Activation Of ICE-like Proteases Research Articles

Antiprion Prophylaxis by Gene Transfer of a Soluble Prion Antagonist

Prion diseases are untreatable neurodegenerative disorders characterized by accumulation of PrP(Sc), an aggregated isoform of the normal prion protein PrP(C). Here, we delivered the soluble prion antagonist PrP-Fc(2) to the brains of mice by lentiviral gene transfer. Although naïve mice developed scrapie at 175 +/- 5 days postintracerebral prion inoculation (dpi), gene transfer before inoculation delayed disease onset by 72 +/- 4 days. At 170 days postintracerebral prion inoculation, PrP(Sc) accumulation and prion infectivity in PrPFc-treated brains were reduced by 3.6 and 4.2 logs, respectively. When PrP-Fc(2) was delivered 30 days after prion inoculation, survival of the treated animals was extended by 25 days. We then used tissue-specific recombination to express PrP-Fc(2) in the entire central nervous system, in only astrocytes, or in only oligodendrocytes. Oligodendrocyte-restricted PrP-Fc(2) expression impaired PrP(Sc) deposition and delayed disease even though oligodendrocytes are completely resistant to prion infection, suggesting that PrP-Fc(2) affords protection via noncell autonomous mechanisms. These results suggest that somatic gene transfer of prion antagonists may be effective for postexposure prophylaxis of prion diseases.

Antitumor mechanism of antisense cantide targeting human telomerase reverse transcriptase.

To investigate the anti-tumor mechanism of antisense oligodeoxynucleotide cantide against hTERT. Tumor cells were cultured overnight and grown to 50-60 % confluence. HepG2 and SMMC-7721 were treated with cantide mixed with lipofectin, or lipofectin alone. After inducted for 6 h at 37 degrees, 10 % FCS in DMEM was replaced in each well. After the treatment repeated twice to three times in each concentration of cantide, hTERT mRNA and protein expression were measured by RT-PCR and Western blot analysis, respectively. Telomerase activity was determined by TRAP-ELISA assay. CPP32- and ICE-like activity was also investigated using CasPACE assay system at 48 h after cantide treatment, and apoptosis was evaluated using the DeadEnd assay at 24, 48 and 72 h after cantide treatment. Compared to the control cells, the cells treated with cantide showed a dose-dependent decrease in hTERT mRNA levels at 24 h and in protein levels at 48 h respectively. The telomerase activity was decreased as the concentration of cantide increased at 48 h. At the concentration of 800 nM, the telomerase activity in the treated HepG2 and SMMC-7721 cells was only 17.1 % (P<0.01) and 20.3 % (P<0.01) of that in untreated cells. The levels of CPP32-like protease activity in HepG2 and SMMC-7721 increased by 2.8- and 3.0-fold (P<0.05) at 48 h, and the levels of ICE-like protease activity also increased by 2.6- and 3.2-fold (P<0.05) respectively. The percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was 63 % and 52 % (P<0.01), respectively. By contrast, 8 % and 9 % of the cells were apoptosis after 72 h treatment with lipofectin alone. Cantide can decrease telomerase activity by inhibiting the expression of hTERT gene and has a rapid anti-tumor effect through inducing the Caspase-dependent apoptosis. The rapid inhibitory effect of cantide on tumor growth demonstrates its feasibility in cancer treatment.

Tributyltin triggers apoptosis in trout hepatocytes: the role of Ca 2+, protein kinase C and proteases

The purpose of the present study was to study the mechanisms involved in the induction of apoptosis and by tributyltin (TBT) in rainbow trout hepatocytes, and to examine the role of intracellular Ca 2+, protein kinase C (PKC) and proteases in the apoptotic process. The intracellular Ca 2+ chelator BAPTA-AM has a suppressive effect on TBT-mediated apoptosis. However, exposure to the ionophore A23187 is not sufficient to induce apoptosis in trout hepatocytes. The results obtained also show that TBT stimulates PKC γ and δ translocation from cytosol to the plasma membrane in trout hepatocytes after 30 min of exposure. However, PKC γ translocation is down-regulated after 90 min of treatment. The addition of protein kinase inhibitors (staurosporine and H-7) not only fails to inhibit apoptosis induced by TBT, but also leads to enhancement of DNA fragmentation. These inhibitors also afford a remarkable protection against the loss of plasma membrane integrity caused by TBT exposure. PMA, a direct activator of PKC, fails to stimulate DNA fragmentation. In addition, Z-VAD.FMK is an extremely potent inhibitor of TBT-induced apoptosis in trout hepatocytes, indicating that the activation of ICE-like proteases is a key event in this process. The cysteine protease inhibitor N-ethylmaleimide also prevented TBT-induced DNA fragmentation. Taken together, these data allow for the first time to suggest a mechanistic model of TBT-induced apoptosis. We propose that TBT could trigger apoptosis through a step involving Ca 2+ efflux from the endoplasmic reticulum or other intracellular pools and by mechanisms involving cysteine proteases, such as calpains, as well as the phosphorylation status of apoptotic proteins such as Bcl-2 homologues.

Fas induces cytoplasmic apoptotic responses and activation of the MKK7-JNK/SAPK and MKK6-p38 pathways independent of CPP32-like proteases.

IL-1beta converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1-like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.

Cytokine suppression of protease activation in wild-type p53-dependent and p53-independent apoptosis.

M1 myeloid leukemic cells overexpressing wild-type p53 undergo apoptosis. This apoptosis can be suppressed by some cytokines, protease inhibitors, and antioxidants. We now show that induction of apoptosis by overexpressing wild-type p53 is associated with activation of interleukin-1beta-converting enzyme (ICE)-like proteases, resulting in cleavage of poly(ADP- ribose) polymerase and the proenzyme of the ICE-like protease Nedd-2. Activation of these proteases and apoptosis were suppressed by the cytokine interleukin 6 or by a combination of the cytokine interferon gamma and the antioxidant butylated hydroxyanisole, and activation of poly(ADP-ribose) polymerase and apoptosis were suppressed by some protease inhibitors. In a clone of M1 cells that did not express p53, vincristine or doxorubicin induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by interleukin 6. In another myeloid leukemia (7-M12) doxorubicin also induced protease activation and apoptosis that were not suppressed by protease inhibitors, but were suppressed by granulocyte-macrophage colony-stimulating factor. The results indicate that (i) overexpression of wild-type p53 by itself or treatment with cytotoxic compounds in wild-type p53-expressing or p53-nonexpressing myeloid leukemic cells is associated with activation of ICE-like proteases; (ii) cytokines exert apoptosis-suppressing functions upstream of protease activation; (iii) the cytotoxic compounds induce additional pathways in apoptosis; and (iv) cytokines can also suppress these other components of the apoptotic machinery.

Apoptosis and metastasis: increased apoptosis resistance of metastatic cancer cells is associated with the profound deficiency of apoptosis execution mechanisms.

Programmed cell death, particularly adhesion-dependent regulation of cell survival and apoptosis, is recognized as one of the main homeostatic mechanisms designed to control cell positioning, eliminate misplaced cells and block metastatic dissemination. Recently we reported that highly metastatic cancer cells exhibit a higher resistance to the programmed cell death compared to their poorly metastatic counterparts (Cancer Lett., 101, 43-51, 1996). However, the molecular and genetic basis for the association of aggressive metastatic phenotype with resistance toward apoptosis remains to be elucidated. Here we extended our investigation on apoptosis and metastasis using a panel of nine murine and human cancer cell lines with different metastatic potential. We examined the relationship of the metastatic ability and the sensitivity to apoptosis as well as determined the status of two major apoptosis execution mechanisms (induction of nuclear Ca2+-dependent endonucleases and activation of ICE-like proteases) in cancer cells with distinct metastatic potential and different sensitivity to apoptosis. We found that high metastatic potential is strictly associated with the increased resistance to apoptosis, diminished level of nuclear Ca2+-dependent endonucleases, and significantly reduced activity of CPP32/Yama death protease. We concluded that high resistance to apoptosis of metastatic cancer cells is associated with and may depend upon the profound deficiency of major apoptosis execution mechanisms.

Intracellular acidification induces apoptosis by stimulating ICE-like protease activity.

ICE-like protease activation and DNA fragmentation are preceded by a decrease in intracellular pH (pHi) during apoptosis in the IL-3 dependent cell line BAF3. Acidification occurs after 7 hours in cells deprived of IL-3 and after 4 hours when cells are treated with etoposide, close to the time of detection of ICE-like protease activity. Increasing extracellular pH reduces ICE-like protease activation and DNA fragmentation. Bcl-2 over-expression both delays acidification and inhibits ICE-like protease activation. Generation of a rapid intracellular pH decrease, using the ionophore nigericin, induces ICE-like protease activation and apoptosis. ZVAD, a cell permeable inhibitor of ICE-like proteases, does not affect acidification but inhibits apoptosis induced by IL-3 removal or nigericin treatment. These data suggest that intracellular acidification triggers apoptosis by directly or indirectly activating ICE-like proteases.

The interleukin 1β-converting enzyme inhibitor CrmA prevents Apo1/Fas- but not glucocorticoid-induced poly(ADP-ribose) polymerase cleavage and apoptosis in lymphoblastic leukemia cells

Glucocorticoids (GC) induce programmed cell death (apoptosis) in immature lymphocytes and are an essential component in the therapy of acute lymphatic leukemia. The mechanism underlying GC-induced apoptosis particularly in leukemia cells is, however, not well understood. Most forms of apoptosis seem to employ a common final effector pathway characterized by specific proteolytic events mediated by interleukin 1β-converting enzyme (ICE) and/or other ICE-like cysteine proteases. These events may result in the morphologic changes characteristic of apoptosis. To determine whether a similar proteolytic pathway is activated during GC-induced leukemia cell apoptosis, we investigated poly(ADP-ribose) polymerase (PARP), a typical target of ICE-like proteases, during GC-induced apoptosis of the human acute T-cell leukemic cell line CEM-C7H2. Our studies showed proteolytic PARP cleavage suggestive of activation of ICE-like proteases that preceeded morphologic signs of apoptosis. We further established stably transfected CEM-C7H2 sublines expressing the cowpox virus protein CrmA that inhibits some, but not all, ICE-like proteases. GC-induced PARP cleavage and apoptosis were neither inhibited nor delayed in crmA-expressing cell lines. In contrast, crmA expression rendered the same lines resistant to Apo1/Fas-induced PARP cleavage and apoptosis. Thus, different proteases might be activated during the effector phases of GC- and Apo1/Fas-induced apoptosis in human leukemia cells.

ICE-protease inhibitors block murine liver injury and apoptosis caused by CD95 or by TNF-α

The two apoptosis receptors of mammalian cells, i.e. the 55 kDa TNF receptor (TNF-R1) and CD95 (Fas/APO1) are activated independently of each other, however, their signaling involves a variety of ICE-related proteases [1]. We used a cell-permeable inhibitor of ICE-like protease activity to examine in vivo whether post-receptor signaling of TNF and CD95 are fully independent processes. Mice pretreated with the inhibitor, Z-VAD-fluoromethylketone (FMK) were dose-dependently protected from liver injury caused by CD95 activation as determined by plasma alanine aminotransferase and also from hepatocyte apoptosis assessed by DNA fragmentation (ID 50=0.1 mg/kg). A dose of 10 mg/kg protected mice also from liver injury induced by TNF-α. Similar results were found when apoptosis was initiated via TNF-α or via CD95 in primary murine hepatocytes (IC 50=1.5 nM) or in various human cell lines. In addition to prevention, an arrest of cell death by Z-VAD-FMK was demonstrated in vivo and in vitro after stimulation of apoptosis receptors. These findings show in vitro and in vivo in mammals that CD95 and the TNF-α receptor share a distal proteolytic apoptosis signal.

Protease Involvement in Fodrin Cleavage and Phosphatidylserine Exposure in Apoptosis

A detailed kinetic analysis of three extranuclear end points of apoptosis, phosphatidylserine exposure, alpha-fodrin degradation, and plasma membrane blebbing, was performed and compared with nuclear fragmentation and the activation of the interleukin-1beta-converting enzyme (ICE)-like proteases in Jurkat T lymphocytes stimulated by anti-Fas monoclonal antibody (anti-Fas mAb) and in monocytic U937 cells stimulated by tumor necrosis factor (TNF) and cycloheximide. Phosphatidylserine exposure was quantitated by plasma clotting time, as well as annexin V-fluorescein isothiocyanate binding, and the ICE-like protease activity was examined by the cleavage of a specific fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin. VAD-chloromethylketone (VAD-cmk), an inhibitor of ICE-like proteases, effectively inhibited ICE-like activity in both cell types studied, whereas the calpain inhibitor calpeptin was ineffective. VAD-cmk also effectively inhibited all three extranuclear events, as well as nuclear fragmentation, in Jurkat cells stimulated by anti-Fas monoclonal antibody, indicating that ICE-like proteases play an important role in the regulation of this apoptotic system. Calpain inhibitors were ineffective in this system. TNF-induced extranuclear and nuclear changes in U937 cells were inhibited by calpeptin but were not as effectively inhibited by VAD-cmk as in Jurkat cells. This suggests that ICE-like enzymes predominate in anti-Fas monoclonal antibody-stimulated Jurkat cells, whereas proteases affected by calpain inhibitors as well as the ICE-like enzymes are involved in the signaling of apoptotic events in TNF-induced U937 cells. Importantly, the two apoptotic systems seem to be regulated by different proteases.

Activation of thereaperGene during Ectopic Cell Killing inDrosophila

The product of thereaper(rpr) gene is required for programmed cell death inDrosophila.We examinedrprexpression during ectopic cell deaths caused by ionizing radiation or aberrant development. In both instances, dramatic induction ofrprexpression was observed. A genomic fragment upstream ofrprconfers this regulatory behavior upon alacZreporter transgene. In a model cell culture system, conditional expression of REAPER is sufficient to induce massive apoptosis that can be prevented by the anti-apoptotic protein p35. Overall, these results suggest that diverse signals converge at, or upstream of,rpr-associated transcriptional regulatory elements that can function to initiate a common apoptotic pathway involving ICE-like protease activity.

Potassium Deprivation-Induced Apoptosis of Cerebellar Granule Neurons: A Sequential Requirement for New mRNA and Protein Synthesis, ICE-Like Protease Activity, and Reactive Oxygen Species

Potassium (K+) deprivation-induced apoptosis of cerebellar granule neurons requires new mRNA and protein synthesis. Using a fluorogenic substrate for interleukin-1beta converting enzyme (ICE), we show that K+ deprivation of cerebellar granule neurons induces cycloheximide-sensitive ICE-like protease activity. A peptide inhibitor of ICE-like protease activity, Ac-YVAD-chloromethylketone (Ac-YVAD-CMK), prevents K+ deprivation-induced apoptosis. Further, reactive oxygen species (ROS) are essential mediators of K+ deprivation-induced apoptosis of cerebellar granule neurons because neuronal death is also blocked by superoxide dismutase, N-acetyl-L-cysteine, and free radical spin traps. Using fluorescent assays, we show that ROS production after K+ deprivation is blocked by actinomycin D, cycloheximide, and Ac-YVAD-CMK, suggesting that ROS act downstream of gene transcription, mRNA translation, and ICE activation. Taken together, we show that new mRNA and protein synthesis, activation of ICE-like proteases, and ROS production are sequential events in K+ deprivation-induced apoptosis of cerebellar granule neurons.

Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors.

Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 1beta converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAF1 and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.

Multiple Proteases Are Involved in Thymocyte Apoptosis

To investigate the involvement of proteases in apoptosis, rat thymocytes were treated with the glucocorticoid hormone methylprednisolone or the topoisomerase II inhibitor etoposide in the presence of selective substrate inhibitors of either interleukin-1β-converting enzyme (ICE), (Z-Val-Ala-Asp-chloromethylketone, VADcmk) or Ca 2+-regulated serine protease (Suc-Ala-Ala-Pro-Phe-chloromethylketone, AAPFcmk). VADcmk protected from lamin proteolysis, chromatin fragmentation, cell shrinkage, and formation of apoptotic nuclei in both methylprednisolone- and etoposide-treated thymocytes when present during the initiation of the apoptotic process. AAPFcmk prevented lamin breakdown, chromatin fragmentation, and apoptotic morphological changes in thymocytes treated with methylprednisolone, but not with etoposide. Both MPS- and etoposide-treated thymocytes exhibited enhanced ICE-like protease activity which was maximal 1 h after treatment. This increase in proteolytic activity was blocked by VADcmk, but not AAPFcmk. Our findings suggest that ICE-like protease activity is critically involved in the early phase of both methylprednisolone- and etoposide-induced apoptosis in thymocytes, whereas the Ca 2+-regulated serine protease is an obligatory component of the proteolytic cascade in methylprednisolone-induced apoptosis.

Bcl-2 blocks glucocorticoid- but not Fas- or activation-induced apoptosis in a T cell hybridoma.

Overexpression of Bcl-2 can prevent or markedly delay cell death induced by a variety of apoptotic stimuli. Although Fas and Fas ligand (FasL) interactions play a major role in the elimination of self-reactive T cells in the periphery, inhibition of Fas-mediated killing by Bcl-2 has not been consistently observed. The mouse T hybridoma 2B4.11 (2B4) has been a useful model to study glucocorticoid- and activation-induced apoptosis, which is mediated through Fas and FasL. Using both stable transfectants and transient transfections, overexpression of Bcl-2 or Bcl-xL readily blocked glucocorticoid-induced but not activation-induced apoptosis of 2B4 cells. Bcl-2 expression did not inhibit Fas-mediated cytotoxicity triggered by cells expressing FasL or by the transient transfection of human Fas. Similarly, overexpression of Bcl-2 in the mouse T hybridoma A1.1 did not block activation-induced/Fas-mediated apoptosis. In Jurkat cells, however, expression of Bcl-2 partially inhibited anti-Fas-induced cell death. A Bcl-2-related protein that can interfere with anti-Fas killing, the adenoviral E1B 19K, also did not block activation-induced/Fas-mediated apoptosis in 2B4 cells. In contrast, expression of CrmA, a cowpox virus protein that inhibits ICE-like protease activity, blocked activation-induced apoptosis in 2B4 cells but had little effect on Dex-mediated cytotoxicity. These results show that: 1) Bcl-2 can have strikingly different anti-cell death activity in the same cell depending upon the apoptotic stimulus, 2) distinct apoptosis signaling pathways may exist with differential sensitivity to Bcl-2 and ICE-like protease inhibitors.