Proteolytic activation of the cell death protease Yama/CPP32 by granzyme B.

The serine protease granzyme B, which is secreted by cytotoxic cells, is one of the major effectors of apoptosis in susceptible targets. To examine the apoptotic mechanism of granzyme B, we have analyzed its effect on purified proteins that are thought to be components of death pathways inherent to cells. We demonstrate that granzyme B processes interleukin 1beta-converting enzyme (ICE) and the ICE-related protease Yama (also known as CPP32 or apopain) by limited proteolysis. Processing of ICE does not lead to activation. However, processing by granzyme B leads directly to the activation of Yama, which is now able to bind inhibitors and cleave the substrate poly(ADP-ribose) polymerase whose proteolysis is a marker of apoptosis initiated by several other stimuli. Thus ICE-related proteases can be activated by serine proteases that possess the correct specificity. Activation of pro-Yama by granzyme B is within the physiologic range. Thus the cytotoxic effect of granzyme B can be explained by its activation of an endogenous protease component of a programmed cell death pathway.     

Differential suppression by protease inhibitors and cytokines of apoptosis induced by wild-type p53 and cytotoxic agents.

Apoptosis induced in myeloid leukemic cells by wild-type p53 was suppressed by different cleavage-site directed protease inhibitors, which inhibit interleukin-1 beta-converting enzyme-like, granzyme B and cathepsins B and L proteases. Apoptosis was also suppressed by the serine and cysteine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone (TPCK) [corrected], but not by other serine or cysteine protease inhibitors including N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), E64, pepstatin A, or chymostatin. Protease inhibitors suppressed induction of apoptosis by gamma-irradiation and cycloheximide but not by doxorubicin, vincristine, or withdrawal of interleukin 3 from interleukin 3-dependent 32D non-malignant myeloid cells. Induction of apoptosis in normal thymocytes by gamma-irradiation or dexamethasone was also suppressed by the cleavage-site directed protease inhibitors, but in contrast to the myeloid leukemic cells apoptosis in thymocytes was suppressed by TLCK but not by TPCK. The results indicate that (i) inhibitors of interleukin-1 beta-converting enzyme-like proteases and some other protease inhibitors suppressed induction of apoptosis by wild-type p53 and certain p53-independent pathways of apoptosis; (ii) the protease inhibitors together with the cytokines interleukin 6 and interferon-gamma or the antioxidant butylated hydroxyanisole gave a cooperative protection against apoptosis; (iii) these protease inhibitors did not suppress induction of apoptosis by some cytotoxic agents or by viability-factor withdrawal from 32D cells, whereas these pathways of apoptosis were suppressed by cytokines; (iv) there are cell type differences in the proteases involved in apoptosis; and (v) there are multiple pathways leading to apoptosis that can be selectively induced and suppressed by different agents.     

Activation of mitochondrial protease OMA1 by Bax and Bak promotes cytochrome c release during apoptosis

Intrinsic apoptotic stimuli initiate mammalian cells’ apoptotic program by first activating the proteins that have only Bcl-2 homology domain 3 (BH3), such as Bcl-2 interacting mediator of cell death (Bim) and truncated BH3 interacting death domain agonist (tBid), which in turn trigger conformational changes in BCL2-associated X (Bax) and BCL2-antagonist/killer (Bak) proteins that enable oligomer formation on the mitochondria, causing cytochrome c and other apoptogenic proteins in the intermembrane space to leak out. Leaked cytochrome c then initiates apoptotic caspase activation through a well-defined biochemical pathway. However, how oligomerized Bax and Bak cause cytochrome c release from mitochondria remains unknown. We report here the establishment of cell lines in which Bim or tBid can be inducibly expressed to initiate apoptosis in a controlled, quantitative manner. We used these cell lines to examine apoptotic events after Bax and Bak oligomerization but before cytochrome c release. The mitochondrial metalloprotease OMA1 was activated in this system in a Bax- and Bak-dependent fashion. Activated OMA1 cleaved the dynamin-like GTPase, optical nerve atrophy 1, an event that is critical for remodeling of mitochondrial cristae. Knockdown or knockout of OMA1 in these cells attenuated cytochrome c release. Thus it is clear that oligomerized Bax and Bak trigger apoptosis by causing both the permeabilization of the mitochondrial outer membrane and activation OMA1.Mitochondria in mammalian cells fulfill multiple functions. They are cells’ bio-energetic center, where reducing agents generated through the Krebs cycle transfer their electrons to oxygen in a manner mediated by the electron transfer chain, a process that builds a proton gradient across the inner membrane of mitochondria. The energy of this gradient is transferred into the high-energy bond of ATP by oxidative phosphorylation of ADP through the F1/F0 ATP synthase. During apoptosis, the sole water-soluble component of the electron transfer chain, cytochrome c, is released from the intermembrane space of mitochondria to the cytosol (1). Cytosolic cytochrome c binds to the Apaf-1 protein to promote the assembly of a heptamer complex named an “apoptosome”; this complex subsequently recruits procaspase-9, which autoactivates once on the apoptosome. The activated caspase-9 then cleaves and activates downstream caspase-3 and caspase-7, which subsequently cleave many intracellular substrates for apoptosis execution (2).In addition to cytochrome c, other proteins that normally are located in the mitochondrial intermembrane space also function in apoptosis. One such protein is second mitochondria-derived activator of caspase (Smac). When Smac is released, it binds to the BIR domain of inhibitors of apoptosis proteins to relieve their inhibition of the caspases directly or to cause their degradation (3, 4). Thus controlling the permeability of mitochondria for these apoptogenic proteins constitutes a key regulatory step for apoptosis.The B-cell leukemia/lymphoma 2 (Bcl-2) family of proteins constitutes a protein network that regulates the release of proteins such as cytochrome c and Smac (5, 6). BCL2-associated X (Bax) and BCL2-antagonist/killer (Bak), the proapoptotic members of the family with multiple Bcl-2 homology (BH) domains, form the core of the mitochondrial membrane permeability machinery that is activated by the proapoptotic proteins that have only the BH3 domain, a process that is inhibited by the proteins whose function is similar to that of Bcl-2 itself (7, 8). In response to apoptotic stimuli, BH3-only proteins, such as Bcl-2 interacting mediator of cell death (Bim), Puma, and truncated BH3 interacting death domain agonist (tBid) directly activate Bax/Bak and lift the inhibition of Bcl-2/Bcl-xl by forming stable heterodimers to sequester them from binding Bak/Bak (7, 9–13). Activated Bax and Bak initially form homodimers and then oligomers on the mitochondrial membrane (14–18). Bax/Bak oligomers are believed to form proteinaceous or lipidic pores on the mitochondrial outer membrane that allow the passage of proteins such as cytochrome c and Smac. Although the results of in vitro liposome leakage experiments support this model, there is no direct in vivo evidence to validate such a straightforward model (19–23).Moreover, increasing evidence indicates that the majority of cytochrome c in the mitochondrial intermembrane space is locked inside cristae by the protein complex containing optical nerve atrophy 1 (OPA1). The cristae must undergo reconfiguration to open up the neck of cristae for the bulk of cytochrome c to be released from the mitochondria after the outer membrane becomes permeable (24–26). The mitochondrial inner membrane fusion factor OPA1, a dynamin-like GTPase, plays a critical role in the remodeling of cristae. OPA1 presents in several spliced and proteolytic forms in mitochondria, and the maintenance of the relative amounts of each of these forms is known to be critical for stabilizing the cristae (27, 28). The longest form, L-OPA1, is cleaved in response to a variety of mitochondrial stresses, leading to the disassembly of OPA1-containing complexes and remodeling of the cristae (24, 26). It has been proposed that several different proteins cleave OPA1; these include the mitochondrial AAA proteases, presenilin-associated rhomboid-like protein (PARL), and the zinc metalloprotease OMA1 (overlapping activity with m-AAA protease) (26, 27, 29–31). However, the relationship between the pore formation on the mitochondrial outer membrane and OPA1 cleavage-mediated cristae remodeling during apoptosis, as well as the precise roles of those mitochondrial proteases in apoptosis, remain to be clarified.To dissect the molecular details of cytochrome c release induced by BH3-only proteins, we generated cell lines in which Bim or tBid can be inducibly expressed by adding doxycycline (Dox) into the culture medium. The expression of these proteins triggers apoptosis in a controlled and synchronized fashion. We used this cell-based system to characterize the mitochondrial response to the induction of Bim and tBid and found that OMA1 activation is an important step for apoptosis induction.     

A comparison of the action of amifostine and melatonin on DNA-damaging effects and apoptosis induced by idarubicin in normal and cancer cells

Amifostine is a well-known cell protector and its actions involve free radical scavenging, which is also considered as a mechanism underlying the protective actions of melatonin, a secretory product of the pineal gland. In this work we compared the action of 14 mM amifostine and 50 microM melatonin on DNA damage and apoptosis induced by idarubicin in normal human lymphocytes, leukemic K562 cells and HeLa cancer cells. We employed the alkaline comet assay and pulse-field gel electrophoresis to estimate DNA damage. Apoptosis was evaluated by caspase 3 activity assay assisted by the comet assay to evaluate DNA fragmentation and DAPI staining for detection of morphological changes in chromatin. We found that idarubicin induced apoptosis in normal and cancer cells and its level was correlated with the extent of DNA strand breaks. Amifostine reduced apoptosis and DNA damage in normal cells, but it potentiated these effects in cancer cells in this in vitro study. Melatonin protected both normal and cancer cells against genotoxic treatment and apoptosis induced by idarubicin. We conclude that despite its recognized potential as an antioxidant, melatonin should be considered with caution when used in combination with cancer chemotherapy agents, especially in the case of leukemias.     

Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins

The paradigm that microtubule-targeting agents (MTAs) cause cell death via mitotic arrest applies to rapidly dividing cells but cannot explain MTA activity in slowly growing human cancers. Many preferred cancer regimens combine a MTA with a DNA-damaging agent (DDA). We hypothesized that MTAs synergize with DDAs by interfering with trafficking of DNA repair proteins on interphase microtubules. We investigated nine proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, p53, 53BP1, and p63. The proteins were sequestered in the cytoplasm by vincristine and paclitaxel but not by an aurora kinase inhibitor, colocalized with tubulin by confocal microscopy and coimmunoprecipitated with the microtubule motor dynein. Furthermore, adding MTAs to radiation, doxorubicin, or etoposide led to more sustained γ-H2AX levels. We conclude DNA damage-repair proteins traffic on microtubules and addition of MTAs sequesters them in the cytoplasm, explaining why MTA/DDA combinations are common anticancer regimens.First developed as anticancer agents in the 1950s, microtubule targeting agents (MTAs) are used in the treatment of a wide variety of malignancies and until now have been thought to kill cells by arresting them in mitosis (1, 2). Although this explanation applies to rapidly dividing cells in preclinical models, it cannot explain the activity of these agents in tumors in humans because these cells divide much more slowly. For the latter situtation, a different paradigm must explain the activity of MTAs, and we have proposed that interfering with microtubule (MT) trafficking in interphase cells is the principal mechanism of MTA action (3–5). In breast, ovarian, lung, and head and neck cancers, as well as in most lymphomas, combination regimens that include a MTA and a DNA-damaging agent (DDA) are preferred (Table S1). Although the frequency with which these combinations are used might be fortuitous, it is likely there is a mechanistic basis for this outcome. We hypothesized that by hampering the trafficking of essential DNA repair proteins, MTAs synergize with DDAs, augmenting their toxicity. To explore this theory further we studied the effects of combining a MTA and a DDA in a number of cell models and examined the distribution and biology of nine different proteins involved in DNA repair. We have confirmed the hypothesis and report our findings.     

Diabetes mellitus and other metabolic disturbances induced by atypical antipsychotic agents

Atypical antipsychotic agents offer significant advantages over older conventional antipsychotic agents. The reduction in antipsychotic drug-associated extrapyramidal symptoms and the potential reduced risk for tardive dyskinesia, compared to conventional drugs, are major advances in the treatment of psychotic patients. However, recent reports of hyperglycemia, new-onset diabetes mellitus, diabetic ketoacidosis, weight gain, and lipid abnormalities associated with atypical antipsychotic agents have emerged. A review of the recent literature and an approach to the evaluation of risk factors to aide in the safe use of atypical antipsychotic agents is presented.     

Attenuation of HIV-1 infection by other microbial agents

Although potentiation of human immunodeficiency virus (HIV) type 1 (HIV-1) infection has been known to occur in coinfection with a variety of pathogens and types of vaccination, there are emerging data on specific infectious agents that may attenuate HIV-1 infection. New literature suggests that certain pathogens are capable of inhibiting HIV-1 replication. These include GB virus C, measles virus, Orientia tsutsugamushi, and human T lymphotropic virus types 1 and 2. In addition, there are conflicting data on the effects of Mycobacterium tuberculosis on the replication of HIV-1, with some suggesting that this organism may inhibit HIV-1 replication. Also remaining controversial are the possible protective effects of HIV type 2 against HIV-1 infection. In this review, we summarize and critically discuss the body of emerging literature concerning infections that may have the ability to attenuate HIV-1 infection.     

In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains.

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in ail known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Kj = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathways.     

Activation of Kupffer cells and caspase-3 involved in rat hepatocyte apoptosis induced by endotoxin.

BACKGROUND/AIMS: Sepsis and lipopolysaccharides (LPS) cause mild to severe hepatic dysfunction. In this study, Kupffer cell activation, involvement of TNFalpha and caspases downstream of the TNFalpha receptor were examined in hepatocyte apoptosis induced by LPS. METHODS: In in vivo experiments, male Sprague-Dawley rats were injected intravenously with LPS, and small amounts of the blood and liver were sampled to evaluate apoptosis. Kupffer cells were inactivated by pretreatment with gadolinium chloride for 2 days. In in vitro experiments, hepatocytes and Kupffer cells were separately isolated from rat livers using collagenase perfusion. RESULTS: LPS induced time-dependent and dose-dependent increases in the number of TUNEL-positive cells, which coincided with the apoptotic features of hepatocytes demonstrated by electron microscopy and DNA ladder. Activation of caspase-3-like proteases was observed with an increase in the number of apoptotic hepatocytes. Immunostaining with activated caspase-3-specific antibody showed that caspase-3 was activated only in the cytoplasm of TUNEL-positive hepatocytes. Inactivation of Kupffer cells by gadolinium chloride was concomitantly accompanied by the prevention of caspase-3 activation, hepatocyte apoptosis and liver injury induced by LPS. The co-culture system of hepatocytes and Kupffer cells, but neither cell culture system, individually, showed LPS-induced hepatocyte apoptosis. Kupffer cell-conditioned medium induced hepatocyte apoptosis, whereas addition of anti-TNFalpha antibody to Kupffer cell-conditioned medium did not. Additions of acetyl-DEVD-CHO, acetyl-YVAD-CHO, and acetyl-IETD-CHO to Kupffer cell-conditioned medium decreased the number of apoptotic hepatocytes. CONCLUSIONS: These results suggest that the activation of Kupffer cells, TNFalpha and caspases downstream of TNFR1 were involved in hepatocyte apoptosis induced by LPS.     

A comparative study on the anti-angiogenic effects of DNA-damaging and cytoskeletal-disrupting agents

The discovery of molecules with anti-angiogenic properties has led to promising new strategies for the treatment of diseases characterized by excessive new vessel growth, such as cancer and haemangioma. We have assessed the effects of DNA-damaging and cytoskeletal-disrupting agents in vitro on several endothelial cell functions. We report that bleomycin, mitomycin C and cytoskeletal-disrupting drugs (2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole, and cytochalasin D) exhibit anti-angiogenic activities of varying potency. Bleomycin and the various cytoskeletal-disrupting drugs inhibited endothelial cell migration, while mitomycin C had a marginal effect. Both DNA-damaging and cytoskeletal-disrupting drugs decreased endothelial cell growth in a dose-dependent manner, and this was accompanied by the induction of apoptosis. The growth inhibitory and apoptotic effects of cytoskeletal-disrupting drugs were the most pronounced. We also show that both classes of drugs inhibited capillary-like tube formation in an assay of in vitro angiogenesis, with cytoskeletal-disrupting agents inhibiting in vitro angiogenesis with greater potency. A targeted approach incorporating several compounds with different mechanisms of action may be useful for the treatment of angiogenesis-dependent diseases such as hemangiomas of infancy.     

Activation of melanocortin 4 receptors reduces the inflammatory response and prevents apoptosis induced by lipopolysaccharide and interferon-gamma in astrocytes

Alpha-MSH exerts an immunomodulatory action in the brain and may play a neuroprotective role acting through melanocortin 4 receptors (MC4Rs). In the present study, we show that MC4Rs are constitutively expressed in astrocytes as determined by immunocytochemistry, RT-PCR, and Western blot analysis. alpha-MSH (5 microm) reduced the nitric oxide production and the expression of inducible nitric oxide synthase (iNOS) induced by bacterial lipopolysaccharide (LPS, 1 microg/ml) plus interferon-gamma (IFN-gamma, 50 ng/ml) in cultured astrocytes after 24 h. alpha-MSH also attenuated the stimulatory effect of LPS/IFN-gamma on prostaglandin E(2) release and cyclooxygenase-2 (COX-2) expression. Treatment with HS024, a selective MC4R antagonist, blocked the antiinflammatory effects of alpha-MSH, suggesting a MC4R-mediated mechanism in the action of this melanocortin. In astrocytes, LPS/IFN-gamma treatment reduced cell viability, increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells and activated caspase-3. alpha-MSH prevented these apoptotic events, and this cytoprotective effect was abolished by HS024. LPS/IFN-gamma decreased Bcl-2, whereas it increased Bax protein expression in astrocytes, thus increasing the Bax/Bcl-2 ratio. Alpha-MSH produced a shift in Bax/Bcl-2 ratio toward astrocyte survival because it increased Bcl-2 expression and also prevented the effect of LPS/IFN-gamma on Bax and Bcl-2 expression. In summary, these findings suggest that alpha-MSH, through MC4R activation, attenuates LPS/IFN-gamma-induced inflammation by decreasing iNOS and COX-2 expression and prevents LPS/IFN-gamma-induced apoptosis of astrocytes by modulating the expression of proteins of the Bcl-2 family.     

Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis.

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.     

Activation of caspase-2 mediates the apoptosis induced by GTP-depletion in insulin-secreting (HIT-T15) cells

This study investigated the possible involvement of a specific caspase(s) (a family of aspartate-specific cysteine proteases) in programmed cell death of islet beta-cells due to sustained GTP depletion. Treatment (up to 48 h) with 3 microg/ml mycophenolic acid (MPA), which specifically depletes intracellular guanine nucleotides, reduced cell-cycle progression from G1 phase into S and G2/M phases (as assessed by flow cytometry) and, subsequently, induced apoptosis of HIT-15 cells (transformed pancreatic beta-cells). The latter was accompanied by a marked increase of caspase-2 activity (+343%) and moderate activation of caspase-9 (+150%) and caspase-3 (+145%). Importantly, only caspase-2 activation preceded induction of apoptosis. There was no change in activity of caspase-1, -4, -5, -6, and -8. Release of the mitochondrial protein cytochrome c into cytosol was also observed at a late stage. Cotreatment of cells with a permeable pan-caspase inhibitor (Z-VAD-FMK) blocked GTP depletion-induced cell death in a dose-dependent manner. A specific caspase-2 inhibitor (Z-VDVAD-FMK), but not a caspase-3 inhibitor (DEVD-CHO), was also capable of restoring cell viability. Interestingly, activation of caspase-2 leads to caspase-3 activation because the caspase-2 inhibitor abrogated caspase-3 activity. Our results indicate that, while activation of multiple caspases are involved in the execution phase of GTP depletion-induced apoptosis, caspase-2 appears to play the major role in the initiation of this program. This study revealed a novel, caspase-2 mediated form of apoptosis that may be consequent to impaired mitogenesis.     

血管紧张素Ⅱ介导的大鼠血管平滑肌细胞STAT1激活与核转位

目的 检测血管紧张素Ⅱ（AngⅡ）介导的大鼠血管平滑肌细胞（VSMC）增殖过程中信号转导和转录活化因子-1（STAT1）的激活与核转位。方法 本文采用Western印迹、非同位素凝胶电泳（EMSA）和免疫荧光染色的方法，观察AngⅡ刺激大鼠主动脉VSMC前后，细胞中STAT1的活化状态与定位。结果 VSMC经AngⅡ干预后，胞内磷酸化的STAT1（P-STAT1）蛋白表达增加（P〈0．01），达峰后随时间梯度逐渐下降，AngⅡ干预15min后检测到胞核内有蛋白．DNA复合物形成。这一反应可被血管紧张素Ⅱ1型受体（AT1）阻滞剂Losartan以及Jak2抑制剂AC-490抑制（P〈0．01）。免疫荧光染色结果也显示AngⅡ干预后P-STAT1主要在胞核内表达。结论AngⅡ可以通过和AT1受体结合，激活Jak／STAT通路，在AngⅡ介导的大鼠VSMC增殖过程中发挥作用。