acetyl-aspartyl-glutamyl-valyl-aspartal and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Smoking can lead to airway inflammation and mucus secretion through the nucleotide-binding domain-like receptor protein 3/caspase-1 pathway. In this study, z-VaD-Ala-Asp-fluoromethyl ketone(Z-VAD), a pan-caspase inhibitor, and acetyl-Asp-Glu-Val-Asp-aldehyde(Ac-DEVD), a caspase-3 inhibitor, were used to investigate the effect of caspase inhibitors on the expression of interleukin(IL)-1β and IL-8, airway inflammation, and mucus secretion in mice exposed to cigarette smoke(CS).. Thirty-two C57BL/6J male mice were divided into a control group, Smoke group, Z-VAD group, and Ac-DEVD group. Except for the control group, the animals were all exposed to CS for three months. After the experiment, lung function was measured and hematoxylin and eosin staining and periodic acid-Schiff staining were performed. The levels of IL-1β, IL-8, and mucin 5ac(Muc5ac) in serum and bronchoalveolar lavage fluid(BALF) were determined by enzyme-linked immunosorbent assay.. Compared with the control group, the lung function of mice exposed to smoke was poorer, with a large number of inflammatory cells infiltrating around the airway, collapse of alveoli, expansion and fusion of distal alveoli, and formation of emphysema. The Z-VAD group was relieved compared with the smoke group. Airway inflammation was also reduced in the Ac-DEVD group compared with the Smoke group, but the degree of emphysema was not significantly improved. Although Z-VAD relieved airway inflammation and emphysema, Ac-DEVD only relieved inflammation. Z-VAD and Ac-DEVD decreased serum IL-1β and IL-8 levels. In BALF, IL-1β was decreased in Z-VAD group and IL-8 was highest in Smoke +Ac-DEVD group compared with control group and Ac-DEVD group. There was no significant difference in the expression of Muc5ac in serum. However, in BALF, levels of Muc5ac were higher in the smoking group and the lowest in the Ac-DEVD group.. Mice exposed to smoke had decreased lung function and significant cilia lodging, epithelial cell shedding, and inflammatory cell infiltration, with significant emphysema formation. The pan-caspase inhibitor, Z-VAD, improved airway inflammation and emphysema lesions in the mice exposed to smoke and reduced IL-1β and IL-8 levels in serum. The caspase-3 inhibitor, Ac-DEVD, reduced airway inflammation, serum IL-1β and IL-8 levels, and Muc5ac levels in BALF, but it did not improve emphysema.

Topics: Animals; Caspase 3; Cigarette Smoking; Inflammation; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Mucus; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema

4-(3',3'-Dimethylallyloxy)-5-methyl-6-methoxy-phthalide (DMMP) has previously been isolated from the endophytic fungus Pestalotiopsis photiniae. Although the cytotoxic activities of DMMP have been reported, little is known concerning the molecular mechanism of its cytotoxic effect. In the present study, we investigated the effect of DMMP on the growth of several types of cancer cell lines and investigated the mechanism of its antiproliferative effect. DMMP caused the growth inhibition of human cancer lines HeLa, MCF7 and MDA-MB-231, but had little antiproliferative effect on MRC5 normal lung cells. DMMP also significantly caused cell cycle arrest in the G1 phase and upregulated the cyclin-dependent kinase inhibitor p27KIPI protein in the HeLa cells. Moreover DMMP was able to induce marked nuclear apoptotic morphology in HeLa cells. DMMP induced apoptosis and loss of mitochondrial membrane potential (ΔΨm) in the HeLa cells. Although the activated forms of caspase-9 and -3 in HeLa cells were detected, pretreatment with caspase inhibitors (Ac-DEVD-CHO and Z-VAD-FMK) failed to attenuate DMMP-induced cell death. In addition, protein levels of the p53 family members, p53 and p73, were upregulated, and DMMP significantly increased the mRNA expression of pro-apoptotic Bcl-2 family genes (PUMA, NOXA, Bax, Bad and Bim). HPV E6-E7 mRNA levels were reduced. In conclusion, DMMP demonstrates potential for use in the treatment of cervical cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; bcl-Associated Death Protein; Benzofurans; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; Female; G1 Phase Cell Cycle Checkpoints; HeLa Cells; Humans; MCF-7 Cells; Membrane Potential, Mitochondrial; Membrane Proteins; Mitochondria; Nuclear Proteins; Oligopeptides; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation; Uterine Cervical Neoplasms; Xylariales

Yersinia species during infection adhere to host immune cells primarily to macrophages and employ its secretary proteins known as Yersinia outer proteins to trigger death in infected cells. In the present study, it is shown that recombinant Yersinia outer protein J (rYopJ) could induce apoptosis in murine peritoneal macrophages in vitro as assessed by morphological features, internucleosomal DNA fragmentation, change in mitochondrial membrane potential (MMP) (Deltapsim), activation of caspases and Annexin V binding. rYopJ-induced cell death was dose and time dependent. Pre-treatment with broad-spectrum caspase inhibitor Z-VAD-FMK, caspase-3 inhibitor Ac-DEVD-CHO and caspase-8 inhibitor Z-IETD-FMK prevented the change in MMP and DNA fragmentation, suggesting caspase-dependent apoptosis of rYopJ-treated macrophages. Blocking the endocytosis by pre-treatment of cells with cytochalasin B did not prevent the rYopJ-induced macrophages apoptosis. The data further suggest that rYopJ-induced apoptosis is mediated by molecules upstream of caspase-8 and relay through mitochondrial pathway involving Bax, Bcl-2, activation of caspase-8 and caspase-3, Bid and polyadenosine diphosphate-ribose polymerase cleavage, cytochrome c release and DNA fragmentation.

Topics: Amino Acid Chloromethyl Ketones; Animals; Annexin A5; Apoptosis; Apoptotic Protease-Activating Factor 1; Bacterial Proteins; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Cytochalasin B; Cytochromes c; Macrophages, Peritoneal; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mitochondria; Oligopeptides; Plague; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Yersinia

The effect of caspase inhibitors on long-term synaptic facilitation induced by nociceptive sensitization (a simple form of learning) was studied on the defensive behavior command neurons (left pleural neuron-1) in edible snail. Acquisition of sensitization under conditions of threatment with caspase-3 or caspase-8 inhibitors selectively inhibits synaptic transmission in the responses of the left pleural neuron-1 to tactile stimulation of the snail head, but not in responses to chemical stimulation of the head or tactile stimulation of the foot. Application of a wide-spectrum caspase inhibitor z-VAD-fmk to neurons of sensitized snails suppressed facilitation of responses evoked by chemical stimulation of the head. Probably, various caspases could be selectively involved into induction of long-term synapse-specific plasticity during learning.

Topics: Amino Acid Chloromethyl Ketones; Animals; Caspase Inhibitors; Helix, Snails; Learning; Neuronal Plasticity; Neurons; Oligopeptides; Synapses; Synaptic Transmission

The critical point at which apoptosis becomes irreversible and how cells attain an anti-apoptotic state remain unknown. Here, we report that apoptotic cells undergoing early-stage dynamic membrane blebbing revive. We examined this phenomenon in cell lines that stably express 2DED2DD, a modified FADD produced by fusing the tandem death effector domains (DEDs) and tandem death domains (DDs). Induction of apoptosis caused rapid blebbing. Eight hours later, most cells shrunk while some detached from the flask. Twenty-four hours later, when activated caspase 3 decreased, more than half the cells revived and appeared normal, probably due to the induction of unidentified anti-apoptotic proteins.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Caspase Inhibitors; Cell Line; Cell Membrane; Fas-Associated Death Domain Protein; Humans; Immunoblotting; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Time Factors

Previous studies reported that the neurotoxin, Crotoxin, isolated from the venom of South American rattlesnake had potent anti-tumor activity. Here, we investigated the involvement of autophagy and apoptosis in the Crotoxin-induced death of chronic myeloid leukemia cell line K562 cells. The neurotoxin dose dependently inhibited the viability of K562 cells. Crotoxin stimulated the autophagic activity as evidenced by the appearance of punctuate monodansylcadaverine (MDC) fluorescence staining in the cytoplasm and increased the formation of autophagosomes. Crotoxin caused the collapse of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-3. Caspase inhibitors attenuated Crotoxin-induced K562 cell death, while blockage of autophagy maturation with 3-methyladenine (3-MA) and NH4Cl potentiated the neurotoxin's cytotoxicity. These results suggest that an apoptotic mechanism contributes to the Crotoxin-induced death of K562 cells, while the activation of autophagy delays neurotoxin-induced apoptosis.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Ammonium Chloride; Apoptosis; Autophagy; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Crotoxin; Cytochromes c; Enzyme Activation; Humans; K562 Cells; Lysosomes; Mitochondria; Oligopeptides; Vacuoles

Development of chemotherapy resistance and evasion from apoptosis in osteosarcoma, a primary malignant bone tumor, is often correlated with constitutive nuclear factor-kappaB (NF-kappaB) activation. Here, we investigated the ability of a polyphenolic fraction of green tea (GTP) that has been shown to have antitumor effects on various malignant cell lines to inhibit growth and induce apoptosis in human osteosarcoma SAOS-2 cells. Treatment of SAOS-2 cells with GTP (20-60 microg/ml) resulted in reduced cell proliferation and induction of apoptosis, which correlated with decreased nuclear DNA binding of NF-kappaB/p65 and lowering of NF-kappaB/p65 and p50 levels in the cytoplasm and nucleus. GTP treatment of cells reduced IkappaB-alpha phosphorylation but had no effect on its protein expression. Furthermore, GTP treatment resulted in the inhibition of IKK-alpha and IKK-beta, the upstream kinases that phosphorylate IkappaB-alpha. The increase in apoptosis in SAOS-2 cells was accompanied with decrease in the protein expression of Bcl-2 and concomitant increase in the levels of Bax. GTP treatment of SAOS-2 cells also resulted in significant activation of caspases as was evident by increased levels of cleaved caspase-3 and caspase-8 in these cells. Treatment of SAOS-2 cells with a specific caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) and general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) rescued SAOS-2 cells from GTP-induced apoptosis. Taken together, these results indicate that GTP is a candidate therapeutic for osteosarcoma that mediates its antiproliferative and apoptotic effects via activation of caspases and inhibition of NF-kappaB.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Electrophoretic Mobility Shift Assay; Flavonoids; Humans; I-kappa B Kinase; I-kappa B Proteins; Immunoblotting; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Oligopeptides; Osteosarcoma; Phenols; Phosphorylation; Poly(ADP-ribose) Polymerases; Polyphenols; Tea; Transcription Factor RelA

In the present study we assessed the neuroprotective effects of the pan-caspase inhibitor z-VAD.fmk [N-benzyloxycarbony-valine-alanine-aspartate-(OMe)-fluoromethylketone], and the caspase-3 inhibitor Ac-DEVD.CHO (acetyl-aspartate-chloromethylketone) in the double-lesion rat model of striatonigral degeneration (SND), the core pathology underlying levodopa-unresponsive parkinsonism associated with multiple system atrophy (MSA). Male Wistar rats were divided into three groups, receiving either Ac-DEVD.CHO, z-VAD.fmk or normal saline before lesion surgery, comprising a sequential unilateral quinolinic acid (QA) lesion of the striatum followed by a 6-hydroxydopamine (6-OHDA) lesion of the ipsilateral medial forebrain bundle. At 2 weeks post lesion, all rats underwent testing of spontaneous nocturnal locomotor behavior in an automated Photobeam Activity System (FlexField). Immunohistochemistry was performed with tyrosine hydroxylase, dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein and glial fibrillary acidic protein antibodies. Morphometry was performed using computerized image analysis. Behavioral and morphological analysis failed to show striatal or nigral protection in caspase inhibitor-treated animals. Our findings suggest that anti-apoptotic strategies are unrewarding in the SND rat model and, therefore, alternative neuroprotective interventions such as anti-glutamatergic agents or inhibitors of microglial activation should be explored instead.

Topics: Amino Acid Chloromethyl Ketones; Analysis of Variance; Animals; Basal Ganglia; Behavior, Animal; Caspase Inhibitors; Cell Count; Cell Size; Disease Models, Animal; Dopamine and cAMP-Regulated Phosphoprotein 32; Functional Laterality; Immunohistochemistry; Male; Motor Activity; Multiple System Atrophy; Nerve Tissue Proteins; Neurons; Neuroprotective Agents; Oligopeptides; Oxidopamine; Phosphoproteins; Quinolinic Acid; Rats; Rats, Wistar; Tyrosine 3-Monooxygenase

Programmed cell death (PCD), or apoptosis, is initiated in response to various stimuli, including virus infection. A number of studies have shown that deregulation of apoptosis is an important feature of virus-induced immunosuppression for various viral diseases. In the present study, CapHV-1 was found to cause apoptosis in mitogen-stimulated as well as nonstimulated caprine peripheral blood mononuclear cells (PBMC). Apoptotic index, as quantified by fluorescent dyes, revealed a significant increase in the percentage of apoptotic cells at 24 and 48 h postinfection as compared to their respective noninfected controls. Apoptosis specific internucleosomal laddering in DNA from CapHV-1 infected PBMC was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control noninfected PBMC. Virus-induced apoptosis was reduced by Z-VAD-FMK, an aspecific caspase inhibitor, by AC-DEVD-CHO (caspase-3-specific) and AC-VEID-CHO (caspase-6-specific) treatment. PCD in CapHV-1 infected peripheral blood mononuclear cells occurs at the G0/G1 phase of the cell cycle. However, penetration of virus particles and infection was not required for PCD, as UV-inactivated CapHV-1 induced apoptosis of mitogen-stimulated bovine peripheral blood mononuclear cells in vitro.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Cycle; DNA Fragmentation; Electrophoresis, Agar Gel; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Goat Diseases; Goats; Herpesviridae Infections; Kinetics; Leukocytes, Mononuclear; Oligopeptides; Varicellovirus

We have investigated the effects of glycyrrhizin (GL) on cell proliferations of human stomach cancer KATO III and promyelotic leukemia HL-60 cells, and on DNA of those cell lines. GL displayed growth inhibitory effect against KATO III and HL-60 cells. Morphological change showing apoptotic bodies was observed in the KATO III and HL-60 cells treated with GL. The fragmentation of DNA by GL to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in both cell lines. Caspase inhibitors such as Z-VAD-FMK and Z-Asp-CH2-DCB suppressed the DNA fragmentation induced by GL. The data of the present study show that the suppression of KATO III and HL-60 cell-growth by GL results from the induction of apoptosis by GL, and that caspase is involved in the induction of apoptosis by GL in these cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Aspartic Acid; Caspase Inhibitors; Cell Line, Tumor; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Glycyrrhizic Acid; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Oligopeptides; Stomach Neoplasms; Time Factors

Monoclonal antibody IDEC-C2B8 (rituximab) has been shown to be highly effective in the treatment of non-Hodgkin's lymphomas (NHL). The present study was designed to investigate relationships between the efficacy of IDEC-C2B8 and expression of CD20, presence of complement, and effects of differently acting chemotherapeutic agents used in lymphoma treatment (doxorubicin, mitoxantrone, cladribine, bendamustine).. DOHH-2, WSU-NHL and Raji lymphoma cell lines and ex vivo cells from patients with chronic lymphocytic leukemia (CLL) (n=17) and leukemic B-cell lymphomas (n=9) were studied. Additionally, the effect of interleukin (IL)-2, IL-4, IL-6, IL-13, granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)alpha on expression of CD20 molecules per cell was determined.. We demonstrate that 10 mg/mL rituximab saturated 80-95% of CD20 molecules per cell in all tested lymphoma samples. Although rituximab induced only a minor increase of apoptosis, combinations of rituximab with different cytotoxic drugs significantly decreased the IC(30)- and IC(50) dosages of the chemotherapeutic agents necessary for induction of apoptosis irrespective of addition of complement, demonstrating a chemosensitizing effect of rituximab in combination with cytotoxic drugs in the neoplastic lymphocytes. This effect seemed to be independent of the percentage of saturated CD20 molecules. After addition of caspase inhibitors to the cell lines incubated with rituximab and cytotoxic agents, caspase-7 and -8 were found, by Western blotting, to be the executioner caspases, possibly explaining the rituximab-sensitized apoptosis. Preincubation of lymphoma cells with cytokines did not alter the expression of CD20; IL-2 and IL-4 even decreased the rate of apoptosis.. We conclude that rituximab sensitizes lymphoma cells to the effect of differently acting cytotoxic drugs used in lymphoma treatment, that this effect does not require complement, and that caspase-7 and -8 may represent the main executioner caspases in chemosensitization by rituximab.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Bendamustine Hydrochloride; Burkitt Lymphoma; Caspase 7; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cladribine; Complement Activation; Complement System Proteins; Cysteine Proteinase Inhibitors; Doxorubicin; Drug Synergism; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Follicular; Mitoxantrone; Neoplasm Proteins; Nitrogen Mustard Compounds; Oligopeptides; Rabbits; Rituximab; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Programmed cell death (PCD) or apoptosis process in a hybridoma cell line induced by the deprivation of one of the main nutrients, glutamine, has been studied. The use of caspase inhibitors has enabled maintenance of cell viability during a significant period of time, when glutamine depletion was maintained in the culture. Two caspase inhibitors partially suppressed the progress of PCD under glutamine deprivation: Ac-DEVD-cho and z-VAD-fmk. Indeed, as a consequence of this protection, the number of viable cells decreased by 10% (for z-VAD-fmk) and by 80% (for Ac-DEVD-cmk) after 36 h of culture, while it decreased by 90% for a control culture in the absence of protective compounds. However, when the culture was exposed to non-apoptotic conditions after this period of time under apoptosis protection conditions, a normal growth pattern was not recovered. Interestingly, the simultaneous use of both inhibitors made the recovery of the cell culture possible even after a period of 36 h under glutamine depletion, indicating that the inhibition of the effector caspases occurs upstream of the point in which hybridoma cells enter into the commitment step of the death programme.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Cell Culture Techniques; Cell Division; Cell Survival; Cysteine Proteinase Inhibitors; Glutamine; Hybridomas; Mice; Oligopeptides

Tissue remodeling during embryonic development and in the adult organism relies on a subtle balance between cell growth and apoptosis. As angiogenesis involves restructuring of preexisting endothelium, we examined the role of apoptosis in new vessel formation. We show that apoptosis occurs before capillary formation but not after vessels have assembled. Using the human umbilical vein endothelial cell (HUVEC) in vitro Matrigel angiogenesis model, we show that vascular-like structure formation requires apoptotic cell death through activation of a caspase-dependent mechanism and mitochondrial cytochrome c release. Vascular-like structure formation was further blocked by caspase inhibitors such as z-VAD or Ac-DEVD-CHO, using HUVEC and human lung microvascular endothelial cells. Overexpression of anti-apoptotic human Bcl-2 or baculovirus p35 genes in HUVEC altered endothelial cell rearrangement during in vitro angiogenesis, causing impaired vessel-like structure formation. Caspase inhibitors blocked VEGF- or bFGF-induced HUVEC angiogenesis on 2- or 3-D collagen gels, respectively, confirming that apoptosis was not the result of nonspecific cell death after seeding on the matrix. In an in vivo angiogenesis assay, caspase inhibitors blocked VEGF-dependent vascular formation at the alignment step, as demonstrated histologically. This evidence indicates that endothelial cell apoptosis may be relevant for precise vascular tissue rearrangement in in vitro and in vivo angiogenesis.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blood Vessels; Caspase 3; Caspase Inhibitors; Caspases; Cells, Cultured; Collagen; Cysteine Proteinase Inhibitors; Cytochrome c Group; Drug Combinations; Endothelial Growth Factors; Endothelium, Vascular; Enzyme Activation; Female; Humans; Infant, Newborn; Jurkat Cells; Laminin; Lymphokines; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mitochondria; Neovascularization, Physiologic; Oligopeptides; Proteoglycans; Rats; Time Factors; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 9; Caspase Inhibitors; Cell Line; Cysteine Proteinase Inhibitors; Humans; Kidney; Kinetics; Oligopeptides; Proteins; Recombinant Proteins; Substrate Specificity; X Chromosome; X-Linked Inhibitor of Apoptosis Protein; Zinc Fingers

Nerve growth factor (NGF) mediates a variety of nerve cell actions through receptor tyrosine kinase TrkA. It has been revealed that the Akt pathway contributes to the prevention of apoptosis. It is thought that Parkinson's disease involves apoptosis, and NGF prevents apoptosis in an in vivo model system. However, there is no evidence that the Akt pathway helps to prevent parkinsonism. Here, we report that NGF prevents apoptosis induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in PC12 cells as an in vitro model system of parkinsonism and that this survival effect diminishes on addition of LY294002, a specific inhibitor of phosphatidylinositol 3-kinase. Immunocytochemical analysis revealed that 1 mM MPTP-treated cells or dominant negative Akt-expressing cells, to which were added NGF and MPTP, undergo apoptosis. Moreover, the caspase-3-like activity is increased by addition of MPTP or MPTP with NGF and LY294002. The importance of another signal pathway is shown by PD98059, a specific inhibitor of MAP kinase (MAPK) kinase, but PD98059 does not alter the survival effect in this model system. These results indicate that the Akt pathway helps to prevent parkinsonism by suppressing caspase-3-like activity, but the MAPK pathway is not involved in the NGF-dependent survival enhancing effect in this model system.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acid Chloromethyl Ketones; Amino Acid Substitution; Animals; Apoptosis; Caspase 3; Caspases; Chromones; Coumarins; Cysteine Proteinase Inhibitors; Dopamine Agents; Drug Evaluation, Preclinical; Enzyme Inhibitors; Flavonoids; Genes, Dominant; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Morpholines; Mutagenesis, Site-Directed; Neoplasm Proteins; Nerve Growth Factor; Oligopeptides; Parkinson Disease; Parkinsonian Disorders; PC12 Cells; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoserine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Recombinant Fusion Proteins; Signal Transduction; Transfection

As transmembrane, Ca2+-dependent cell-cell adhesion molecules, cadherins play a central role in tissue morphogenesis and homeostasis. Stable adhesion is dependent on interactions of the cytoplasmic domain of the cadherins with a group of intracellular proteins, the catenins. In the present study, we have detected the expression of alpha-, beta-, and gamma-catenins in human osteoblasts, which assemble with cadherins to form two distinct complexes containing cadherin and alpha-catenin, with either beta- or gamma-catenin. In osteoblasts undergoing apoptosis, proteolytic cleavage of N-cadherin and beta- and gamma- catenins but not alpha-catenin was associated with the activation of caspase-3 and prevented by the caspase inhibitor Z-VAD-fmk. The pattern of cadherin/catenin cleavage detected in apoptotic osteoblasts was reproduced in vitro by recombinant caspase-3. The presence of a 90-kDa extracellular domain fragment of N-cadherin in conditioned medium from apoptotic cells indicates that additional extracellular or membrane-associated proteases also are activated. Disruption of N-cadherin-mediated cell-cell adhesion with function-blocking antibodies induced osteoblast apoptosis, activation of caspases, and cleavage of beta-catenin. These findings provide compelling evidence that N-cadherin-mediated cell-cell adhesion promotes osteoblast survival and suggest that the underlying mechanism may involve activation of beta-catenin signaling.

Topics: alpha Catenin; Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; beta Catenin; Cadherins; Caspase 1; Caspase 3; Caspase Inhibitors; Caspases; Cell Adhesion; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Desmoplakins; gamma Catenin; Humans; Oligopeptides; Osteoblasts; Peptide Fragments; Trans-Activators

Sodium nitroprusside (SNP) induces apoptosis in H9C2 cardiac muscle cells. Treatment with an exogenous NO donor SNP (2 mM) to H9C2 cells resulted in apoptotic morphological changes; a bright blue-fluorescent condensed nuclei and chromatin fragmentation by fluorescence microscope of Hoechst 33258-staining. The activity of caspase-3 like protease was increased during SNP-induced cell death. However, the activity of caspase-1 like protease was not affected by SNP. Pretreatment with Z-VAD-FMK (a pan-caspase inhibitor) or Ac-DEVD-CHO (a specific caspase-3 inhibitor) abrogated the SNP-induced cell death. SNP markedly activated three MAP kinases (JNK/SAPK, ERK and p38 MAP kinase) in the cardiac muscle cells. In this study, selective inhibition of the ERK or p38 MAPK pathway (by PD98059 or SB203580, respectively) had no effect on the extent of SNP-induced apoptosis in cardiac muscle cells. In contrast, inhibition of the JNK pathway by transfection of a dominant negative mutant of JNK markedly reduced the extent of SNP-induced cell death. Taken together, we suggest that JNK/SAPK will be related to SNP-induced apoptosis of H9C2 cardiac muscle cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 1; Caspase 3; Caspase Inhibitors; Caspases; Cell Line; Cysteine Proteinase Inhibitors; Cytochrome c Group; Enzyme Activation; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Mutation; Myocardium; Nitric Oxide Donors; Nitroprusside; Oligopeptides; Rats; Transfection

Infection of humans with influenza A virus (IAV) results in a severe transient leukopenia. The goal of these studies was to analyze possible mechanisms behind this IAV-induced leukopenia with emphasis on the potential induction of apoptosis of lymphocytes by the virus. Analysis of lymphocyte subpopulations after exposure to IAV showed that a portion of CD3(+), CD4(+), CD8(+), and CD19(+) lymphocytes became apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling positive). The percentage of cells that are infected was shown to be less than the percentage of apoptotic cells, suggesting that direct effects of cell infection by the virus cannot account fully for the high level of cell death. Removal of monocytes-macrophages after IAV exposure reduced the percent of lymphocytes that were apoptotic. Treatment of virus-exposed cultures with anti-tumor necrosis factor alpha did not reduce the percentage of lymphocytes that were apoptotic. In virus-exposed cultures treated with anti-FasL antibody, recombinant soluble human Fas, Ac-DEVD-CHO (caspase-3 inhibitor), or Z-VAD-FMK (general caspase inhibitor), apoptosis and production of the active form of caspase-3 was reduced. The apoptotic cells were Fas-high-density cells while the nonapoptotic cells expressed a low density of Fas. The present studies showed that Fas-FasL signaling plays a major role in the induction of apoptosis in lymphocytes after exposure to IAV. Since the host response to influenza virus commonly results in recovery from the infection, with residual disease uncommon, lymphocyte apoptosis likely represents a part of an overall beneficial immune response but could be a possible mechanism of disease pathogenesis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Fas Ligand Protein; fas Receptor; Humans; Influenza A virus; Lymphocytes; Membrane Glycoproteins; Oligopeptides

Dlk/ZIP kinase is a member of the Death Associated Protein (DAP) kinase family of pro-apoptotic serine/threonine kinases that have been implicated in regulation of apoptosis and tumour suppression. Expression of both Dlk/ZIP kinase and its interaction partner Par-4 is maintained in four medulloblastoma cell lines investigated, whereas three of seven neuroblastoma cell lines have lost expression of Par-4. Overexpression of a constitutively pro-apoptotic deletion mutant of Dlk/ZIP kinase induced significant apoptosis in D283 medulloblastoma cells. Cell death was characterized by apoptotic membrane blebbing, and a late stage during which the cells had ceased blebbing and were drastically shrunken or disrupted into apoptotic bodies. Over-expression of the anti-apoptotic Bcl-xL protein had no effect on Dlk/ZIP kinase-induced membrane blebbing, but potently inhibited Dlk/ZIP kinase-induced cytochrome c release and transition of cells to late stage apoptosis. Treatment with caspase inhibitors delayed, but did not prevent entry into late stage apoptosis. These results demonstrate that Dlk/ZIP kinase-triggered apoptosis involves the mitochondrial apoptosis pathway. However, cell death proceeded in the presence of caspase inhibitors, suggesting that Dlk/ZIP kinase is able to activate alternative cell death pathways. Alterations of signal transduction pathways leading to Dlk/ZIP kinase induced apoptosis or loss of expression of upstream activators could play important roles in tumour progression and metastasis of neural tumours.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Calcium-Calmodulin-Dependent Protein Kinases; Carrier Proteins; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Cytochrome c Group; Death-Associated Protein Kinases; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Intracellular Membranes; Intracellular Signaling Peptides and Proteins; Luminescent Proteins; Medulloblastoma; Microscopy, Fluorescence; Mitochondria; Mutation; Neuroblastoma; Oligopeptides; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Signal Transduction; Transfection; Tumor Cells, Cultured

We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin.

Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; Aspartic Acid; Calcium-Binding Proteins; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; fas Receptor; Humans; Jurkat Cells; Leupeptins; Oligopeptides; Poly(ADP-ribose) Polymerases; Recombinant Proteins; Tumor Necrosis Factor-alpha

Excessive apoptosis may have a role in the ineffective hematopoiesis and cytopenias observed in myelodysplastic syndromes. The goals of this study were 1) to quantify apoptosis in patients with "early stage" myelodysplasia [including patients with refractory anemia (RA), RA with ringed sideroblasts (RARS), RA with excess blasts and with less than 10% blasts (RAEB(<10))], and in patients with "late stage" myelodysplasia [including RAEB with more than 10% blasts (RAEB(>10)), RAEB in transformation (RAEB-t), and acute myeloid leukemia secondary to myelodysplasia (LAM2)]; 2) to study the activation of the caspase-3/CPP32 enzyme, a major "effector" caspase in hematopoiesis, in patients with "early stage" myelodysplasia, and 3) to evaluate the effect of caspase inhibition on the apoptotic phenotype and clonogenicity of hematopoietic progenitors in vitro in these patients.. Fifty-four patients with myelodysplastic syndromes, including 30 with "early stage" myelodysplasia and 24 with "late stage" myelodysplasia were studied. Study of apoptosis: TUNEL assay performed on bone marrow smears and/or quantification of annexin V positive bone marrow mononuclear cells by flow cytometric analysis. Caspacse-3/CPP32 activity: Quantitative measurement of caspase-3/CPP32 activity on total bone marrow mononuclear cells using a fluorogenic substrate. Effect of the caspase-inhibitor Z-VAD-FMK: 1) on the apoptotic phenotype of total bone marrow mononuclear cells and 2) on the clonogenicity of hematopoietic progenitor cells.. The group of 30 patients with "early stage" myelodysplasia had statistically increased apoptosis compared to the group of 24 patients with "late stage" myelodysplasia (44.1% +/- 4.8 vs 21.8% +/- 3.6; p = 0.02) using the TDT-mediated dUTP nick-end labeling (TUNEL) assay. In the group of patients with RAEB, those with MDS(RAEB<10) had excessive apoptosis compared to those with MDS(RAEB>10) (44.0% +/- 3.5% vs 29.5% +/- 3.6%;p = 0.042) The median caspase-3 activity in 20 "early stage" myelodysplasia patients was 19,000 U (range 3,460-41,000) and significantly increased compared to normal individuals (4,256 U, range 3,200-5,200; p = 0.032) Bone marrow mononuclear cells from 12 "early stage" MDS patients (including 11 from the 20 studied for caspase-3 activity) were incubated with or without the broad-spectrum caspase inhibitor Z-VAD-FMK. In 4 of 9 evaluable patients (44.4%) with excessive apoptosis, the number of annexin V positive cells decreased in a dose-dependent manner in the presence of Z-VAD-FMK. However, in none of these patients was caspase inhibition with Z-VAD-FMK able to enhance colony formation in vitro.. These results confirm that a major characteristic of patients with "early stage" myelodysplasia is increased apoptosis. The results also indicate that excessive apoptosis in these patients is accompanied by increased caspase-3/CPP32 activity. However, caspase inhibition with the broad-spectrum inhibitor Z-VAD-FMK cannot improve hematopoiesis in this group of patients, even when apoptosis is attenuated.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cells, Cultured; Colony-Forming Units Assay; Cysteine Proteinase Inhibitors; Hematopoietic Stem Cells; Humans; Myelodysplastic Syndromes; Oligopeptides; Time Factors

APP is a precursor of beta amyloid deposited in Alzheimer's disease (AD). Although genetic studies established that mutations in APP cause familial AD (FAD), the mechanism for neuronal death by FAD mutants has not been well understood. We established neuronal cells (F11/EcR/V642I cells) in which V642I APP was inducibly expressed by ecdysone. Treatment with ecdysone, but not vehicle, killed most cells within a few days, with rounding, shrinkage, and detachment as well as nuclear fragmentation. Death was suppressed by Ac-DEVD-CHO and pertussis toxin. Electron microscopic analysis revealed that apoptosis occurred in ecdysone-treated cells. V642I-APP-induced death was suppressed by the anti-AD factors estrogen and apoE2. These data demonstrate not only that expression of this FAD gene causes neuronal apoptosis, but that F11/EcR/V642I cells, the first neuronal cells with inducible FAD gene expression, provide a useful model system in investigating AD disorders.

Topics: Alzheimer Disease; Amino Acid Chloromethyl Ketones; Amino Acid Substitution; Amyloid beta-Protein Precursor; Animals; Apolipoprotein E2; Apolipoprotein E4; Apolipoproteins E; Apoptosis; Cell Line; Cell Survival; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Ecdysone; Estradiol; Gene Expression; Hybrid Cells; Mice; Models, Biological; Neurons; Oligopeptides; Pertussis Toxin; Rats; Receptors, Steroid; Recombinant Proteins; Transfection; Virulence Factors, Bordetella

Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Cytochrome c Group; Fenretinide; Free Radicals; Humans; Mitochondria; Neuroblastoma; Oligopeptides; Poly(ADP-ribose) Polymerases; Receptors, Retinoic Acid; Tumor Cells, Cultured

Tamoxifen (TAM) is widely used in the treatment of breast cancer. The cytostatic effects of TAM have been attributed to the antagonism of estrogen receptor (ER) and inhibition of estrogen-dependent proliferative events. However, the mechanism by which TAM is also effective against certain ER-negative breast tumors remains to be elucidated. Here we report that TAM induced the activity of caspase-3-like proteases in ER-negative breast cancer cell lines MDA-MB-231 and BT-20, as evidenced by the cleavage of fluorogenic tetrapeptide substrate and of poly(ADP-ribose) polymerase. The activation of caspase-3-like proteases preceded TAM-induced chromatin condensation and nuclear fragmentation, the typical apoptotic morphologies. Pretreatment of cells with a specific inhibitor of caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde, or with a general inhibitor of caspases, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, prevented TAM-induced apoptosis. TAM also stimulated c-Jun NH2-terminal kinase (JNK) 1 activity, and interfering with the JNK pathway by over-expressing a DN JNK1 mutant attenuated TAM-induced apoptosis. In addition, treatment of cells with a lipid-soluble antioxidant vitamin E blocked TAM-induced caspase-3 and JNK1 activation as well as apoptosis, whereas water-soluble antioxidants N-acetyl L-cysteine and glutathione had little effect. Thus, this study demonstrates that TAM induces apoptosis in ER-negative breast cancer cells through caspase-3 and JNK1 pathways, which are probably initiated at the cell membrane by an oxidative mechanism.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Drug Interactions; Enzyme Activation; Enzyme Induction; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Oligopeptides; Tamoxifen; Tumor Cells, Cultured

Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is believed to be due, in part, to the production of reactive oxygen species (ROS) and/or an inhibition of mitochondrial function. However, little is known about the ensuing intracellular events which ultimately result in cell death. Here we show that exposure to relatively low concentrations of 6-OHDA induces apoptosis of cerebellar granule neurons (CGN). 6-OHDA-induced apoptosis of CGN is associated with activation of a caspase-3-like protease. Western blots of cytosolic extracts from 6-OHDA-treated CGN reveal a translocation of cytochrome c from mitochondria to the cytosol, which precedes activation of the protease detected by Ac-DEVD-pNA. DNA laddering can be blocked by caspase inhibitors zVAD-FMK and Ac-DEVD-CHO, however cell death can only be attenuated for a short time period in the presence of these inhibitors. Our data suggest that 6-OHDA-induced apoptosis of CGN involves activation of a caspase-3-like protease. In contrast to the neurotoxicity induced by MPP+, however, the peptide inhibitors zVAD-FMK and Ac-DEVD-CHO can only attenuate early neuronal death induced by 6-OHDA. At later time points, neuronal death lacking DNA laddering occurs even in the presence of the peptide inhibitor zVAD-FMK or Ac-DEVD-CHO.

Topics: 1-Methyl-4-phenylpyridinium; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspases; Cells, Cultured; Cerebellum; Cysteine Proteinase Inhibitors; Cytochrome c Group; DNA Fragmentation; Dopamine Agents; Mitochondria; Neurons; Oligopeptides; Oxidopamine; Parkinson Disease, Secondary; Rats; Rats, Sprague-Dawley; Sympatholytics

Caspases have been implicated in the induction of apoptosis in most systems studied. The importance of caspases for apoptosis was further investigated using the system of didemnin B-induced apoptosis. We found that benzyloxycarbonyl-VAD-fluoromethylketone, a general caspase inhibitor, inhibits didemnin B-induced apoptosis in HL-60 and Daudi cells. Acetyl-YVAD-chloromethylketone, a caspase-1-like activity inhibitor, inhibits didemnin B-induced apoptosis in Daudi cells, whereas the caspase-3-like activity inhibitor, acetyl-DEVD-aldehyde, has no effect. Using immunoblots to investigate cleavage of caspases-1 and -3, we found that both caspases are activated in both cell lines. We showed that the caspase substrate poly(ADP-ribose)polymerase is cleaved in these cells after didemnin B treatment. In both cell lines, poly(ADP-ribose)polymerase cleavage is inhibited by benzyloxycarbonyl-VAD-fluoromethylketone and also by acetyl-YVAD-chloromethylketone in Daudi cells. These results indicate that a caspase(s) other than caspase-3 is required for didemnin B-induced apoptosis. We show that caspases may be activated during apoptosis that are not required for the progression of apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 1; Caspase 3; Caspases; Cell Line; Depsipeptides; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Immunoblotting; Oligopeptides; Peptides, Cyclic; Poly(ADP-ribose) Polymerases; Purines; Roscovitine; Time Factors

We have previously demonstrated that calpain is responsible for the cleavage of Bax, a proapoptotic protein, during drug-induced apoptosis of HL-60 cells (Wood, D. E., Thomas, A., Devi, L. A., Berman, Y., Beavis, R. C., Reed, J. C., and Newcomb, E. W. (1998) Oncogene 17, 1069-1078). Here we show the sequential activation of caspases and calpain during drug-induced apoptosis of HL-60 cells. Time course experiments using the topoisomerase I inhibitor 9-amino-20(S)-camptothecin revealed that cleavage of caspase-3 substrates poly(ADP-ribose) polymerase (PARP) and the retinoblastoma protein as well as DNA fragmentation occurred several hours before calpain activation and Bax cleavage. Pretreatment with the calpain inhibitor calpeptin blocked calpain activation and Bax cleavage but did not inhibit PARP cleavage, DNA fragmentation, or 9-amino-20(S)-camptothecin-induced morphological changes and cell death. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) inhibited PARP cleavage, DNA fragmentation, calpain activation, and Bax cleavage and increased cell survival by 40%. Interestingly, Z-VAD-fmk-treated cells died in a caspase- and calpain-independent manner that appeared morphologically distinct from apoptosis. Our results suggest that excessive or uncontrolled calpain activity may play a role downstream of and distinct from caspases in the degradation phase of apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Calpain; Caspase 3; Caspases; Coumarins; Cysteine Proteinase Inhibitors; Dipeptides; DNA Fragmentation; Enzyme Activation; HL-60 Cells; Humans; Oligopeptides

To date, eight neurodegenerative disorders, including Huntington's disease and dentatorubral-pallidoluysian atrophy, have been identified to be caused by expansion of a CAG repeat coding for a polyglutamine (polyQ) stretch. It is, however, unclear how polyQ expansion mediates neuronal cell death observed in these disorders. Here, we have established a tetracycline-regulated expression system producing 19 and 56 repeats of glutamine fused with green fluorescent protein. Induced expression of the 56 polyQ, but not of the 19 polyQ stretch caused marked nuclear aggregation and apoptotic morphological changes of the nucleus. In vitro enzyme assays and Western blotting showed that polyQ56 expression sequentially activated initiator and effector caspases, such as caspase-8 or -9, and caspase-3, respectively. Furthermore, using cell-permeable fluorogenic substrate, the activation of caspase-3-like proteases was demonstrated in intact cells with aggregated polyQ. This is the first direct evidence that the expression of extended polyQ activates caspases and together with the previous findings that some of the products of genes responsible for CAG repeat diseases are substrates of caspase-3 indicates an important role of caspases in the pathogenesis of these diseases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Nucleus; Enzyme Activation; Female; Green Fluorescent Proteins; Humans; Luminescent Proteins; Neurodegenerative Diseases; Oligopeptides; Ovarian Neoplasms; Peptides; Poly(ADP-ribose) Polymerases; Protein Binding; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured

p27[KIP1] (p27) is a cyclin dependent kinase inhibitor, involved in the negative regulation of G1 progression in response to a number of anti-proliferative signals. In this study we show, in growing mouse hybridoma (7TD1) and human myeloma (U266) cell lines, that p27 is highly expressed but slightly upregulated when cells are arrested, regardless to the phases of the cell cycle. In contrast, the specific blockade of these cells in early G1 phase reveals the induction of a protein of 23 kDa (p23) specifically recognized by polyclonal anti-p27 antibodies raised against the NH2 terminal part of p27 but not by anti-p21[CIP1] antibodies. Experiments using caspase inhibitors strongly suggest that p23 results from the proteolysis of p27 by a 'caspase-3-like' protease. This cleavage leads to the cytosolic sequestration of p23 but does not alter its binding properties to CDK2 and CDK4 kinases. Indeed, p23 associated in vivo with high molecular weight complexes and coprecipitated with CDK2 and CDK4. We demonstrate by transfection experiments in SaOS-2 cells that p23 induces a G1 phase growth arrest by inhibition of cyclin/CDK2 activity. In summary we describe here a caspase-cleaved form of p27, induced in absence of detectable apoptosis and likely involved in cell cycle regulation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cell Line; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Cysteine Proteinase Inhibitors; Cytosol; Dimethyl Sulfoxide; G1 Phase; Humans; Hybridomas; Interleukin-6; Mice; Microtubule-Associated Proteins; Molecular Weight; Multiple Myeloma; Oligopeptides; Peptide Fragments; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Proteins

Hinokitiol, a potent iron chelator, has been reported to induce differentiation in teratocarcinoma F9 cells with a reduction of viable cells. In this study, we examined the steps leading to eventual cell death by hinokitiol during differentiation. Hinokitiol induced DNA fragmentation of F9 cells in a concentration- and time-dependent manner. This effect was also observed in a cell-free system using the nuclei from intact cells and the cytosols from hinokitiol-treated cells. In contrast, hinokitiol methyl ether and hinokitiol-Fe (III) complex, which are deficient in iron-chelating activity, showed no DNA fragmentation activity in both cell culture and cell-free systems. These results suggest that iron deprivation by hinokitiol may be involved in the induction of apoptosis of F9 cells. Caspase-3, one of the key enzymes in the apoptotic cascade, was specifically activated by hinokitiol treatment, but not by the other two derivatives. In addition, its specific inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, strongly blocked hinokitiol-induced DNA fragmentation. These results indicate that iron deprivation by hinokitiol can induce apoptosis of F9 cells through the activation of caspase-3.

Topics: Amino Acid Chloromethyl Ketones; Aniline Compounds; Apoptosis; Caspase 3; Caspases; Cell-Free System; Cysteine Proteinase Inhibitors; DNA Fragmentation; Embryonal Carcinoma Stem Cells; Enzyme Activation; Iron Chelating Agents; Monoterpenes; Neoplastic Stem Cells; Oligopeptides; Teratocarcinoma; Tropolone

Studies with peptide-based and macromolecular inhibitors of the caspase family of cysteine proteases have helped to define a central role for these enzymes in inflammation and mammalian apoptosis. A clear interpretation of these studies has been compromised by an incomplete understanding of the selectivity of these molecules. Here we describe the selectivity of several peptide-based inhibitors and the coxpox serpin CrmA against 10 human caspases. The peptide aldehydes that were examined (Ac-WEHD-CHO, Ac-DEVD-CHO, Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO) included several that contain the optimal tetrapeptide recognition motif for various caspases. These aldehydes display a wide range of selectivities and potencies against these enzymes, with dissociation constants ranging from 75 pM to >10 microM. The halomethyl ketone benzyloxycarbonyl-VAD fluoromethyl ketone is a broad specificity irreversible caspase inhibitor, with second-order inactivation rates that range from 2.9 x 10(2) M-1 s-1 for caspase-2 to 2.8 x 10(5) M-1 s-1 for caspase-1. The results obtained with peptide-based inhibitors are in accord with those predicted from the substrate specificity studies described earlier. The cowpox serpin CrmA is a potent (Ki < 20 nM) and selective inhibitor of Group I caspases (caspase-1, -4, and -5) and most Group III caspases (caspase-8, -9, and -10), suggesting that this virus facilitates infection through inhibition of both apoptosis and the host inflammatory response.

Topics: Aldehydes; Amino Acid Chloromethyl Ketones; Caspases; Cysteine Proteinase Inhibitors; Humans; Peptides; Recombinant Proteins; Serpins; Substrate Specificity; Viral Proteins

We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.

Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Apoptosis; Bridged-Ring Compounds; Caspase 1; Caspase 3; Caspases; Ceramides; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytochalasins; fas Receptor; Histocompatibility Antigens Class I; Humans; Mitochondria; Models, Biological; Norbornanes; Okadaic Acid; Oligopeptides; Proton-Motive Force; Signal Transduction; T-Lymphocytes; Thiocarbamates; Thiones

Trophic factor deprivation induces neuronal nitric oxide synthase (NOS) and apoptosis of rat embryonic motor neurons in culture. We report here that motor neurons constitutively express endothelial NOS that helps support the survival of motor neurons cultured with brain-derived neurotrophic factor (BDNF) by activating the nitric oxide-dependent soluble guanylate cyclase. Exposure of BDNF-treated motor neurons to nitro-L-arginine methyl ester (L-NAME) decreased cell survival 40-50% 24 hr after plating. Both low steady-state concentrations of exogenous nitric oxide (<0.1 microM) and cGMP analogs protected BDNF-treated motor neurons from death induced by L-NAME. Equivalent concentrations of cAMP analogs did not affect cell survival. Inhibition of nitric oxide-sensitive guanylate cyclase with 2 microM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced the survival of BDNF-treated motor neurons by 35%. cGMP analogs also protected from ODQ-induced motor neuron death, whereas exogenous nitric oxide did not. In all cases, cell death was prevented with caspase inhibitors. Our results suggest that nitric oxide-stimulated cGMP synthesis helps to prevent apoptosis in BDNF-treated motor neurons.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Brain-Derived Neurotrophic Factor; Cell Survival; Cells, Cultured; Cyclic GMP; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Fetus; Guanylate Cyclase; Motor Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oligopeptides; Oxadiazoles; Quinoxalines; Rats; Solubility

Multiple aspartate-specific cysteine proteases have been identified and specific members of this family have been implicated in the apoptotic death of many mammalian cell types. Caspase-3-like proteases seem to play a pivotal role in neuronal apoptosis since mice with germline inactivation of the caspase-3 gene manifest profound alterations in neurogenesis. Moreover, inhibitors of caspase-3-related proteases have been shown to inhibit neuronal apoptosis. Here we extend recent work from our laboratory on the mechanisms mediating the neurotoxic actions of 1-methyl-4-phenylpyridinium using ventral mesencephalon cultures containing dopamine neurons. We demonstrate that low concentrations of 1-methyl-4-phenylpyridinium induce apoptosis in dopamine neurons by morphological and biochemical criteria. Moreover, pretreatment of ventral mesencephalon cultures with the tetrapeptide inhibitors of the caspase-3-like proteases zVAD-FMK or Ac-DEVD-CHO specifically inhibit death of dopamine neurons induced by low concentrations of 1-methyl-4-phenylpyridinium, whereas the caspase-1-like inhibitor Ac-YVAD-CHO was without effect. Our data indicate that exposure of cultured ventral mesencephalon dopamine neurons to low concentrations of 1-methyl-4-phenylpyridinium results in apoptotic death and that caspase-3-like proteases may mediate the neurotoxic apoptotic actions of 1-methyl-4-phenylpyridinium.

Topics: 1-Methyl-4-phenylpyridinium; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspases; Cell Death; Cells, Cultured; Cysteine Proteinase Inhibitors; Dopamine; Fetus; Mesencephalon; Mice; Neurons; Oligopeptides; Rats

Granulysin is a newly described cytolytic molecule released by CTL and NK cells via granule-mediated exocytosis. It shares homology with saposin-like proteins, including NK-lysin and amoebapores, and has been implicated in the lysis of tumor cells and microbes. In the present study we show that recombinant granulysin alone induces apoptosis of Jurkat cells. This apoptosis is associated with a sixfold increase in the ceramide/sphingomyelin ratio, implicating the activation of sphingomyelinases. Granulysin- and ceramide-induced apoptosis are similar in that they both are only minimally inhibited by the more selective cysteine protease p32 (caspase 3)-like caspase inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde, while they are significantly inhibited by the more general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). Nevertheless, while Z-VAD-fmk almost completely inhibits ceramide-induced apoptosis, a Z-VAD-fmk-resistant component was observed using granulysin. Granulysin also causes apoptosis in cells depleted of sphingomyelin by prolonged treatment with the ceramide synthase inhibitor fumonisin B1. These data indicate that granulysin induces target cell death by both ceramide- and caspase-dependent and -independent pathways.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antigens, Differentiation, T-Lymphocyte; Apoptosis; Carboxylic Acids; Cattle; Ceramides; Cysteine Proteinase Inhibitors; Cytotoxicity, Immunologic; Fumonisins; Humans; Jurkat Cells; Oligopeptides; Oxidoreductases; Recombinant Proteins; Sphingomyelins

Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspases; Cisplatin; Cytochrome c Group; Deoxyadenine Nucleotides; Etoposide; Eukaryotic Initiation Factor-4G; HeLa Cells; Humans; Kinetics; Oligopeptides; Peptide Initiation Factors; Protease Inhibitors; Protein Biosynthesis; Recombinant Proteins

Glucocorticoids (GCs) are essential therapeutic reagents for the treatment of lymphomas and leukemias. GCs cause cell death in certain types of lymphoid cells mediated by the process known as apoptosis. This cell death is completely inhibited by Bcl-2. Here we report that Bcl-2 and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), a broad spectrum caspase inhibitor, prevent loss of mitochondrial membrane potential (delta psi m) and the production of reactive oxygen species (ROS) caused by GC, while acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), an inhibitor of the caspase-3 family proteases, does not. This suggests that the inhibition by Bcl-2 and activation of some initiator caspases are upstream events of mitochondrial damage, whereas the activation of caspase-3 family proteases occurs downstream of mitochondrial changes. We also demonstrate that caspase-6 but not caspase-3 is cleaved and activated during GC-mediated apoptosis and that poly(ADP-ribose) polymerase (PARP), a substrate of caspases, also undergoes proteolysis. In addition, we provide the evidence that DNA fragmentation is markedly inhibited by Ac-DEVD-CHO, while cell death, assessed by the damage of the plasma membrane, is marginally inhibited or merely delayed.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspase 6; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Dexamethasone; DNA Fragmentation; Enzyme Precursors; Gene Expression Regulation, Enzymologic; Glucocorticoids; Humans; Leukemia, B-Cell; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tumor Cells, Cultured

Multicellular organisms can employ a number of defences to combat viral replication, the most dramatic being implementation of a cell autonomous apoptotic process. The overall cost to the viability of an organism of losing infected cells by apoptosis may be small if the dying cells can be substituted. In contrast, suicide of irreplaceable cells such as highly specialised neurons may have a more dramatic, even fatal consequence. Previous in vitro approaches to understanding whether neurotropic viruses cause neurons to apoptose have utilised transformed cell lines. These are not in the appropriate state of differentiation to provide an accurate indication of events in vivo. We have chosen to characterise the ability of a model CNS disease-causing virus, Semliki Forest virus (SFV), to infect and trigger apoptosis in primary cultures of nerve growth factor (NGF)-dependent sensory neurons. These cells are known to die when deprived of NGF and constitute a useful indicator of apoptosis. We observe that infection causes cell death which bears the morphological hallmarks of apoptosis, this occurs even in the present of survival promoting NGF and is concomitant with new virus production. Using the TUNEL (transferase dUTP nick end labelling) technique we show that SFV-induced apoptosis involves DNA fragmentation and requires caspase (CED-3/ICE cysteine protease) activation, as does apoptosis induced by NGF-deprivation. Extensive areas of apoptosis, as defined using a combination of ultrastructural analysis and TUNEL occur in infected neonatal mouse brains. The novel evidence that infection of primary neurons with SFV induces apoptosis with activation of one or more caspases defines a system for the further anlaysis of apoptosis regulation in physiologically relevant neurons.

Topics: Alphavirus Infections; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Brain; Caspase Inhibitors; Caspases; Cell Nucleus; Cysteine Proteinase Inhibitors; DNA Fragmentation; Encephalitis, Viral; Female; In Situ Nick-End Labeling; Mice; Microscopy, Electron; Nerve Growth Factors; Neurons; Oligopeptides; Pregnancy; Semliki forest virus; Serine Proteinase Inhibitors; Tosyllysine Chloromethyl Ketone

PC12 cells are a useful model system for studying neuronal apoptosis. Like neurons, they undergo apoptosis when deprived of trophic support. Involvement of caspases [interleukin 1beta-converting enzyme (ICE)-related proteases] has been implicated in apoptosis induced by various stimuli in many cell types, including neurons. In the present study we investigated the need for caspases participation in apoptosis induced by growth factor deprivation in naive and neuronal PC12 cells. For this purpose we generated PC12 cell lines that consistently express the viral caspases inhibitor genes p35 or crmA, and analyzed their susceptibility to trophic factor deprivation. We also examined the effects of cell-permeable peptide inhibitors of caspases. Our results showed that broad-spectrum inhibitors of the caspases, namely the baculovirus p35 gene and the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, effectively inhibit the death of both naive and neuronal PC12 cells. However, caspase-1 (ICE)-specific inhibitors, namely the peptides Ac-Try-Val-Ala-Asp-chloromethylketone and Ac-Try-Val-Ala-Asp-aldehyde, as well as crmA, were much less effective. These findings demonstrate that caspases, but not caspase-1, are needed for apoptosis induced by trophic factor deprivation in both naive and neuronal PC12 cells. Northern and Western blot analyses showed that PC12 cells express caspase-3. We therefore examined the involvement of caspase-3 in the death process of trophic factor-deprived PC12 cells. Our results showed that the pro-caspase-3 and its substrate poly-(ADP-ribose) polymerase are cleaved at similar rates in serum-deprived PC12 cells. Moreover, cell lysates prepared from these cells possess caspase-3-like activity, as determined by their ability to cleave the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin. These findings strongly suggest that caspase-3 or caspase-3-like proteases are activated in trophic factor-deprived PC12 cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Baculoviridae; Caspase 1; Caspase 3; Caspases; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Viral; Inhibitor of Apoptosis Proteins; Nerve Growth Factors; Neurons; Oligopeptides; PC12 Cells; Rats; Recombinant Proteins; RNA, Messenger; Serpins; Viral Proteins

Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Apoptosis; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Leukemia, Monocytic, Acute; Leukemia, T-Cell; Molecular Sequence Data; Oligopeptides; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protease Inhibitors; Tumor Cells, Cultured