Poly (ADP-ribose) polymerase cleavage monitored in situ in apoptotic cells

During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.     

Inhibition of apoptosis and clonogenic survival of cells expressing crmA variants: optimal caspase substrates are not necessarily optimal inhibitors.

To study the role of various caspases during apoptosis, we have designed a series of caspase inhibitors based on the cowpox virus cytokine response modifier A (crmA) protein. Wild-type crmA inhibits caspases 1 and 8 and thereby protects cells from apoptosis triggered by ligation of CD95 or tumour necrosis factor (TNF) receptors, but it does not protect against death mediated by other caspases. By replacing the tetrapeptide pseudosubstrate region of crmA (LVAD) with tetrapeptides that are optimal substrates for the different families of caspases, or with the four residues from the cleavage site of the baculovirus protein p35 (DQMD), we have generated a family of caspase inhibitors that show altered ability to protect against cell death. Although DEVD is the optimal substrate for caspase 3, crmA DEVD was degraded rapidly and was a weaker inhibitor than crmA DQMD, which was not degraded. Unlike wild-type crmA and crmA DEVD, crmA DQMD was able to inhibit apoptosis caused by direct activation of caspase 3 and protected lymphoid cells from death induced by radiation and dexamethasone. Significantly, the protected cells were capable of sustained growth.     

Notch1 antiapoptotic activity is abrogated by caspase cleavage in dying T lymphocytes

Excessive signaling via the Notch1 receptor inhibits apoptosis in T lymphocytes. Since several antiapoptotic proteins are cleaved by caspases during cell death, we investigated whether Notch1 was a caspase substrate. Results demonstrate that the intracellular domain of Notch1 (NICD) is cleaved into six fragments during apoptosis in Jurkat cells or peripheral T lymphocytes. Notch1 cleavage is prevented by the caspase inhibitors DEVD-fmk and VEID-fmk or by Bcl-2 expression. Caspase-3 and caspase-6 cleave the NICD into six fragments using sites located within the NF-kappaB binding domain, the ankyrin repeats and the transactivation domain. Notch1 cleavage correlates with the loss of HES-1 expression in apoptotic T cells. Notch1 fragments cannot inhibit activation-induced cell death in a T-cell hybridoma, confirming the abrogation of Notch1 antiapoptotic activity by caspases. The ability of the NICD but not the fragments to antagonize Nur77 activity supports a role for this factor in Notch1 antiapoptotic function.     

p53-Independent caspase-mediated apoptosis in human leukaemic cells is induced by a DNA minor groove binder with antineoplastic activity

Treatment of HL60 and Jurkat leukaemic cell lines, both not expressing p53, with the new non-covalent DNA minor groove binder -bromoacryloyl-distamycin (PNU 151807), induces apoptosis as shown either morphologically or by DNA laddering formation. We evaluated the p53-independent mechanisms of activation of apoptosis in these cell systems, by determining the levels of different caspases at different times after treatment with PNU 151807. Through Western blotting analysis we could measure the cleavage of the 110 Kd form of PARP in both HL60 and Jurkat cells and correspondingly the activation of CPP32-caspase 3. The levels of caspase-1 did not change at any time after drug treatment. By using the tetrapeptidic sequence recognized by caspase-3 (DEVD-AMC) or by caspase-1 (YVAD-AMC) linked to fluorogenic substrate, we also demonstrated that only the DEVD sequence was recognized and cleaved after drug treatment, while no significant changes were found for YVAD peptides. PNU 151807-induced DNA fragmentation and DEVD-cleavage were both inhibited by concomitant treatment with the specific inhibitor DEVD-CHO, but not by YVAD-CHO, clearly demonstrating the direct activation of caspase-3-like caspases in the induction of programmed cell death in these cell lines after minor groove binder exposure.     

Type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.     

Caspases induce cytochrome c release from mitochondria by activating cytosolic factors.

We investigated the ability of caspases (cysteine proteases with aspartic acid specificity) to induce cytochrome c release from mitochondria. When Jurkat cells were induced to undergo apoptosis by Fas receptor ligation, cytochrome c was released from mitochondria, an event that was prevented by the caspase inhibitor, zVAD-fmk (zVal-Ala-Asp-CH2F). Purified caspase-8 triggered rapid cytochrome c release from isolated mitochondria in vitro. The effect was indirect, as the presence of cytosol was required, suggesting that caspase-8 cleaves and activates a cytosolic substrate, which in turn is able to induce cytochrome c release from mitochondria. The cytochrome c releasing activity was not blocked by caspase inhibition, but was antagonized by Bcl-2 or Bcl-xL. Caspase-8 and caspase-3 cleaved Bid, a proapoptotic Bcl-2 family member, which gains cytochrome c releasing activity in response to caspase cleavage. However, caspase-6 and caspase-7 did not cleave Bid, although they initiated cytochrome c release from mitochondria in the presence of cytosol. Thus, effector caspases may cleave and activate another cytosolic substrate (other than Bid), which then promotes cytochrome c release from mitochondria. Mitochondria significantly amplified the caspase-8 initiated DEVD-specific cleavage activity. Our data suggest that cytochrome c release, initiated by the action of caspases on a cytosolic substrates, may act to amplify a caspase cascade during apoptosis.     

Caspase-3 mediated cleavage of HsRad51 at an unconventional site.

The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41-kDa full-length protein into a 33-kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S-100 extract. In this study, site-directed mutagenesis was used for mapping of the cleavage site to AQVD274 downward arrow G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine-treated apoptotic MCF-7 cells, which lack caspase-3, indicates that caspase-3 is essential for HsRad51 cleavage in vivo. Cleavage into the 33-kDa fragment was generated by recombinant caspase-3 and -7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 downward arrow G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33-kDa product by recombinant caspase-3, whereas caspase-7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 downward arrow G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 downward arrow N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 downward arrow G and DVLD187 downward arrow N cleavage products of HsRad51 appeared at equal concentrations of caspase-3. Moreover both fragments were generated by induction of apoptosis in MDA-MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo.     

Inositol 1,4,5-trisphosphate receptor type 1 is a substrate for caspase-3 and is cleaved during apoptosis in a caspase-3-dependent manner

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.     

Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways

Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.     

Requirement of Apaf-1 for mitochondrial events and the cleavage or activation of all procaspases during genotoxic stress-induced apoptosis

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.     

Lack of correlation between caspase activation and caspase activity assays in paclitaxel-treated MCF-7 breast cancer cells.

MCF-7 human breast cancer cells are widely utilized to study apoptotic processes. Recent studies demonstrated that these cells lack procaspase-3. In the present study, caspase activation and activity were examined in this cell line after treatment with the microtubule poison paclitaxel. When cells were harvested 72 h after the start of a 24-h treatment with 100 nm paclitaxel, 37 +/- 5% of the cells were nonadherent and displayed apoptotic morphological changes. Although mitochondrial cytochrome c release and caspase-9 cleavage were detectable by immunoblotting, assays of cytosol and nuclei prepared from the apoptotic cells failed to demonstrate the presence of activity that cleaved the synthetic caspase substrates LEHD-7-amino-4-trifluoromethylcoumarin (LEHD-AFC), DEVD-AFC, and VEID-AFC. Likewise, the paclitaxel-treated MCF-7 cells failed to cleave a variety of caspase substrates, including lamin A, beta-catenin, gelsolin, protein kinase Cdelta, topoisomerase I, and procaspases-6, -8, and -10. Transfection of MCF-7 cells with wild type procaspase-3 partially restored cleavage of these polypeptides but did not result in detectable activities that could cleave the synthetic caspase substrates. Immunoblotting revealed that caspase-9, and -3, which were proteolytically cleaved in paclitaxel-treated MCF-7/caspase-3 cells, were sequestered in a salt-resistant sedimentable fraction rather than released to the cytosol. Immunofluorescence indicated large cytoplasmic aggregates containing cleaved caspase-3 in these apoptotic cells. These observations suggest that sequestration of caspases can occur in some model systems, causing tetrapeptide-based activity assays to underestimate the amount of caspase activation that has occurred in situ.     

Activation of intrinsic and extrinsic pathways in apoptotic signaling during UV-C-induced death of Jurkat cells: the role of caspase inhibition

We have examined UV irradiation-induced cell death in Jurkat cells and evaluated the relationships that exist between inhibition of caspase activity and the signaling mechanisms and pathways of apoptosis. Jurkat cells were irradiated with UV-C light, either with or without pretreatment with the pan-caspase inhibitor, z-VAD-fmk (ZVAD), or the more selective caspase inhibitors z-IETD-fmk (IETD), z-LEHD-fmk (LEHD), and z-DEVD-fmk (DEVD). Flow cytometry was used to examine alterations in viability, cell size, plasma membrane potential (PMP), mitochondrial membrane potential (DeltaPsi(mito)), intracellular Na(+) and K(+) concentrations, and DNA degradation. Processing of pro-caspases 3, 8, and 9 and the pro-apoptotic protein Bid was determined by Western blotting. UV-C irradiation of Jurkat cells resulted in characteristic apoptosis within 6 h after treatment and pretreatment of cells with ZVAD blocked these features. In contrast, pretreatment of the cells with the more selective caspase inhibitors under conditions that effectively blocked DNA degradation and inhibited caspase 3 and 8 processing as well as Bid cleavage had little protective effect on the other apoptotic characteristics examined. Thus, both intrinsic and extrinsic pathways are activated during UV-induced apoptosis in Jurkat cells and this redundancy appears to assure cell death during selective caspase inhibition.     

Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

Activation of poly(ADP-ribose) polymerase (PARP) by DNA breaks catalyzes poly(ADP-ribosyl)ation and results in depletion of NAD+ and ATP, which is thought to induce necrosis. Proteolytic cleavage of PARP by caspases is a hallmark of apoptosis. To investigate whether PARP cleavage plays a role in apoptosis and in the decision of cells to undergo apoptosis or necrosis, we introduced a point mutation into the cleavage site (DEVD) of PARP that renders the protein resistant to caspase cleavage in vitro and in vivo. Here, we show that after treatment with tumor necrosis factor alpha, fibroblasts expressing this caspase-resistant PARP exhibited an accelerated cell death. This enhanced cell death is attributable to the induction of necrosis and an increased apoptosis and was coupled with depletion of NAD+ and ATP that occurred only in cells expressing caspase-resistant PARP. The PARP inhibitor 3-aminobenzamide prevented the NAD+ drop and concomitantly inhibited necrosis and the elevated apoptosis. These data indicate that this accelerated cell death is due to NAD+ depletion, a mechanism known to kill various cell types, caused by activation of uncleaved PARP after DNA fragmentation. The present study demonstrates that PARP cleavage prevents induction of necrosis during apoptosis and ensures appropriate execution of caspase-mediated programmed cell death.     

Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis

Apoptosis is an important process involved in diverse developmental pathways, homeostasis, and response to therapy for a variety of diseases. Thus, noninvasive methods to study regulation and to monitor cell death in cells and whole animals are desired. To specifically detect apoptosis in vivo, a novel cell-permeable activatable caspase substrate, TcapQ647, was synthesized and Km, kcat, and Ki values were biochemically characterized. Specific cleavage of TcapQ647 by effector caspases was demonstrated using a panel of purified recombinant enzyme assays. Of note, caspase 3 was shown to cleave TcapQ647 with a kcat 7-fold greater than caspase 7 and 16-fold greater than caspase 6. No evidence of TcapQ647 cleavage by initiator caspases was observed. In KB 3-1 or Jurkat cells treated with cytotoxic agents or C6-ceramide, TcapQ647 detected apoptosis in individual- and population-based fluorescent cell assays in an effector caspase inhibitor-specific manner. Further, only background fluorescence was observed in cells incubated with dTcapQ647, a noncleavable all d-amino acid control peptide. Finally, in vivo experiments demonstrated the utility of TcapQ647 to detect parasite-induced apoptosis in human colon xenograft and liver abscess mouse models. Thus, TcapQ647 represents a sensitive, effector caspase-specific far-red "smart" probe to noninvasively monitor apoptosis in vivo.     

Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7.

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks. During almost all forms of apoptosis, PARP is cleaved by caspases, suggesting the crucial role of its inactivation. A few studies have also reported a stimulation of PARP during apoptosis. However, the role of PARP stimulation and cleavage during this cell death process remains poorly understood. Here, we measured the stimulation of endogenous poly(ADP-ribose) synthesis during VP-16-induced apoptosis in HL60 cells and found that PARP was cleaved by caspases at the time of its poly(ADP-ribosyl)ation. In vitro experiments showed that PARP cleavage by caspase-7, but not by caspase-3, was stimulated by its automodification by long and branched poly(ADP-ribose). Consistently, caspase-7 exhibited an affinity for poly(ADP-ribose), whereas caspase-3 did not. In addition, caspase-7 was activated and accumulated in the nucleus of HL60 cells in response to the VP-16 treatment. Furthermore, caspase-7 activation was concommitant with PARP cleavage in the caspase-3-deficient cell line MCF-7 in response to staurosporine treatment. These results strongly suggest that, in vivo, it is caspase-7 that is responsible for PARP cleavage and that poly(ADP-ribosyl)ation of PARP accelerates its proteolysis. Cleavage of the active form of caspase substrates could be a general feature of the apoptotic process, ensuring the rapid inactivation of stress signaling proteins.     

Inhibition of caspases inhibits the release of apoptotic bodies: Bcl-2 inhibits the initiation of formation of apoptotic bodies in chemotherapeutic agent-induced apoptosis

During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as "apoptotic bodies." We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.     

Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.     

Activation of caspase 3 in HL-60 cells exposed to hydrogen peroxide

Recent studies have suggested that hydrogen peroxide (H2O2), a reactive compound formed endogenously in the breakdown of superoxide, may mediate the induction of apoptosis in various cell types in response to external stimuli. However, the role of H2O2 in the apoptotic pathway has not been clearly established. The purpose of this study was to determine if H2O2 treatment could induce apoptosis through the activation of caspases. Doses of H2O2 ranging from 10 microM to 100 microM, when added to HL-60 cells, resulted in the cleavage of poly(ADP-ribose) polymerase (PARP) from its native 113 Kd form to a processed 89 Kd fragment, indicative of cells undergoing apoptosis. PARP was predominantly in the fragmented form when doses of 20 microM and greater were used. A time course study of changes in PARP processing in H2O2-treated cells revealed that 10 and 50 microM H2O2 required 6 and 3 h, respectively, to specifically degrade PARP, suggesting that the H2O2-induced PARP cleavage is both time and concentration dependent. Since PARP is cleaved by CPP32 (caspase-3), we next determined if H2O2 was capable of effecting changes in CPP32 activity. The caspase activity was assayed using a colorimetric substrate, DEVD-pNa. Results of these experiments showed that H2O2 increased caspase activity at 3 h, corresponding to the time of appearance of fragmented PARP. Also, CPP32 activity and PARP processing were both significantly suppressed by caspase-3 inhibitors. Taken together, these results suggest that H2O2 mediates specific cleavage of PARP and possibly apoptosis by activating caspase 3.     

The adenomatous polyposis coli protein and retinoblastoma protein are cleaved early in apoptosis and are potential substrates for caspases

Apoptosis in human monocytic THP.1 tumour cells, induced by diverse stimuli, was accompanied by proteolytic cleavage of the adenomatous polyposis coli gene product (APC) and by sequential cleavage of the retinoblastoma susceptibility gene product (Rb). Cleavage of poly(ADP-ribose) polymerase (PARP), APC and the initial cleavage of Rb at the carboxy terminal region all occurred at a similar time, early in the apoptotic process. Subsequently, Rb underwent a secondary cleavage to 43 kDa and 30 kDa protein fragments. Two caspase inhibitors, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.FMK) and acetyl-Tyr-Val-Ala-Asp chloromethyl ketone (YVAD.CMK), had markedly different effects on the induction of apoptosis. Z-VAD.FMK inhibited the primary and secondary cleavage of Rb, cleavage of APC and PARP, and apoptosis assessed by flow cytometry. In marked contrast, YVAD.CMK inhibited cleavage of APC and the secondary cleavage of Rb to the 43 kDa and 30 kDa protein fragments but did not inhibit the primary carboxy terminal cleavage of Rb, PARP proteolysis or apoptosis assessed by flow cytometry. These results suggest that different caspases are responsible for the cleavage of different substrates at different stages during the apoptotic process and that a caspase may either cleave APC directly or may be involved in the pathway leading to APC proteolysis. This is the first report suggesting that a cytoplasmic tumour suppressor gene (APC) may be cleaved by a caspase during apoptosis.     

Caspase sensitive gold nanoparticle for apoptosis imaging in live cells

We developed a new apoptosis imaging probe with gold nanoparticles (AuNPs). A near-infrared fluorescence dye was attached to AuNP surface through the bridge of peptide substrate (DEVD). The fluorescence was quenched in physiological conditions due to the quenching effect of AuNP, and the quenched fluorescence was recovered after the DEVD had been cleaved by caspase-3, the enzyme involved in apoptotic process. The adhesion of DEVD substrates on AuNP surface was accomplished by conjugation of the 3,4-dihydroxy phenylalanine (DOPA) groups which are adhesive to inorganic surface and rich in mussels. This surface modification with DEVD substrates by DOPA groups resulted in increased stability of AuNP in cytosol condition for hours. Moreover, the cleavage of substrate and the dequenching process are very fast, and the cells did not need to be fixed for imaging. Therefore, the real-time monitoring of caspase activity could be achieved in live cells, which enabled early detection of apoptosis compared to a conventional apoptosis kit such as Annexin V-FITC. Therefore, our apoptosis imaging has great potential as a simple, inexpensive, and efficient apoptosis imaging probe for biomedical applications.