Proteasome inhibition activates the mitochondrial pathway of apoptosis in human CD4+ T cells

We have previously shown that inhibition of the proteolytic activity of the proteasome induces apoptosis and suppresses essential functions of activated human CD4⁺ T cells, and we report now the detailed mechanisms of apoptosis following proteasome inhibition in these cells. Here we show that proteasome inhibition by bortezomib activates the mitochondrial pathway of apoptosis in activated CD4⁺ T cells by disrupting the equilibrium of pro‐apoptotic and anti‐apoptotic proteins at the outer mitochondrial membrane (OMM) and by inducing the generation of reactive oxygen species (ROS). Proteasome inhibition leads to accumulation of pro‐apoptotic proteins PUMA, Noxa, Bim and p53 at the OMM. This event provokes mitochondrial translocation of activated Bax and Bak homodimers, which induce loss of mitochondrial membrane potential (ΔΨm). Breakdown of ΔΨm is followed by rapid release of pro‐apoptotic Smac/DIABLO and HtrA2 from mitochondria, whereas release of cytochrome c and AIF is delayed. Cytoplasmic Smac/DIABLO and HtrA2 antagonize IAP‐mediated inhibition of partially activated caspases, leading to premature activation of caspase‐3 followed by activation of caspase‐9. Our data show that proteasome inhibition triggers the mitochondrial pathway of apoptosis by activating mutually independent apoptotic pathways. These results provide novel insights into the mechanisms of apoptosis induced by proteasome inhibition in activated T cells and underscore the future use of proteasome inhibitors for immunosuppression. J. Cell. Biochem. 108: 935–946, 2009. © 2009 Wiley‐Liss, Inc.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Glucosamine sulfate–induced apoptosis in chronic myelogenous leukemia K562 cells is associated with translocation of cathepsin D and downregulation of Bcl-xL

Cathepsin D (cat D) reportedly plays an important role in certain apoptotic processes, the downstream pathways of which involve release of cytochrome c (cyt c) from mitochondria and activation of the caspase cascade. Previous studies revealed that the B-cell lymphoma 2 (Bcl-2) family members Bax or Bid play important roles in apoptotic signal transduction between cat D and mitochondria. Here, we show that glucosamine sulfate (GS) inhibits the proliferation and induces apoptosis of human chronic myelogenous leukemia K562 cells in vitro. GS interfered with the maturation of cat D. Activation of caspase-3, cleavage of poly-(ADP-ribose)-polymerase, release of cyt c, and downregulation of Bcl-xL accompanied GS-induced apoptosis, and these processes were inhibited by the cat D inhibitor pepstatin A. However, we did not detect any altered gene expression of Bcl-2, Bax, or Bid during apoptosis. Translocation of cat D from the lysosome to the cytosol was observed in GS-treated K562 cells. These findings suggest that GS-induced K562 cell apoptosis involves the translocation of cat D from the lysosome to the cytosol. Furthermore, our findings suggest that downregulation of Bcl-xL (but not Bcl-2, Bax, or Bid) connects cat D and the mitochondrial pathway, which causes the release of cyt c and activation of the caspase cascade during GS-induced apoptosis of K562 cells.     

Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F<Subscript>0</Subscript>-F<Subscript>1</Subscript> ATP synthase complex

The number of genes that are up regulated or down regulated during apoptosis is large and still increasing. In an attempt to characterize differential gene expression during serum factor induced apoptosis in AK-5 cells (a rat histiocytoma), we found subunit 6 and subunit 8 of the transmembrane proton channel and subunit alpha of the catalytic core of the mitochondrial F₀-F₁ ATP synthase complex to be up regulated during apoptosis. The increase in the expression levels of these subunits was concomitant with a transient increase in the intracellular ATP levels, suggesting that the increase in cellular ATP content is a result of the increase in the expression of ATP synthase subunits' gene and de novo protein synthesis. Depleting the cellular ATP levels with oligomycin inhibited apoptosis significantly, pointing to the requirement of ATP during apoptosis. Caspase 1 and caspase 3 activity and the loss of mitochondrial membrane potential were also inhibited by oligomycin during apoptosis in these cells, suggesting that the oligomycin induced inhibition of apoptosis could be due to inhibition of caspase activity and inhibition of mitochondrial depolarization. However, cytochrome C release during apoptosis was found to be completely independent of intracellular ATP content. Besides the ATP synthase complex genes, other mitochondrial genes like cytochrome C oxidase subunit II and III also showed elevated levels of expression during apoptosis. This kind of a mitochondrial gene expression profile suggests that in AK-5 cells, these genes are upregulated in a time-linked manner to ensure sufficient intracellular ATP levels and an efficient functioning of the mitochondrial respiratory chain for successful completion of the apoptotic pathway.     

Ozone alleviates ischemia/reperfusion injury by inhibiting mitochondrion‐mediated apoptosis pathway in SH‐SY5Y cells

Cerebral ischemia/reperfusion (I/R) injuries are common and often cause severe complications. Ozone has been applied for protecting I/R injury in animal models of several organs including cerebra, but the detailed mechanism remains unclear. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase measurement were used to determine the influence of ozone on cell activity and damage of SH‐SY5Y cells. Some redox items such as catalase (CAT), malondialdehyde (MDA), glutathione peroxidase (GSH‐Px), and superoxide dismutase (SOD) were measured by enzyme‐linked immunosorbent assay. The mitochondrial membrane potential (ΔΨ_m) was determined by JC‐1 assay. Cytochrome‐c (cyt‐c) level in the cytoplasm and mitochondrion was measured by western blotting. Apoptosis was determined by flow cytometry, and some apoptosis‐related molecules were detected by quantitative real‐time polymerase chain reaction and western blotting. Ozone alleviated oxidative damage by increasing GSH‐Px, SOD, CAT, and decreasing MDA. Ozone decreased mitochondrial damage caused by I/R injury and inhibited the release of cyt‐c from mitochondrion to cytoplasm in SH‐SY5Y cells. The cell apoptosis caused by I/R was inhibited by ozone, and ozone could decrease apoptosis by increasing the ratio of Bcl‐2/Bax and inhibiting caspase signaling pathway in SH‐SY5Y cells. Ozone has the ability of maintaining redox homeostasis, decreasing mitochondrion damage, and inhibiting neurocytes apoptosis induced by I/R. Therefore, ozone may be a promising protective strategy against cerebral I/R injury.     

Separation of cytochrome c-dependent caspase activation from thiol-disulfide redox change in cells lacking mitochondrial DNA

Release of mitochondrial cytochrome c (cyt c) is an early and common event during apoptosis. Previous studies showed that the loss of cyt c triggered superoxide production by mitochondria and contributed to the oxidation of cellular thiol-disulfide redox state. In this study, we tested whether loss of the functional electron transport chain due to depleting mitochondrial DNA (mtDNA) would affect this redox-signaling mechanism during apoptosis. Results showed that cyt c release and caspase activation in response to staurosporine treatment were preserved in cells lacking mitochondrial DNA (rho0 cells). However, unlike the case with rho+ cells, in which a dramatic oxidation of intracellular glutathione (GSH) occurred after mitochondrial cyt c release, the thiol-disulfide redox state in apoptotic rho0 cells remained largely unchanged. Thus, mitochondrial signaling of caspase activation can be separated from the bioenergetic function, and mitochondrial respiratory chain is the principal source of ROS generation in staurosporine-induced apoptosis.     

The Temporal Relationship between Protein Phosphatase, Mitochondrial CytochromecRelease, and Caspase Activation in Apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochromec.Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochromecin apoptosis induced by the Fas receptor. We demonstrate that cytochromecis released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochromec.The protein phosphatase inhibitor calyculin A prevented cytochromecrelease and apoptosis induced by both agents, suggesting that release of cytochromecis required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochromec.The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochromecand apoptosis induced by both insults.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

The Nitric Oxide Prodrug JS‐K Induces Ca2+‐Mediated Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

Hepatocellular carcinoma is one of the most common and deadly forms of human malignancies. JS‐K, O²‐(2, 4‐dinitrophenyl) 1‐ [(4‐ethoxycarbonyl) piperazin‐1‐yl] diazen‐1‐ium‐1, 2‐diolate, has the ability to induce apoptosis of tumor cell lines. In the present study, JS‐K inhibited the proliferation of HepG2 cells in a time‐ and concentration‐dependent manner and significantly induced apoptosis. JS‐K enhanced the ratio of Bax‐to‐Bcl‐2, released of cytochrome c (Cyt c) from mitochondria and the activated caspase‐9/3. JS‐K caused an increasing cytosolic Ca²⁺ and the loss of mitochondrial membrane potential. Carboxy‐PTIO (a NO scavenger) and BAPTA‐AM (an intracellular Ca²⁺ chelator) significantly blocked an increasing cytosolic Ca²⁺ in JS‐K‐induced HepG2 cells apoptosis, especially Carboxy‐PTIO. Meanwhile, Carboxy‐PTIO and BAPTA‐AM treatment both attenuate JS‐K‐induced apoptosis through upregulation of Bcl‐2, downregulation of Bax, reduction of Cyt c release from mitochondria to cytoplasm and inactivation of caspase‐9/3. In summary, JS‐K induced HepG2 cells apoptosis via Ca²⁺/caspase‐3‐mediated mitochondrial pathway.     

Delay in Apoptosome Formation Attenuates Apoptosis in Mouse Embryonic Stem Cell Differentiation

Differentiation is an inseparable process of development in multicellular organisms. Mouse embryonic stem cells (mESCs) represent a valuable research tool to conduct in vitro studies of cell differentiation. Apoptosis as a well known cell death mechanism shows some common features with cell differentiation, which has caused a number of ambiguities in the field. The research question here is how cells could differentiate these two processes from each other. We have investigated the role of the mitochondrial apoptotic pathway and cell energy level during differentiation of mESCs into the cardiomyocytes and their apoptosis. p53 expression, cytochrome c release, apoptosome formation, and caspase-3/7 activation are observed upon induction of both apoptosis and differentiation. However, remarkable differences are detected in time of cytochrome c appearance, apoptosome formation, and caspase activity upon induction of both processes. In apoptosis, apoptosome formation and caspase activity were observed rapidly following the cytochrome c release. Unlike apoptosis, the release of cytochrome c upon differentiation took more time, and the maximum caspase activity was also postponed for 24 h. This delay suggests that there is a regulatory mechanism during differentiation of mESCs into cardiomyocytes. The highest ATP content of cells was observed immediately after cytochrome c release 6 h after apoptosis induction and then decreased, but it was gradually increased up to 48 h after differentiation. These observations suggest that a delay in the release of cytochrome c or delay in ATP increase attenuate apoptosome formation, and caspase activation thereby discriminates apoptosis from differentiation in mESCs.     

Cytochrome <Emphasis Type="Italic">c</Emphasis> is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa₃) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be ≈62% oxidized before MOMP and became ≈70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc₁ with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s.     

Swainsonine Induces Caprine Luteal Cells Apoptosis via Mitochondrial‐Mediated Caspase‐Dependent Pathway

Swainsonine (SW) is an indolizidine alkaloid isolated from a number of poisonous plants. We have previously reported that SW inhibited luteal cell progesterone production by inducing caprine luteal cell apoptosis in vitro; however, the molecular mechanism of this phenomenon remains unclear. In this study, SW‐treated luteal cells showed apoptosis characteristics, including nuclear fragmentation, DNA ladder formation, and phosphatidylserine externalization. Further studies showed that SW activated caspase‐9 and caspase‐3, which subsequently cleaved poly(ADP‐ribose) polymerase. SW also increased in Bax/BcL‐2 ratios, promoted Bax translocation from the cytosol to mitochondria, and triggered the release of cytochrome c from mitochondria into the cytoplasm. However, Fas and Fas ligand induction or caspase‐8 activity did not appear any significant changes. Additional analysis also showed that pan‐caspase inhibitor, caspase‐9 inhibitor, or caspase‐3 inhibitor almost completely protected the cells from SW‐induced apoptosis, but not caspase‐8 inhibitor. Overall, these data demonstrated that SW induced luteal cells apoptosis through a mitochondrial‐mediated caspase‐dependent pathway.     

Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases’ cascade

Antizymes delicately regulate ornithine decarboxylase (ODC) enzyme activity and polyamine transportation. One member of the family, antizyme-1, plays vital roles in molecular and cellular functions, including developmental regulation, cell cycle, proliferation, cell death, differentiation and tumorigenesis. However, the question of how does it participate in the cell apoptotic mechanism is still unsolved. To elucidate the contribution of human antizyme-1 in haematopoietic cell death, we examine whether inducible overexpression of antizyme enhances apoptotic cell death. Antizyme reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells, acute T leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G₁ appearance, loss of mitochondrial membrane potential (Δψ _m ), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following conditional antizyme overexpression, all protein levels of cyclin-dependent kinases (Cdks) and cyclins are not significantly reduced, except cyclin D, before their entrance into apoptotic cell death. However, introduced cyclin D1 into Jurkat T tetracycline (Tet)-On cell system still couldn’t rescue cells from apoptosis. Antizyme doesn’t influence the expression of tumor suppressor p53 and its downstream p21, but it interferes in the expressions of Bcl-2 family. Inducible antizyme largely enters mitochondria resulting in cytochrome c release from mitochondria to cytosol following Bcl-xL decrease and Bax increase. According to these data, we suggest that antizyme induces apoptosis mainly through mitochondria-mediated and cell cycle-independent pathway. Furthermore, antizyme induces apoptosis not only by Bax accumulation reducing the function of the Bcl-2 family, destroying the Δψ _m , and releasing cytochrome c to cytoplasm but also by the activation of apoptosomal caspase cascade.     

Helicobacter hepaticus Cytolethal Distending Toxin Causes Cell Death in Intestinal Epithelial Cells via Mitochondrial Apoptotic Pathway

Background: Helicobacter hepaticus, the prototype for enterohepatic Helicobacter species, colonizes the lower intestinal and hepatobiliary tracts of mice and causes typhlocolitis, hepatitis, and hepatocellular carcinoma in susceptible mouse strains. Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus. CDT of several Gram‐negative bacteria is associated with double‐stranded DNA breaks resulting in cell cycle arrest and death of a wide range of eukaryotic cells in vitro. We previously observed H. hepaticus CDT (HhCDT) mediated apoptosis in INT407 cells. However, the exact mechanism for the induction of the apoptotic pathway by HhCDT is unknown. The objective of this study was to identify the apoptotic signaling pathway induced by HhCDT in INT407 cells. Materials and Methods: INT407 cells were incubated with or without recombinant HhCDT for 0–72 hours. H2AX phosphorylation and apoptotic parameters were analyzed. Results: H2AX was phosphorylated 24 hours postexposure to HhCDT. Expression of pro‐apoptotic Bax protein was upregulated after 24 hours, while Bcl₂ expression decreased. Cytochrome c was released from mitochondria after 12–24 hours of exposure. Concurrently, caspase 3/7 and 9 were activated. However, pretreatment of INT407 cells with caspase inhibitor (Z‐VAD‐FMK) inhibited the activation of caspase 3/7 and 9. Significant activity of caspase 8 was not observed in toxin treated cells. Activation of caspase 3/7 and caspase 9 confirms the involvement of the mitochondrial apoptotic pathway in HhCDT‐treated cells. Conclusion: These findings show, for the first time, the ability of HhCDT to induce apoptosis via the mitochondrial pathway.     

Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS‐dependent mitochondrial signalling pathway

Oxidative stress is an important molecular mechanism underlying lung fibrosis. The mitochondrion is a major organelle for oxidative stress in cells. Therefore, blocking the mitochondrial signalling pathway may be the best therapeutic manoeuver to ameliorate lung fibrosis. Astaxanthin (AST) is an excellent antioxidant, but no study has addressed the pathway of AST against pulmonary oxidative stress and free radicals by the mitochondrion‐mediated signalling pathway. In this study, we investigated the antioxidative effects of AST against H₂O₂‐ or bleomycin (BLM)‐induced mitochondrial dysfunction and reactive oxygen species (ROS) production in alveolar epithelial cells type II (AECs‐II) in vivo and in vitro. Our data show that AST blocks H₂O₂‐ or BLM‐induced ROS generation and dose‐dependent apoptosis in AECs‐II, as characterized by changes in cell and mitochondria morphology, translocation of apoptotic proteins, inhibition of cytochrome c (Cyt c) release, and the activation of caspase‐9, caspase‐3, Nrf‐2 and other cytoprotective genes. These data suggest that AST inhibits apoptosis in AECs‐II cells through the ROS‐dependent mitochondrial signalling pathway and may be of potential therapeutic value in lung fibrosis treatment.     

Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-x<Subscript>L</Subscript> subcellular distribution

Plasminogen Kringle 5(K5) is a proteolytic fragment of plasminogen, which displays potent anti-angiogenic activities. K5 has been shown to induce apoptosis in proliferating endothelial cells; however the exact mechanism has not been well explored. The present study was designed to elucidate the possible molecular mechanism of K5-induced endothelial cell apoptosis. Our results showed that K5 inhibited basic fibroblast growth factors activated in human umbilical vein endothelial cells (HUVECs), indicating proliferation in a dose-dependent manner and induced endothelial cell death via apoptosis. K5 exposure activated caspase 7, 8 and 9. These results suggested that both the intrinsic mitochondrial apoptosis pathway and extrinsic pathway might be involved in K5-induced apoptosis. K5 reduced mitochondrial membrane potential (MMP) of HUVECs, demonstrating mitochondrial depolarization in HUVECs. K5 increased the ratio of Bak to Bcl-x_L on mitochondria, decreased the ratio in cytosol, and had no effect on the total amounts of these proteins. K5 also did not effect on Bax/Bcl-2 distribution. K5 increased the ratio of Bak to Bcl-x_L on mitochondrial that resulted in mitochondrial depolarization, cytochrome c release and consequently the cleavage of caspase 9. These results suggested that K5 induces endothelial cell apoptosis at least in part via activating mitochondrial apoptosis pathway. The regulation of K5 on Bak and Bcl-x_L distribution may play an important role in endothelial cell apoptosis. These results provide further insight into the anti-angiogenesis roles of K5 in angiogenesis-related ocular diseases and solid tumors.     

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

Impairment of mitochondria function and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9–20)-, mid-age (48–60)-, and >60 (62–76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H₂O₂ toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca²⁺ [Ca²⁺]_c, and mitochondrial Ca²⁺ [Ca²⁺]_m levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5′,6,6′-tetrachloro 1,1′3,3′-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca²⁺]_c, and increased sequestration of [Ca²⁺]_m in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.     

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.     

Distinct Alterations in Mitochondrial Mass and Function Characterize Different Models of Apoptosis

Recent studies have shown that reduction in mitochondrial membrane potential (ΔΨ_m) and generation of reactive oxygen species are early events in apoptosis. In this study, we present two different models of apoptotic cell death, Chinese hamster ovary (CHO) cells treated with aphidicolin and dexamethasone-treated 2B4 T-cell hybridoma cells, which display opposing mitochondrial changes. CHO cells arrested at G1/S with aphidicolin have a progressive increase in mitochondria mass and number, assessed by flow cytometry and fluorescent microscopy with mitochondria-specific probes. The increase in mitochondrial mass was not accompanied by a gain in net cellular mitochondrial membrane potential, consistent with an accumulation of relatively depolarized mitochondria. Fluorescent microscopy demonstrated an increased content of low ΔΨ_mmitochondria in aphidicolin-treated CHO cells, but high ΔΨ_mmitochondria were also present and remained stable in number. Mitochondrial mass correlated with decreased clonogenicity of aphidicolin-treated CHO cells. Cycloheximide prevented both the proliferation of mitochondria and subsequent cell death. In contrast, dexamethasone treatment of 2B4 T-cell hybridoma cells caused a decrease in ΔΨ_mwithout mitochondrial proliferation. Cycloheximide and Bcl-2 overexpression inhibited the loss of ΔΨ_m, as well as apoptosis. In both models, cell death was associated with a decrease in mitochondrial potential relative to mitochondrial mass, suggesting that an accumulation of damaged or dysfunctional mitochondria had occurred.