhibit cell death by blocking opening of mitochondrial permeability transition (MPT) pores, mitochondria from heat-preconditioned rat livers were isolated by differential centrifugation. Heat shock inhibited MPT pore opening induced by 50 microm CaCl(2) plus 5 microm HgCl(2) or 1 microm mastoparan and by 200 microm CaCl(2) alone. Half-maximal swelling was delayed 15 min or more after heat shock compared with control. Heat shock also increased the threshold of unregulated (Ca(2+)-independent and cyclosporin A-insensitive) MPT pore opening induced by higher doses of HgCl(2) and mastoparan. Heat shock treatment decreased mitochondrial reactive oxygen species formation by 27% but did not change mitochondrial respiration, membrane potential, Ca(2+) uptake, or total glutathione in mitochondrial and cytosolic extracts of liver. Western blot analysis showed that mitochondrial Hsp25 increased, whereas Hsp10, Hsp60, Hsp70, Hsp75, cyclophilin D, and voltage-dependent anion channel did not change after heat shock. These results indicate that heat shock causes resistance to opening of MPT pores, which may contribute to heat shock protection against cellular injury.     

Heat shock induces apoptosis independently of any known initiator caspase-activating complex

Adaptive responses to mild heat shock are among the most widely conserved and studied in nature. More intense heat shock, however, induces apoptosis through mechanisms that remain largely unknown. Herein, we present evidence that heat shock activates an apical protease that stimulates mitochondrial outer membrane permeabilization and processing of the effector caspase-3 in a benzyloxycarbonyl-VAD-fluoromethyl ketone (polycaspase inhibitor)- and Bcl-2-inhibitable manner. Surprisingly, however, neither FADD.caspase-8 nor RAIDD.caspase-2 PIDDosome (p53-induced protein with a death domain) complexes were detected in dying cells, and neither of these initiator caspases nor the endoplasmic reticulum stress-activated caspases-4/12 were required for mitochondrial outer membrane permeabilization. Similarly, although cytochrome c was released from mitochondria following heat shock, functional Apaf-1.caspase-9 apoptosome complexes were not formed, and caspase-9 was not essential for the activation of caspase-3 or the induction of apoptosis. Thus, heat shock does not require any of the known initiator caspases or their activating complexes to promote apoptotic cell death but instead relies upon the activation of an apparently novel apical protease with caspase-like activity.     

Tumor necrosis factor induces apoptosis in hepatoma cells by increasing Ca(2+) release from the endoplasmic reticulum and suppressing Bcl-2 expression

Tumor necrosis factor (TNF) plays an import role in the control of apoptosis. The most well known apoptotic pathway regulated by TNF involves the TNFR1-associated death domain protein, Fas-associated death domain protein, and caspase-8. This study examines the mechanism of TNF-induced apoptosis in FaO rat hepatoma cells. TNF treatment significantly increased the percentage of apoptotic cells. TNF did not activate caspase-8 but activated caspase-3, -10, and -12. The effect of TNF on the expression of different members of the Bcl-2 family in these cells was studied. We observed no detectable changes in the steady-state levels of Bcl-X(L), Bax, and Bid, although TNF suppresses Bcl-2 expression. Dantrolene suppressed the inhibitory effect of TNF on Bcl-2 expression. TNF induced release of Ca(2+) from the endoplasmic reticulum (ER) that was blocked by dantrolene. Importantly, the expression of Bcl-2 blocked TNF-induced apoptosis and decreased TNF-induced Ca(2+) release. These results suggest that TNF induces apoptosis by a mechanism that involves increasing Ca(2+) release from the ER and suppression of Bcl-2 expression.     

Dual anticancer activity of 8-Cl-cAMP: inhibition of cell proliferation and induction of apoptotic cell death

8-Cl-cAMP induces apoptotic cell death in human cancer cells. To look at this more closely, we examined the changes in the levels of Bcl-2 family proteins during 8-Cl-cAMP-induced apoptosis of SH-SY5Y human neuroblastoma cells. Following the treatment with 8-Cl-cAMP, Bcl-2 was transiently down-regulated and Bad was increased continuously up to day 5. In addition, overexpression of Bcl-2 efficiently blocked the 8-Cl-cAMP-induced apoptosis, suggesting Bcl-2 family proteins may be involved in the 8-Cl-cAMP-induced apoptosis. The contribution of the apoptotic cell death and the inhibition of cell proliferation in the 8-Cl-cAMP-induced growth inhibition was closely monitored in the Bcl-2-overexpressing cells. Though the apoptosis was reduced significantly, no significant difference was observed in the inhibition of cell proliferation up to day 2 of 8-Cl-cAMP treatment. These results suggest that 8-Cl-cAMP exerts anticancer activity by two distinct mechanisms, i.e. , through the inhibition of cell proliferation as well as the induction of apoptosis. Supporting this notion was the observations that (1) suppression of apoptosis by zVAD did not abrogate 8-Cl-cAMP-induced inhibition of cell proliferation, and (2) 8-Cl-cAMP did not show additive inhibition of cell proliferation in RIIbeta-overexpressing cells.     

Enhanced cell transplantation: preventing apoptosis increases cell survival and ventricular function

Cell transplantation prevents cardiac dysfunction after myocardial infarction. However, because most implanted cells are lost to ischemia and apoptosis, the benefits of cell transplantation on heart function could be improved by increasing cell survival. To examine this possibility, male Lewis rat aortic smooth muscle cells (SMCs; 4 x 10(6)) were pretreated with antiapoptotic Bcl-2 gene transfection or heat shock and then implanted into the infarcted myocardium of anesthetized, syngenic female rats (n = 23 per group). On the first day after transplantation, apoptotic SMCs were quantified by using transferase-mediated dUTP nick-end labeling staining. On days 7 and 28, grafted cell survival was quantified by using real-time PCR, and heart function was assessed with the use of echocardiography and the Langendorff apparatus. SMCs given antiapoptotic pretreatments exhibited improvements in each measure relative to controls. Apoptosis was reduced in Bcl-2-treated cells relative to all other groups (P < 0.05), whereas survival (P < 0.01) was increased. Heat shock also significantly decreased apoptosis and increased survival relative to control groups (P < 0.05 for group effect), although these effects were less pronounced than in the Bcl-2-treated group. Further, scar areas were reduced in both Bcl-2- and heat shock-treated groups relative to controls (P < 0.05), and fractional area change and cardiac function were greater (P < 0.05 for both measures). These results indicate that antiapoptosis pretreatments reduced grafted SMC loss after transplantation and enhanced grafted cell survival and ventricular function, which was directly related (r = 0.72; P = 0.002) to the number of surviving engrafted cells.