Genistein induces apoptosis of RPE-J cells by opening mitochondrial PTP

Although previous studies demonstrated that genistein-induced apoptosis of various cell types including RPE-J cells, the involvement of mitochondrial events in such types of apoptosis has not been demonstrated to date. In this investigation of genistein-induced apoptosis of RPE-J cells, genistein induced the reduction of the mitochondrial membrane potential and the release of cytochrome c to cytosol. A mitochondrial permeability transition pore (PTP) blocker bongkrekic acid prevented the reduction of the mitochondrial membrane potential and cytochrome c release, and consequently abolished caspase-3 activation, nuclear condensation, and DNA fragmentation. On the other hand, zVAD-fmk did not inhibit the mitochondrial event such as the reduction of the mitochondrial membrane potential and cytochrome c release although it prevented caspase-3 activation, nuclear condensation, and DNA fragmentation. Taken together, genistein induces apoptosis of RPE-J cells by opening the mitochondrial PTP, and the mitochondrial event in this type of apoptosis is caused independently of caspase.     

Helicobacter pylori vacuolating cytotoxin enters cells, localizes to the mitochondria, and induces mitochondrial membrane permeability changes correlated to toxin channel activity

The Helicobacter pylori vacuolating cytotoxin (VacA) intoxicates mammalian cells resulting in reduction of mitochondrial transmembrane potential (Delta Psi m reduction) and cytochrome c release, two events consistent with the modulation of mitochondrial membrane permeability. We now demonstrate that the entry of VacA into cells and the capacity of VacA to form anion-selective channels are both essential for Delta Psi m reduction and cytochrome c release. Subsequent to cell entry, a substantial fraction of VacA localizes to the mitochondria. Neither Delta Psi m reduction nor cytochrome c release within VacA-intoxicated cells requires cellular caspase activity. Moreover, VacA cellular activity is not sensitive to cyclosporin A, suggesting that VacA does not induce the mitochondrial permeability transition as a mechanism for Delta Psi m reduction and cytochrome c release. Time-course and dose-response studies indicate that Delta Psi m reduction occurs substantially before and at lower concentrations of VacA than cytochrome c release. Collectively, these results support a model that VacA enters mammalian cells, localizes to the mitochondria, and modulates mitochondrial membrane permeability by a mechanism dependent on toxin channel activity ultimately resulting in cytochrome c release. This model represents a novel mechanism for regulation of a mitochondrial-dependent apoptosis pathway by a bacterial toxin.     

Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x(L) in osteosarcoma cells

We explored the role of low mitochondrial membrane potential (DeltaPsim) and the lack of oxidative phosphorylation in apoptosis by assessing the susceptibility of osteosarcoma cell lines with and without mitochondrial DNA to staurosporine-induced death. Our cells without mitochondrial DNA had low DeltaPsim and no functional oxidative phosphorylation. Contrary to our expectation, these cells were more resistant to staurosporine-induced death than were the parental cells. This reduced susceptibility was associated with decreased activation of caspase 3 but not with the mitochondrial permeability transition pore or cytochrome c release from the mitochondria. Apoptosis in both cell lines was associated with an increase in DeltaPsim. Bcl-x(L) could protect both cell types against caspase 3 activation and apoptosis by a mechanism that does not appear to be mediated by mitochondrial function or modulation of DeltaPsim. Nevertheless, we found that Bcl-x(L) expression can stimulate cell respiration in cells with mitochondrial DNA. Our results showed that the lack of functional oxidative phosphorylation and/or low mitochondrial membrane potential are associated with an antiapoptotic effect, possibly contributing to the development of some types of cancer. It also reinforces a model in which Bcl-x(L) can exert an antiapoptotic effect by stimulating oxidative phosphorylation and/or inhibiting caspase activation.     

Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization.

Mitochondrial cytochrome c, which functions as an electron carrier in the respiratory chain, translocates to the cytosol in cells undergoing apoptosis, where it participates in the activation of DEVD-specific caspases. The apoptosis inhibitors Bcl-2 or Bcl-xL prevent the efflux of cytochrome c from mitochondria. The mechanism responsible for the release of cytochrome c from mitochondria during apoptosis is unknown. Here, we report that cytochrome c release from mitochondria is an early event in the apoptotic process induced by UVB irradiation or staurosporine treatment in CEM or HeLa cells, preceding or at the time of DEVD-specific caspase activation and substrate cleavage. A reduction in mitochondrial transmembrane potential (Deltapsim) occurred considerably later than cytochrome c translocation and caspase activation, and was not necessary for DNA fragmentation. Although zVAD-fmk substantially blocked caspase activity, a reduction in Deltapsim and cell death, it failed to prevent the passage of cytochrome c from mitochondria to the cytosol. Thus the translocation of cytochrome c from mitochondria to cytosol does not require a mitochondrial transmembrane depolarization.     

Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.     

Caspase inhibition extends the commitment to neuronal death beyond cytochrome c release to the point of mitochondrial depolarization

Nerve growth factor (NGF) deprivation induces a Bax-dependent, caspase-dependent programmed cell death in sympathetic neurons. We examined whether the release of cytochrome c was accompanied by the loss of mitochondrial membrane potential during sympathetic neuronal death. NGF- deprived, caspase inhibitor-treated mouse sympathetic neurons maintained mitochondrial membrane potential for 25-30 h after releasing cytochrome c. NGF- deprived sympathetic neurons became committed to die, as measured by the inability of cells to be rescued by NGF readdition, at the time of cytochrome c release. In the presence of caspase inhibitor, however, this commitment to death was extended beyond the point of cytochrome c release, but only up to the subsequent point of mitochondrial membrane potential loss. Caspase-9 deficiency also arrested NGF-deprived sympathetic neurons after release of cytochrome c, and permitted these neurons to be rescued with NGF readdition. Commitment to death in the NGF-deprived, caspase- 9-deficient sympathetic neurons was also coincident with the loss of mitochondrial membrane potential. Thus, caspase inhibition extended commitment to death in trophic factor-deprived sympathetic neurons and allowed recovery of neurons arrested after the loss of cytochrome c, but not beyond the subsequent loss of mitochondrial membrane potential.     

p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release

The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 death signals lead to caspase activation; however, the mechanism of caspase activation by p53 still is unclear. Expressing wild type p53 by means of an adenoviral expression vector, we were able to induce apoptotic cell death, as characterized by morphological changes, phosphatidylserine externalization, and internucleosomal DNA fragmentation, in p53(null) Saos-2 cells. This cell death was accompanied by caspase activation as well as by cleavage of caspase substrates and was preceded by mitochondrial cytochrome c release. The addition of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transduction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcysteine, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduced with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of zVAD-fmk. When Bax was immunodepleted from the cytosolic extracts of p53-expressing cells before incubation with isolated mitochondria, the in vitro cytochrome c release was abolished. Thus, we could demonstrate in cells and in vitro that p53 activates the apoptotic machinery through induction of the release of cytochrome c from the mitochondrial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bax for this cytochrome c-releasing activity of p53 in Saos-2 cells.     

Mitochondrial damage as death inducer in heart-derived H9c2 cells: more than one way for an early demise

The release of cytochrome c from mitochondria induced by 10 μM thapsigargin was linked to rapid loss of the mitochondrial membrane potential whereas that induced by 50 nM staurosporine was mediated by Bax activation and occurred in polarized mitochondria. Similar levels of cytochrome c were observed when induced by either thapsigargin or staurosporine indicating that the release magnitude was independent of the mechanism involved in membrane permeabilization. In any case caspase 3 activation was subsequent to cytochrome c release. Mitochondrial dysfunction and release of cytochrome c occurred earlier when induced by thapsigargin even though morphological alteration of the cell and chromatin condensation were developed earlier in the presence of staurosporine. In addition, a general and irreversible caspase inhibitor did not protect against chromatin condensation induced by staurosporine. It is also shown that earlier mitochondrial damage does not always correlate with earlier cell demise. This can be attributed to the existence of alternative caspase-independent cell death programmes.     

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.     

Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process

During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.     

Retroactive pathway involving mitochondria in electroloaded cytochrome c-induced apoptosis. Protective properties of Bcl-2 and Bcl-XL

Cytochrome c release is thought to play an important role in the initiation of apoptosis. The nature of the control exerted by Bcl-2 and Bcl-XL on such a pathway is not precisely known. We addressed this issue by square-wave pulse electroloading of exogenous cytochrome c into Jurkat cells. Three hours after cytochrome c loading into the cells, characteristic phenotypes of apoptosis were observed. However, a significant drop in the mitochondrial membrane potential (Deltapsim) was also observed, while cytochrome c was generally considered to act downstream from the mitochondria. Related to the Deltapsim drop, there was a release of proapoptotic proteins such as AIF and Smac from the mitochondria. This release, as well as NAD(P)H and cardiolipids oxidation, are linked to previous caspase activation. Cytochrome c-linked caspase activation also led to potassium efflux out of the cell. Overexpression of Bcl-2 and Bcl-XL or N-acetyl-DEVD-aldehyde treatment not only prevented the mitochondrial membrane potential decrease, but also protected cells from the apoptosis directly induced by cytochrome c electroloading. Bcl-2 and Bcl-XL protection is based on the inhibition of the caspase-dependent retroactive pathway affecting the mitochondrial compartment.     

A comparison of three flow cytometry methods for evaluating mitochondrial damage during staurosporine-induced apoptosis in Jurkat cells.

Measuring cytochrome c release during apoptosis provides valuable information about the nature and extent of apoptosis. Several years ago a flow cytometric method (based on selective permeabilization of the plasma membrane with digitonin) was developed that has advantages over other techniques. These experiments describe a comprehensive evaluation of that method. Apoptosis was triggered in Jurkat cells with staurosporine and then flow cytometry was used to measure three aspects of mitochondrial damage: (1) cytochrome c release (with the digitonin assay and a commercially available kit based on the same principle), using a DNA-binding dye to define cell cycle stage; (2) loss of mitochondrial cardiolipin, assessed by a decrease in 10 N-nonyl acridine orange (NAO) binding; and (3) loss of mitochondrial membrane potential, assessed by a decrease in tetramethylrhodamineethylester (TMRE) binding. The results from these three assays were compared with an antibody-based assay for cleaved caspase 3. The digitonin assay and the commercially available kit gave comparable results, showing that staurosporine caused cytochrome c release in all phases of the cell cycle and clearly defining those cells that had lost DNA due to internucleosomal DNA fragmentation. The pattern of fluorescence demonstrated that the mitochondrial apoptotic pathway was either the sole or the predominant pathway to be activated and that cytochrome c release in an individual cell was all-or-nothing. However, comparison with the other assays showed that the cytochrome c release assay underestimated the true extent of apoptosis. This was caused by the selective loss of some digitonin-treated apoptotic cells. The flow cytometry assay for cytochrome c release provides valuable information but it underestimates the percentage of apoptotic cells.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.     

Activation of caspases and cleavage of Bid are required for tyrosine and phenylalanine deficiency-induced apoptosis of human A375 melanoma cells

Deprivation of tyrosine (Tyr) and phenylalanine (Phe) inhibits growth and induces programmed cell death (apoptosis) of human A375 melanoma cells. Herein, we found that activation of caspases and release of mitochondrial cytochrome c are required for this process. Culturing A375 cells in Tyr/Phe-free medium, containing 10% dialyzed fetal bovine serum, results in activation of caspase-3-like activity. This is accompanied by decreased cell viability and increased apoptosis. Tyr/Phe deprivation also stimulates proteolytic cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP). Western blot analysis showed that caspases 3, 7, 8, and 9 are activated by deprivation of Tyr/Phe. Tyr/Phe deprivation decreases mitochondrial membrane potential, induces cleavage of Bid, increases translocation of Bax from the cytosol to mitochondria, and results in release of cytochrome c from the mitochondria to the cytosol. Apoptosis due to Tyr/Phe deprivation is almost completely inhibited by the broad-spectrum cell-permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z.VAD.fmk). This inhibitor suppresses the cleavage of Bid, the release of cytochrome c from the mitochondria to the cytosol, and the cleavage of PARP. Decylubiquinone, a mitochondrial permeability transition pore inhibitor, does not suppress the activation of caspase 8 but suppresses release of cytochrome c, activation of caspase 9, and induction of apoptosis. These results indicate that activation of caspases, cleavage of Bid, and mitochondrial release of cytochrome c are required for apoptosis induced by Tyr/Phe deprivation.     

Caspase-dependent cytosolic release of cytochrome c and membrane translocation of Bax in p53-induced apoptosis

Activation of p53 induces apoptosis in various cell types. However, the mechanism by which p53 induces apoptosis is still unclear. We reported previously that the activation of a temperature-sensitive mutant p53 (p53(138Val)) induced activation of caspase 3 and apoptosis in Jurkat cells. To elucidate the pathway linking p53 and downstream caspases, we examined the activation of caspases 8 and 9 in apoptotic cells. The results showed that both caspases were activated during apoptosis as judged by the appearance of cleavage products from procaspases and the caspase activities to cleave specific fluorogenic substrates. The significant inhibition of apoptosis by a tetrapeptide inhibitor of caspase 8 and caspase 9 suggested that both caspases are required for apoptosis induction. In addition, the membrane translocation of Bax and cytosolic release of cytochrome c, but not loss of mitochondrial membrane potential, were detected at an early stage of apoptosis. Moreover, Bax translocation, cytochrome c release, and caspase 9 activation were blocked by the broad-spectrum caspase inhibitor, Z-VAD-fmk and the caspase 8-preferential inhibitor, Ac-IETD-CHO, suggesting that the mitochondria might participate in apoptosis by amplifying the upstream death signals. In conclusion, our results indicated that activation of caspase 8 or other caspase(s) by p53 triggered the membrane translocation of Bax and cytosolic release of cytochrome c, which might amplify the apoptotic signal by activating caspase 9 and its downstream caspases.     

Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition (IC50) of MCF-7 cells at 26.4% 0.7% M over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with 100 microM acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/ JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.     

The PKC delta inhibitor, rottlerin, induces apoptosis of haematopoietic cell lines through mitochondrial membrane depolarization and caspases' cascade

Rottlerin is a widely selective protein kinase C delta (PKCdelta) inhibitor isolated from Mallotus philippinensis. It shown to be effective against several human tumor cell lines and in potentiating chemotherapy-induced cytotoxcicity. Using the trypan blue exclusion assay, we demonstrated that rottlerin reduced the viability in a dose- and time-dependent manner of human leukemia HL60 cells, human acute T cell leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. Rottlerin caused apoptosis and the apaptotic processing was inhibited by a caspase inhibitor, z-VAD-fmk, in these haematopoietic cells. The apoptosis-inducing activities were determined by nuclear condensation, sub-G1 appearance, DNA fragmentation, loss of mitochondrial membrane potential (Deltapsim), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Expression of PKCdelta and Bcl-2 protein inhibited Deltapsim change and repressed cell death. These studies suggest that the cytotoxic effects of rottlerin through inhibition of PKCdelta cause mitochondrial dysfunction, cytochrome c release from mitochondria into cytoplasm and the activation of caspases' cascade.     

DPI induces mitochondrial superoxide-mediated apoptosis

The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.     

Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.     

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.