Caspases: their intracellular localization and translocation during apoptosis.

The activation of the caspase family of proteases has been detected in numerous cell systems and appears to function as a common pathway through which apoptotic mechanisms may operate. Caspases are synthesized as precursors (pro-caspases) and are converted into mature enzymes by apoptotic signals. The effects of caspases in apoptosis are accomplished by the cleavage of numerous proteins located in different intracellular compartments. In the present study we have addressed the question of the subcellular localization of different pro- and active caspases as well as several other proteins, such as Apaf-1, calpain and DFF, which also play important roles in the apoptotic process. We found that at least three pro-caspases (pro-caspases-2, -3 and -9) were present in both the mitochondrial and cytosolic fractions of untreated Jurkat T lymphocytes. Only pro-caspase-2 was found in the nuclear fraction. Pro-caspases-7 and -8 were found only in the cytosolic fraction. In apoptotic cells, caspases-3, -8 and -9 were present in the cytosolic fraction, whereas caspases-3 and -9 were also found in the mitochondrial fraction and caspase-7 in the microsomal fraction. Caspases-2 and -3 were present in the nuclear fraction. The selective localization of pro-caspases in different subcellular compartments may play an important, but yet unknown, role in their activation. The translocation of active caspases to other subcellular compartments appears to be critical for the development of the apoptotic process.     

Trail-induced apoptosis in Type I leukemic cells is not enhanced by overexpression of bax

We have previously shown that Bax translocation was crucial in TNFalpha or etoposide-induced apoptosis. Overexpression of Bax sensitized chronic myeloid leukemic K562 cells to etoposide-induced apoptosis. Treatment with TNF-related apoptosis-inducing ligand (TRAIL) induces a loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondria, activation of caspases-8, -9, and -3, and cleavage of Bid in the K562 cell line. Bax failed to sensitize K562 cells to TRAIL-induced apoptosis. TRAIL did not induce Bax expression and/or translocation from cytosol to mitochondria in the K562 cell line. However, 100 microM Z-VAD.fmk, a pan caspase inhibitor, completely blocked TRAIL-initiated mitochondrial alterations and cleavages of caspases and Bid. We propose that TRAIL-induced apoptosis in K562 cells is via Type I apoptotic signal pathway. Bax translocation is not essential for TRAIL-induced cytochrome c release and DeltaPsim collapse in the Type I cells.     

Resveratrol induces mitochondria-mediated AIF and to a lesser extent caspase-9-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

Resveratrol (RV), a natural plant polyphenol widely present in foods such as grapes, wine, and peanuts, has an ability to inhibit various stages of carcinogenesis in vitro and in vivo. In this report, we explored the roles of intrinsic and extrinsic apoptotic pathways during RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. After exposure of cells to different concentrations of RV, we found that RV induced concentration-dependent apoptosis. Fluorometric substrates assay and western blotting (WB) analysis showed that caspase-8 was not activated, which was further verified by monitoring the cleavage of Bid to tBid using fluorescence resonance energy transfer (FRET) microscopy imaging inside single living cells, indicating that extrinsic apoptotic pathway was not involved in RV-induced apoptosis. In addition, inhibition of caspases-3 or -9 but not caspase-8 using the specific inhibitors of caspases modestly but significantly attenuated RV-induced apoptosis. Moreover, flow cytometry (FCM) analysis showed that RV treatment induced time-dependent loss of mitochondrial membrane potential (?ψ(m)), in combination with the activation of caspases-3 and -9; we therefore concluded that RV-induced apoptosis involved the intrinsic apoptotic pathway. It is noteworthy that RV treatment induced translocation of AIF from mitochondria to nucleus in a time dependent manner, and that knockdown of AIF remarkably attenuated RV-induced apoptosis. Collectively, our findings demonstrate that RV induces caspase-8-independent apoptosis via AIF and to a lesser extent caspase-9-dependent mitochondrial pathway in ASTC-a-1 cells.     

Mitochondria connects the antigen receptor to effector caspases during B cell receptor-induced apoptosis in normal human B cells.

We have previously reported that CD40 stimulation sensitizes human memory B cells to undergo apoptosis upon subsequent B cell receptor (BCR) ligation. We have proposed that activation stimuli connect the BCR to an apoptotic pathway in mature B cells and that BCR-induced apoptosis of activated B cells could serve a similar function as activation-induced cell death in the mature T cell compartment. Although it has been reported that caspases are activated during this process, the early molecular events that link the Ag receptor to these apoptosis effectors are largely unknown. In this study, we report that acquisition of susceptibility to BCR-induced apoptosis requires entry of memory B cells into the S phase of the cell cycle. We also show that transduction of the death signal via the BCR sequentially proceeds through a caspase-independent and a caspase-dependent phase, which take place upstream and downstream of the mitochondria, respectively. Furthermore, our data indicate that the BCR-induced alterations of the mitochondrial functions are involved in activation of the caspase cascade. We have found both caspases-3 and -9, but not caspase-8, to be involved in the BCR apoptotic pathway, thus supporting the notion that initiation of the caspase cascade could be under the control of the caspase-9/Apaf-1/cytochrome c multimolecular complex. Altogether, our findings establish the mitochondria as the connection point through which the Ag receptor can trigger the executioners of apoptotic cell death in mature B lymphocytes.     

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.     

Caspase-8 and Apaf-1-independent caspase-9 activation in Sendai virus-infected cells

Apoptotic cell death is of central importance in the pathogenesis of viral infections. Activation of a cascade of cysteine proteases, i.e. caspases, plays a key role in the effector phase of virus-induced apoptosis. However, little is known about pathways leading to the activation of initiator caspases in virus-infected host cells. Recently, we have shown that Sendai virus (SeV) infection triggers apoptotic cell death by activation of the effector caspase-3 and initiator caspase-8. We now investigated mechanisms leading to the activation of another initiator caspase, caspase-9. Unexpectedly we found that caspase-9 cleavage is not dependent on the presence of active caspases-3 or -8. Furthermore, the presence of caspase-9 in mouse embryonic fibroblast (MEF) cells was a prerequisite for Sendai virus-induced apoptotic cell death. Caspase-9 activation occurred without the release of cytochrome c from mitochondria and was not dependent on the presence of Apaf-1 or reactive oxygen intermediates. Our results therefore suggest an alternative mechanism for caspase-9 activation in virally infected cells beside the well characterized pathways via death receptors or mitochondrial cytochrome c release.     

Activation of the intrinsic and extrinsic pathways in high pressure-induced apoptosis of murine erythroleukemia cells

We previously demonstrated that caspase-3, an executioner of apoptosis, is activated in the pressure-induced apoptosis of murine erythroleukemia (MEL) cells (at 100 MPa). Here, we examined the pathway of caspase-3 activation using peptide substrates and caspase inhibitors. Using the substrates of caspases-8 and -9, it was found that both are activated in cells under high pressure. The production of nuclei with sub-G1 DNA content in 100 MPa-treated MEL cells was suppressed by inhibitors of caspases-8 and -9, and pan-caspase. In 100 MPa-treated cells, pan-caspase inhibitor partially prevented the cytochrome c release from the mitochondria and the breakdown of mitochondrial membrane potential. These results suggest that the intrinsic and extrinsic pathways are activated in apoptotic signaling during the high pressure-induced death of MEL cells.     

Delineation of the caspase-9 signaling cascade

In the intrinsic apoptosis pathway, mitochondrial disruption leads to the release of multiple apoptosis signaling molecules, triggering both caspase-dependent and -independent cell death. The release of cytochrome c induces the formation of the apoptosome, resulting in caspase-9 activation. Multiple caspases are activated downstream of caspase-9, however, the precise order of caspase activation downstream of caspase-9 in intact cells has not been completely resolved. To characterize the caspase-9 signaling cascade in intact cells, we employed chemically induced dimerization to activate caspase-9 specifically. Dimerization of caspase-9 led to rapid activation of effector caspases, including caspases-3, -6 and -7, as well as initiator caspases, including caspases-2, -8 and -10, in H9 and Jurkat cells. Knockdown of caspase-3 suppressed caspase-9-induced processing of the other caspases downstream of caspase-9. Silencing of caspase-6 partially inhibited caspase-9-mediated processing of caspases-2, -3 and -10, while silencing of caspase-7 partially inhibited caspase-9-induced processing of caspase-2, -3, -6 and -10. In contrast, deficiency in caspase-2, -8 or -10 did not significantly affect the caspase-9-induced caspase cascade. Our data provide novel insights into the ordering of a caspase signaling network downstream of caspase-9 in intact cells during apoptosis.     

Polypeptide from Chlamys farreri protects HaCaT cells from UVB-induced apoptosis

A natural polypeptide from marine Chlamys farreri (a kind of scallop) (PCF), has been recently been found to be an effective photoprotective agent against ultraviolet rays B (UVB)-induced mitochondria damage in normal human fibroblasts. To investigate whether PCF has the antiapoptotic effect on human keratinocytes, in the present study, we established an apoptotic model on HaCaT cell line by means of UVB radiance of 30 mJ/cm(2) and compared the effect of different PCF treatments on UVB-radiated cells. Flow cytometry analyses showed that PCF treatment before UVB-irradiation inhibited UVB-induced apoptosis, the loss of mitochondrial membrane potential (Deltapsim) and the increase of free Ca(2+) level in HaCaT cells. In parallel with these results, UVB-irradiation enhanced activities of caspases-3, 8, 9, while this enhancement was inhibited by PCF treatment prior to irradiation. PCF added after irradiation neither reduced UVB-induced activities of the three caspases nor synergized the effect of pre-added PCF. Cellular ultrastructural features obtained from transmission electron microscopy further confirmed the antiapoptotic effect of PCF pre-treatment. It is concluded that the antiapoptotic effect of PCF is not therapeutic but prophylactic. Caspases-3, 8, 9, Deltapsim and calcium are involved in UVB-induced apoptosis, while prophylactic PCF inhibits apoptosis of UVB-irradiated HaCaT cells by blocking the caspases activities, the Deltapsim lost and the elevation of intracellular free Ca(2+) level.     

Granulysin delivered by cytotoxic cells damages endoplasmic reticulum and activates caspase-7 in target cells

Granulysin is a human cytolytic molecule present in cytotoxic granules with perforin and granzymes. Recombinant 9-kDa granulysin kills a variety of microbes, including bacteria, yeast, fungi, and parasites, and induces apoptosis in tumor cells by causing intracellular calcium overload, mitochondrial damage, and activation of downstream caspases. Reasoning that granulysin delivered by cytotoxic cells may work in concert with other molecules, we crossed granulysin transgenic (GNLY(+/-)) mice onto perforin (perf)- or granzyme B (gzmb)-deficient mice to examine granulysin-mediated killing in a more physiologic whole-cell system. Splenocytes from these animals were activated in vitro with IL-15 to generate cytolytic T cells and NK cells. Cytotoxic cells expressing granulysin require perforin, but not granzyme B, to cause apoptosis of targets. Whereas granzyme B induces mitochondrial damage and activates caspases-3 and -9 in targets, cytotoxic cell-delivered granulysin induces endoplasmic reticulum stress and activates caspase-7 with no effect on mitochondria or caspases-3 and -9. In addition, recombinant granulysin and cell-delivered granulysin activate distinct apoptotic pathways in target cells. These findings suggest that cytotoxic cells have evolved multiple nonredundant cell death pathways, enabling host defense to counteract escape mechanisms employed by pathogens or tumor cells.     

Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa

We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8, and -9 activity, intact mitochondrial membrane potential (Deltapsim), and DNA fragmentation in human spermatozoa. Eleven pools of cryopreserved and eight pools of fresh semen samples were examined. Mature and immature fractions were separated on a two-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and its binding to annexin V-labeled superparamagnetic microbeads (ANMB). Levels of activated caspases were assessed using fluorescein-labeled inhibitors of caspases (FLICA), Deltapsim using a lipophilic cationic dye, and DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Cryopreservation was significantly associated with activation of caspases-3, -8, and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA fragmentation. In mature sperm, caspase activation was only detected in the ANMB+ fraction, whereas in immature sperm, both ANMB+ and ANMB- fractions showed activated caspase levels. In ANMB+ immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB- immature sperm, apoptosis was induced by activation of caspase-9 following loss of intact Deltapsim. These results demonstrate that selection of annexin V-negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during the cryopreservation process.     

A novel lignan composition from Cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia Molt-4 and HL-60 cells.

AP9-cd, a standardized lignan composition from Cedrus deodara consisting of (-)-wikstromal, (-)-matairesinol, and dibenzyl butyrolactol, showed cytotoxicity in several human cancer cell lines reported earlier. An attempt was made in this study to investigate the mechanism of cell death in human leukemia Molt-4 and HL-60 cells. It inhibited Molt-4 cell proliferation with 48-h IC(50) of approximately 15 microg/ml, increased sub-G0 cell fraction with no mitotic block, produced apoptotic bodies and induced DNA ladder formation. Flow cytometric analysis of annexinV-FITC/PI-stained cells showed time-related increase in apoptosis and post-apoptotic necrosis. All these biological end-points indicated cell death by apoptosis. Further, initial events involved massive nitric oxide (NO) formation within 4 h with subsequent late appearance of peroxides in cells; measured by flow cytometry using specific fluorescent probes. Persistently high levels of NO and peroxide appeared to decrease mitochondrial membrane potential (Psi(mt)) which was recovered by cyclosporin A in Molt-4 cells. AP9-cd caused 2-fold activation of caspase-3 in Molt-4 and 5-fold activation in HL-60 cells. Also caspases-8 and -9 were activated in HL-60 cells. Ascorbate suppressed the enhanced caspases activities indicating a pro-oxidant effect of AP9-cd. Further, caspase-3 activation correlated with NO generation that was partially impaired by nitric oxide synthase (NOS) inhibitors and ascorbate suggesting a role of pro-oxidant species in caspase-3 activation. AP9-cd produced no cytotoxicity in primary rat hepatocyte culture at the concentrations used. The studies indicated that AP9-cd mediated early NO formation leads to caspases activation, peroxide generation, and mitochondrial depolarization which may be responsible for mitochondrial-dependent and -independent apoptotic pathways involved in the killing of leukemia cells by AP9-cd.     

Mitochondria from TRAIL-resistant prostate cancer cells are capable of responding to apoptotic stimuli

TNFalpha-related apoptosis inducing ligand (TRAIL) has been shown to induce apoptosis in prostate cancer cells. However, some prostate cancer cells, such as LNCaP are resistant to TRAIL. In addition to the involvement of several pathways in the TRAIL-resistance of LNCaP, it has been shown that mitochondrial response to TRIAL is low in these cells. Therefore, in this study, using in vitro cell free and reconstitution models, we have demonstrated that mitochondria from these cells are capable of responding to apoptotic stimuli. Furthermore, experiments to determine the influence of cytochrome c on apoptotic response noted that incubation of cytosol with exogenous cytochrome c induced truncation of Bid. We have demonstrated that truncation of Bid by exogenous cytochrome c is mediated through the activation of caspases-9 and -3. Incubation of cytosol with recombinant caspases-9 and -3 in the absence or presence of inhibitors showed that activation of caspase-9, leading to the activation of caspase-3 was necessary for the truncation of Bid. Published results indicate that in apoptotic cells cytochrome c is released from the mitochondria in two installments, an early small amount and a late larger amount. Our results suggest that the initial release of cytochrome generates tBid that is capable of translocation into the mitochondria causing further release of cytochrome c. Thus, in addition to providing functional explanation for the biphasic release of cytochrome c from mitochondria, we demonstrate the presence of a feedback amplification of mitochondrial apoptotic signal.     

Ordering the Cytochrome c–initiated Caspase Cascade: Hierarchical Activation of Caspases-2, -3, -6, -7, -8, and -10 in a Caspase-9–dependent Manner

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1–mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c–inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.     

Caspase-dependent cytochrome c release and cell death in chick cardiomyocytes after simulated ischemia-reperfusion

We recently demonstrated that reperfusion rapidly induces the mitochondrial pathway of apoptosis in chick cardiomyocytes after 1 h of simulated ischemia. Here we tested whether ischemia-reperfusion (I/R)-induced apoptosis could be initiated by caspase-dependent cytochrome c release in this model of cardiomyocyte injury. Fluorometric assays of caspase activity showed little, if any, activation of caspases above baseline levels induced by 1 h of ischemia alone. However, these assays revealed rapid activation of caspase-2, yielding a 2.95 +/- 0.52-fold increase (over ischemia only) within the 1st h of reperfusion, whereas activities of caspases-3, -8, and -9 increased only slightly from their baseline levels. The rapid and prominent activation of caspase-2 suggested that it could be an important initiator caspase in this model, and using specific caspase inhibitors given only at the point of reperfusion, we tested this hypothesis. The caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-CH(2)F was the only caspase inhibitor that significantly inhibited cytochrome c release from mitochondria. This inhibitor also completely blocked activation of caspases-3, -8, and -9. The caspase-3/7 inhibitor transiently and only partially blocked caspase-2 activity and was less effective in blocking the activities of caspases-8 and -9. The caspase-8 inhibitor failed to significantly block caspase-2 or -3, and the caspase-9 inhibitor blocked only caspase-9. Furthermore, the caspase-2 inhibitor protected against I/R-induced cell death, but the caspase-8 inhibitor failed to do so. These data suggest that active caspase-2 initiates cytochrome c release after reperfusion and that it is critical for the I/R-induced apoptosis in this model.     

Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells

Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.     

Caspase-independent photoreceptor apoptosis in vivo and differential expression of apoptotic protease activating factor-1 and caspase-3 during retinal development

Apoptosis is the mode of photoreceptor cell death in many retinal dystrophies. Exposure of Balb/c mice to excessive levels of light induces photoreceptor apoptosis and represents an animal model for the study of retinal degenerations. Caspases have emerged as central regulators of apoptosis, executing this tightly controlled death pathway in many cells. Previously we have reported that light-induced photoreceptor apoptosis occurs independently of one the key executioners of apoptosis, caspase-3. This present study extends these results reporting on the lack of activation of other caspases in this model including caspases-8, -9, -7, and -1. Furthermore, photoreceptor apoptosis cannot be inhibited with the broad range caspase inhibitor zVAD-fmk indicating that light-induced retinal degeneration is caspase-independent. We demonstrate that cytochrome c does not translocate from mitochondria to the cytosol during photoreceptor apoptosis. We also show that during retinal development apoptotic protease activating factor (Apaf-1) protein levels are markedly decreased and this is associated with the inability to activate the mitochondrial caspase cascade in the mature retina. In addition, there is also a significant reduction in expression of caspases-3 and -9 during retinal maturation and these levels do not increase following light exposure. Finally, we show that the calcium-dependent proteases calpains are active during light-induced retinal degeneration and establish that the calcium channel blocker D-cis-diltiazem completely inhibits photoreceptor apoptosis.     

Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide-induced apoptosis

Recently, caspase-2 was shown to act upstream of mitochondria in stress-induced apoptosis. Activation of caspase-8, a key event in death receptor-mediated apoptosis, also has been demonstrated in death receptor-independent apoptosis. The regulation of these initiator caspases, which trigger the mitochondrial apoptotic pathway, is unclear. Here we report a potential regulatory role of caspase-2 on caspase-8 during ceramide-induced apoptosis. Our results demonstrate the sequential events of initiator caspase-2 and caspase-8 activation, Bid cleavage and translocation, and mitochondrial damage followed by downstream caspase-9 and -3 activation and cell apoptosis after ceramide induction in T cell lines. The expression of truncated Bid (tBid) and the reduction in mitochondrial transmembrane potential were blocked by caspase-2 or caspase-8, but not caspase-3, knockdown using an RNA interference technique. Ceramide-induced caspase-8 activation, mitochondrial damage, and apoptosis were blocked in caspase-2 short interfering RNA-expressing cells. Therefore, caspase-2 acts upstream of caspase-8 during ceramide-induced mitochondrial apoptosis. Similarly, sequential caspase-2 and caspase-8 activation upstream of mitochondria was also observed in etoposide-induced apoptosis. These data suggest sequential initiator caspase-2 and caspase-8 activation in the mitochondrial apoptotic pathway induced by ceramide or etoposide.     

Apoptosis induced by dithiothreitol in HL-60 cells shows early activation of caspase 3 and is independent of mitochondria

Previous studies have shown that under certain conditions some thiol-containing compounds can cause apoptosis in a number of different cell lines. Herein, we investigated the apoptotic pathways in HL-60 cells triggered by dithiothreitol (DTT), used as a model thiol compound, and tested the hypothesis that thiols cause apoptosis via production of hydrogen peroxide (H2O2) during thiol oxidation. The results show that, unlike H2O2, DTT does not induce apoptosis via a mitochondrial pathway. This is demonstrated by the absence of early cytochrome c release from mitochondria into the cytosol, the lack of mitochondrial membrane depolarization at early times, and the minor role of caspase 9 in DTT-induced apoptosis. The first caspase activity detectable in DTT-treated cells is caspase 3, which is increased significantly 1 - 2 h after the start of DTT treatment. This was shown by following the cleavage of both a natural substrate, DFF-45/ICAD, and a synthetic fluorescent substrate, z-DEVD-AFC. Cleavage of substrates of caspases 2 and 8, known as initiator caspases, does not start until 3 - 4 h after DTT exposure, well after caspase 3 has become active and at a time when apoptosis is in late stages, as shown by the occurrence of DNA fragmentation to oligonucleosomal-sized pieces. Although oxidizing DTT can produce H2O2, data presented here indicate that DTT-induced apoptosis is not mediated by production of H2O2 and occurs via a novel pathway that involves activation of caspase 3 at early stages, prior to activation of the common 'initiator' caspases 2, 8 and 9.     

Caspase-3 mediated feedback activation of apical caspases in doxorubicin and TNF-α induced apoptosis

Aberrant apoptosis has been associated with the development and therapeutic resistance of cancer. Recent studies suggest that caspase deficiency/downregulation is frequently detected in different cancers. We have previously shown that caspase-3 reconstitution significantly sensitized MCF-7 cells to doxorubicin and etoposide. In contrast to the well established role of caspase-3 as an effector caspase, the focus of this study is to delineate caspase-3 induced feedback activation of the apical caspases-2, -8, -9 and -10A in doxorubicin and TNF-α induced apoptosis. Using cell-free systems we show that caspases-9 and 2 are the most sensitive, caspase-8 is less sensitive and caspase-10A is the least sensitive to caspase-3 mediated-cleavage. When apoptosis is induced by doxorubicin or TNF-α in an intact cell model, cleavage of caspases-8 and -9, but not caspase-2, was markedly enhanced by caspase-3. Caspase-3 mediated-feedback and activation of caspase-8 and -9 in MCF-7/C3 cells is further supported by an increase in the cleavage of caspase-8 and 9 substrates and cytochrome c release. These data indicate that, in addition to its function as an effector caspase, caspase-3 plays an important role in maximizing the activation of apical caspases and crosstalk between the two major apoptotic pathways. The significant impact of caspase-3 on both effector and apical caspases suggests that modulation of caspase-3 activity would be a useful approach to overcome drug resistance in clinical oncology. XiaoHe Yang: This work was supported in part by the Career Development Award DAMD17-99-1-9180 from Department of Defense to X.H.Y.