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1.
Cell Death Differ ; 12(12): 1622-35, 2005 Dec.
Article in English | MEDLINE | ID: mdl-15933739

ABSTRACT

High-risk strains of human papillomavirus, including HPV 16, cause human cervical carcinomas, due in part to the activity of their E6 oncogene. E6 interacts with a number of cellular proteins involved in host-initiated apoptotic responses. Paradoxically, literature reports show that E6 can both protect cells from and sensitize cells to tumor necrosis factor (TNF). To examine this apparent contradiction, E6 was transfected into U2OS cells and stable clones were treated with TNF. Intriguingly, clones with a high level of E6 expression displayed an increased sensitivity to TNF by undergoing apoptosis, while those with low expression were resistant. Furthermore, TNF treatment of cells in which the expression of E6 was regulated by the addition of doxycycline demonstrated clearly that while low levels of E6 protect cells from TNF, high levels sensitize cells. Together, these results demonstrate that virus-host interactions can be complex and that both quantitative and qualitative aspects are important in determining outcome.


Subject(s)
Apoptosis/physiology , Oncogene Proteins, Viral/metabolism , Repressor Proteins/metabolism , Tumor Necrosis Factors/pharmacology , Apoptosis/drug effects , Caspases/metabolism , Cell Line, Tumor , Doxycycline/pharmacology , Enzyme Activation , Humans , Tumor Suppressor Protein p53/metabolism
2.
Cell Death Differ ; 9(9): 915-25, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12181742

ABSTRACT

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a nuclear autoantigen in atopic disorders implicated in cellular protection against stress-induced apoptosis. We observed that LEDGF/p75 was cleaved during apoptosis into fragments of 65 and 58 kD generated by caspases-3 and -7 cleaving at three sites: DEVPD30/G, DAQD486/G and WEID85/N. Sequence analysis revealed that the DEVPD30/G and WEID85/N sites lie within the highly conserved HATH (homologous to amino terminus of hepatoma-derived growth factor) region, also known as PWWP domain. Alignment of proteins containing this domain failed to reveal conservation of the DEVPD30/G and WEID85/N sites, suggesting that the HATH/PWWP domain of LEDGF/p75 may be specifically targeted by caspases. Overexpression of LEDGF/p75 protected HepG2 cells from serum starvation-induced cell death, whereas expression of the 65 kD fragment failed to protect. The apoptotic cleavage of LEDGF/p75 may contribute to the pathogenesis of atopic disorders by abrogating its pro-survival function and enhancing its immunogenicity.


Subject(s)
Apoptosis/immunology , Autoimmune Diseases/immunology , Caspases/immunology , Cell Survival/immunology , Hypersensitivity, Immediate/immunology , Intercellular Signaling Peptides and Proteins/immunology , Apoptosis/genetics , Asthma/enzymology , Asthma/genetics , Asthma/immunology , Autoimmune Diseases/enzymology , Autoimmune Diseases/genetics , Binding Sites/genetics , Caspase 3 , Caspase 7 , Caspases/metabolism , Culture Media, Serum-Free/pharmacology , Dermatitis, Atopic/enzymology , Dermatitis, Atopic/genetics , Dermatitis, Atopic/immunology , Gene Expression Regulation/genetics , HeLa Cells , Humans , Hypersensitivity, Immediate/enzymology , Hypersensitivity, Immediate/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Jurkat Cells , Molecular Sequence Data , Mutation/physiology , Peptide Fragments/genetics , Peptide Fragments/immunology , Peptide Fragments/metabolism , Peptide Hydrolases/metabolism , Rhinitis/enzymology , Rhinitis/genetics , Rhinitis/immunology , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
3.
Arthritis Rheum ; 44(11): 2642-52, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11710720

ABSTRACT

OBJECTIVE: Specific autoantigens targeted in systemic autoimmunity undergo posttranslational modifications, such as cleavage, during cell death that could potentially enhance their immunogenicity. In light of the increasing interest in the immunologic consequences of defective clearance of apoptotic cells, we sought to determine whether autoantigens cleaved during apoptosis undergo an additional wave of proteolysis as apoptosis progresses to secondary necrosis in the absence of phagocytosis. METHODS: Apoptosis was induced in Jurkat cells with etoposide, anti-Fas antibody, or staurosporine (STS), and in HeLa cells with STS. Progression to secondary necrosis was assessed morphologically and quantified by trypan blue uptake. Autoantigen proteolysis during cell death was examined by immunoblotting of cell lysates using highly specific human autoantibodies as detecting probes. RESULTS: Cells treated with the different apoptosis inducers underwent a rapid apoptosis that gradually progressed to secondary necrosis. During the initial apoptotic stages, several autoantigens, including poly(ADP-ribose) polymerase, topoisomerase I (or Scl-70), SSB/La, and U1-70 kd, were cleaved into their signature apoptotic fragments. Progression of apoptosis to secondary necrosis was associated with additional proteolysis of these and other autoantigens in a caspase-independent manner. Some autoantigens (e.g., ribosomal RNP, Ku, and SSA/Ro) appeared to be resistant to proteolysis during cell death. CONCLUSION: In the absence of phagocytosis, apoptotic cells may undergo secondary necrosis, a process associated with additional proteolytic degradation of specific autoantigens. Secondary necrosis may occur in vivo in autoimmune disorders associated with impaired clearance of apoptotic cells and serve as a source of modified forms of specific autoantigens that might stimulate autoantibody responses under proinflammatory conditions.


Subject(s)
Apoptosis/immunology , Autoantigens/metabolism , Necrosis , Amino Acid Chloromethyl Ketones/pharmacology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Murine-Derived , Apoptosis/drug effects , Caspases/metabolism , DNA Topoisomerases, Type I/metabolism , Drug Combinations , Etoposide/pharmacology , HeLa Cells/drug effects , HeLa Cells/metabolism , HeLa Cells/pathology , Humans , Jurkat Cells/drug effects , Jurkat Cells/metabolism , Jurkat Cells/pathology , Poly(ADP-ribose) Polymerases/metabolism , Ribonucleoprotein, U1 Small Nuclear/metabolism , Staurosporine/pharmacology
4.
J Periodontol ; 72(5): 641-50, 2001 May.
Article in English | MEDLINE | ID: mdl-11394400

ABSTRACT

BACKGROUND: The protease-induced cytotoxicity of P. gingivalis may partly result from alteration of the extracellular matrix and/or surface receptors that mediate interaction between the host cells and their matrix. While P. gingivalis-induced degradation of E-cadherin has been documented, there is no information on the effects of P. gingivalis proteases on other members of this family of cell adhesion proteins. METHODS: Human epithelial KB cells were exposed to protease-active extracellular protein preparations from isogenic mutants of P. gingivalis. Quantification of apoptosis was performed by visualization of nuclei stained with 4,6'-diamidino-2-phenylindole. Alteration of cell adhesion proteins was examined by immunoblotting of cell lysates using monoclonal antibodies to those proteins. RESULTS: Treated cells exhibited loss of cell adhesion properties with apoptotic cell death subsequently observed. These effects correlated with the different levels of cysteine-dependent proteolytic activities of the isogenic mutants tested. Cleavage of N-cadherin was observed in immunoblots of lysates from detached cells. There was a direct correlation between the kinetics of N-cadherin cleavage and loss of cell adhesion properties. Loss of cell adhesion, as well as N-cadherin cleavage, could be inhibited by preincubation of P. gingivalis protease active extracellular protein preparations with the cysteine protease inhibitor TLCK. In control experiments, the cleavage of N-cadherin was detected after treatment of KB cells with trypsin but not after cell dissociation by a non-enzymatic method. CONCLUSIONS: These results suggest that extracellular proteases from P. gingivalis can induce degradation of N-cadherin, which could have implications for the pathogenicity of this bacterium.


Subject(s)
Apoptosis/drug effects , Cadherins/metabolism , Endopeptidases/pharmacology , Porphyromonas gingivalis/enzymology , Analysis of Variance , Antibodies, Monoclonal , Cell Adhesion/drug effects , Cell Nucleus/drug effects , Cysteine Endopeptidases/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Epithelial Cells/drug effects , Fluorescent Dyes , Humans , Immunoblotting , Indoles , KB Cells/drug effects , Mutation/genetics , Porphyromonas gingivalis/genetics , Porphyromonas gingivalis/pathogenicity , Tosyllysine Chloromethyl Ketone/pharmacology , Trypsin/pharmacology
5.
Head Neck ; 23(2): 104-12, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11303627

ABSTRACT

BACKGROUND: Antibodies against cancer-related genes have been detected in human cancers including head and neck cancers. High titers of c-Myc autoantibodies have been linked to gene amplification and tumor progression. Centromere protein-F (CENP-F) autoantibodies have been detected in patients with various cancers, suggesting similar gene alteration. METHODS: CENP-F and c-MYC amplification was assessed in 72 head and neck squamous cell carcinoma (HNSCC) patients. Tumor and matched mucosa from 22 patients were analyzed for CENP-F mRNA levels by RT-PCR. RESULTS: The larynx was the site most altered by amplification of either gene. CENP-F and c-MYC were amplified in 11% and 17% of the tumors, respectively. Coamplification was found in 7% of the tumors, most of which showed regional node involvement. CENP-F mRNA was overexpressed in 36% of tumors, and 23% of paired mucosa. CONCLUSION: Our results provide the first evidence that CENP-F gene is amplified and overexpressed in HNSCC. No correlation was noted between CENP-F amplification and clinicopathologic parameters. However, CENP-F overexpression correlated with nodal metastasis.


Subject(s)
Carcinoma, Squamous Cell/genetics , Chromosomal Proteins, Non-Histone/genetics , Head and Neck Neoplasms/genetics , Adult , Aged , Centromere , Female , Gene Amplification , Gene Expression Regulation, Neoplastic , Genes, myc/genetics , Humans , Male , Microfilament Proteins , Middle Aged , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
6.
J Pharmacol Exp Ther ; 295(3): 896-903, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11082422

ABSTRACT

Cocaine induces apoptosis in coronary artery endothelial cells. Yet the cellular and molecular mechanisms are not clear. Given that cocaine has profound toxic effects on the mitochondria, the present study examined the role of mitochondrial cytochrome c in cocaine-mediated apoptosis. Using cultured bovine coronary artery endothelial cells, we found that cocaine-induced apoptosis was dose dependently inhibited by cyclosporin A with IC(50) of 0.2 microM. The maximum of 65% inhibition was obtained with 3 microM cyclosporin A. Cocaine induced a translocation of cytochrome c from the mitochondria to the cytosol with a 1.8-fold increase in cytosolic cytochrome c levels, and a corresponding decrease in mitochondrial cytochrome c. In accordance with its inhibition of cocaine-induced apoptosis, cyclosporin A blocked cocaine-induced cytochrome c translocation. Correspondingly, cocaine-induced activation of caspase-9 preceded that of caspase-3. Caspase-8 was not activated. Cocaine also produced a dose-dependent decrease in Bcl-2 protein levels, but had no effect on Bax protein levels. The cocaine-induced decrease in the Bcl-2 protein was not affected by cyclosporin A but was partially blocked by caspase-3 inhibitor Ac-DEVD-CHO. Collectively, these data indicate that the release of cytochrome c from the mitochondria and the subsequent activation of caspase-9 and caspase-3 play a key role in cocaine-induced apoptosis in these cells. Furthermore, the down-regulation of the Bcl-2 protein may play an important role in cocaine-induced release of cytochrome c.


Subject(s)
Apoptosis/drug effects , Cocaine/pharmacology , Coronary Vessels/drug effects , Cytochrome c Group/physiology , Endothelium, Vascular/drug effects , Mitochondria/enzymology , Animals , Caspases/metabolism , Cattle , Cells, Cultured , Coronary Vessels/pathology , Dose-Response Relationship, Drug , Endothelium, Vascular/pathology , Mitochondria/physiology , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins c-bcl-2/analysis , bcl-2-Associated X Protein
8.
Mod Pathol ; 12(1): 69-74, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9950165

ABSTRACT

CENP-F is a newly characterized cell cycle-associated nuclear antigen that is expressed in low amounts in G0/G1 cells and that accumulates in the nuclear matrix during S phase with a maximal expression in G2/M cells. CENP-F can be analyzed by flow cytometry and used as a proliferation marker. In the present study, therefore, we characterized the expression of CENP-F in non-Hodgkin's lymphoma by immunohistochemical techniques to detect potential dysregulation of the protein or to establish CENP-F as a reliable proliferation marker. A polyclonal rabbit antibody reacting with CENP-F was prepared and used for immunohistochemical analyses after antigen retrieval. The rabbit antibody produced immunofluorescence patterns, flow cytometric profiles, and Western blot reactivity identical to those of the human autoantibody used in earlier studies. The percentage of CENP-F-positive and Ki-67-positive cells, as well as the labeling index, S-phase time, and potential doubling time, derived from in vivo iododeoxyuridine incorporation, were evaluated in 41 non-Hodgkin's lymphomas. Aggressive lymphomas showed higher CENP-F values than did indolent cases (10.1 vs. 3.4%). The percentage of CENP-F-positive cells correlated significantly to the S-phase fraction (r(s) = 0.68), the Ki-67 index (r(s) = 0.56) and the labeling index of iododeoxyuridine (r(s) = 0.47), as well as to S-phase time and potential doubling time (r(s) = 0.34 and -0.40). A lower fraction of CENP-F-positive cells was found, compared with the Ki-67 index (4.9 vs. 9.4%), supporting previous observations that CENP-F was expressed in a fraction of actively growing cells. These correlative data indicate that CENP-F expression defines a specific subpopulation of growing cells and that no clear evidence for dysregulation was found. Accordingly, CENP-F seems to be a useful proliferation marker for formalin-fixed and paraffin-embedded material.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Lymphoma, Non-Hodgkin/metabolism , Animals , Biomarkers , Cell Division/physiology , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Lymphoma, Non-Hodgkin/therapy , Microfilament Proteins , Rabbits , Treatment Outcome
9.
J Cell Biol ; 144(2): 281-92, 1999 Jan 25.
Article in English | MEDLINE | ID: mdl-9922454

ABSTRACT

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


Subject(s)
Caspases/metabolism , Cytochrome c Group/metabolism , Animals , Apoptosis , Apoptotic Protease-Activating Factor 1 , Caspase 10 , Caspase 2 , Caspase 3 , Caspase 6 , Caspase 7 , Caspase 8 , Caspase 9 , Cell Extracts , Enzyme Activation , Humans , Jurkat Cells , Protein Processing, Post-Translational , Proteins/metabolism , Rabbits
10.
J Immunol ; 161(9): 5061-9, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9794444

ABSTRACT

Access of intracellular Ags SSA/Ro and SSB/La to cognate maternal autoantibodies is unexplained despite their strong association with congenital heart block. To investigate the hypothesis that apoptosis facilitates surface accessibility of these Ags, human fetal cardiac myocytes from 16- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusing the aorta with collagenase. Confirmation of cardiac myocytes included positive staining with antisarcomeric alpha-actinin and contractility induced by 1.8 mM calcium. Incubation with 0.5 microM staurosporine or 0.3 mM 2,3-dimethoxy-1,4-naphthoquinone induced the characteristic morphologic and biochemical changes of apoptosis. The cellular topology of Ro and La was evaluated with confocal microscopy and determined in nonapoptotic and apoptotic cardiocytes by indirect immunofluorescence. In permeabilized nonapoptotic cardiocytes, Ro and La were predominantly nuclear, and propidium iodide (PI) stained the nucleus. In early apoptotic cardiocytes, condensation of the PI- and Ro- or La-stained nucleus was observed, accompanied by Ro/La fluorescence around the cell periphery. In later stages of apoptosis, nuclear Ro and La staining became weaker, and PI demonstrated nuclear fragmentation. Ro/La-stained blebs emerged from the cell membrane, a finding observed in nonpermeabilized cells, supporting an Ab-Ag interaction at the cell surface. In summary, induction of apoptosis in cultured cardiocytes results in surface translocation of Ro/La and recognition by Abs. Although apoptotic cells are programmed to die and do not characteristically evoke inflammation, binding of maternal Abs and subsequent influx of leukocytes could damage surrounding healthy fetal cardiocytes.


Subject(s)
Apoptosis , Autoantibodies/immunology , Autoantigens/immunology , Autoimmune Diseases/immunology , Fetal Heart/immunology , Fetal Proteins/immunology , Heart Block/congenital , Immunity, Maternally-Acquired , Myocardium/immunology , Pregnancy Complications/immunology , RNA, Small Cytoplasmic , Ribonucleoproteins/immunology , Antigen-Antibody Reactions , Antigens, Surface/immunology , Aorta/embryology , Autoantibodies/blood , Biological Transport , Cell Nucleus/immunology , Cell Separation/methods , Cells, Cultured , Collagenases/pharmacology , Enzyme Inhibitors/pharmacology , Female , Fetal Heart/cytology , Fetal Heart/drug effects , Heart Block/etiology , Humans , Muscle Proteins/immunology , Myocardium/cytology , Naphthoquinones/pharmacology , Pregnancy , Protein Isoforms/immunology , Staurosporine/pharmacology , SS-B Antigen
11.
Cell Death Differ ; 5(2): 183-90, 1998 Feb.
Article in English | MEDLINE | ID: mdl-10200463

ABSTRACT

A central mechanism in apoptosis is the activation of proteases of the caspase (cysteine aspartases) family. Protease activation has also been implicated in necrosis, but its role in this cell death process and the identity of the proteases involved and their substrates, are unknown. Using human autoantibodies to well characterized cellular proteins as detecting probes in immunoblotting, we observed that a defined and somewhat similar set of nuclear proteins, including poly (ADP-ribose) polymerase (PARP) and DNA topoisomerase I (Topo I), were selectively cleaved during both apoptosis and necrosis of cultured cells induced by various stimuli. The resulting cleavage products were distinctively different in the two cell death pathways. In contrast to apoptosis, the cleavages of PARP and Topo I during necrosis were not blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). These findings suggest that different proteases act in apoptosis and necrosis, and that although both cell death processes result in selective cleavage of almost identical cellular proteins, they can be distinguished immunochemically on the basis of their cleavage products.


Subject(s)
Apoptosis/physiology , Autoantibodies/pharmacology , Cell Nucleus/metabolism , Nuclear Proteins/immunology , Nuclear Proteins/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Autoantigens/metabolism , Cell Nucleus/pathology , Cell Nucleus/ultrastructure , Cysteine Proteinase Inhibitors/pharmacology , Detergents , HL-60 Cells , Humans , Jurkat Cells , Microscopy, Electron , Necrosis , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases , Proteins/metabolism , fas Receptor/analysis , fas Receptor/immunology , fas Receptor/metabolism
12.
Clin Invest Med ; 20(5): 308-19, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9336656

ABSTRACT

OBJECTIVE: To study the clinical features of patients with autoantibodies to centromere protein CENP-F and the frequency of CENP-F autoantibodies in patients with various diseases. DESIGN: Retrospective clinical and serologic study. METHODS: Thirty-six patients with anti-CENP-F were identified by a characteristic pattern of indirect immunofluorescence (IIF) on HEp-2 cells. Fifty patients with melanoma, 50 with breast cancer, 10 with lung cancer, 354 with systemic sclerosis, 120 with systemic lupus erythematosus and 50 with rheumatoid arthritis were also studied. Recombinant proteins were produced from 5 CENP-F cDNA clones representing amino acids 2192-3317 (p-F1), 5561-7126 (p-F2), 5892-6883 (p-F3), 7538-10,116 (p-F4) and 9242-10,096 (p-F5). The presence of CENP-F antigen was studied in a breast carcinoma cell line, cryosections of breast carcinoma, normal breast tissue and tonsils. RESULTS: Twenty-two of 36 patients with CENP-F antibodies had neoplasms; breast (9/22) and lung (5/22) cancer were the most common diagnoses. Thirty-three sera were available for further study; when tested for reactivity to the recombinant peptides, the sera of 21 of 21 patients with neoplasms and 5 of 12 patients with other diseases bound the C-terminal p-F4 peptide. When the terminal third of the p-F4 peptide (p-F5) was studied, a significant difference in pattern of reactivity was not detected. By comparison, the frequency of reactivity with peptides representing other domains of CENP-F was less than that with p-F4 (p-F2 > p-F3 > p-F1). CENP-F autoantibodies were not found in any of the control sera from patients with systemic lupus erythematosus, rheumatoid arthritis or systemic sclerosis or in unselected sera from various malignancies. CENP-F antigens were identified in breast carcinoma tissue but were rarely observed in normal tissues. CONCLUSIONS: A high proportion of individuals with CENP-F antibodies have neoplasia, and there is a bias among their sera for reactivity with determinants in the carboxy terminal domain of CENP-F. CENP-F antigens appear to be highly expressed in malignant tissues.


Subject(s)
Autoantibodies/blood , Centromere/immunology , Chromosomal Proteins, Non-Histone/immunology , Neoplasms/immunology , Adult , Aged , Autoantigens/immunology , Breast Neoplasms/immunology , Carcinoma, Small Cell/immunology , Female , Fluorescent Antibody Technique, Indirect , Humans , Lung Neoplasms/immunology , Male , Melanoma/immunology , Microfilament Proteins , Middle Aged , Retrospective Studies , Rheumatic Diseases/immunology
13.
J Exp Med ; 186(8): 1323-31, 1997 Oct 20.
Article in English | MEDLINE | ID: mdl-9334372

ABSTRACT

We report that the serine protease granzyme B (GrB), which is crucial for granule-mediated cell killing, initiates apoptosis in target cells by first maturing caspase-10. In addition, GrB has a limited capacity to mature other caspases and to cause cell death independently of the caspases. Compared with other members, GrB in vitro most efficiently processes caspase-7 and -10. In a human cell model, full maturation of caspase-7 does not occur unless caspase-10 is present. Furthermore, GrB matured caspase-3 with less efficiency than caspase-7 or caspase-10. With the caspases fully inactivated by peptidic inhibitors, GrB induced in Jurkat cells growth arrest and, over a delayed time period, cell death. Thus, the primary mechanism by which GrB initiates cell death is activation of the caspases through caspase-10. However, under circumstances where caspase-10 is absent or dysfunctional, GrB can act through secondary mechanisms including activation of other caspases and direct cell killing by cleavage of noncaspase substrates. The redundant functions of GrB ensure the effectiveness of granule-mediated cell killing, even in target cells that lack the expression or function (e.g., by mutation or a viral serpin) of one or more of the caspases, providing the host with overlapping safeguards against aberrantly replicating, nonself or virally infected cells.


Subject(s)
Apoptosis/immunology , Caspases , Cytoplasmic Granules/enzymology , Cytoplasmic Granules/immunology , Cytotoxicity, Immunologic , Serine Endopeptidases/pharmacology , Apoptosis/drug effects , Breast Neoplasms , Caspase 3 , Caspase 7 , Cell Death/drug effects , Cell Death/immunology , Cysteine Endopeptidases/metabolism , Cytotoxicity, Immunologic/drug effects , Enzyme Activation/drug effects , Granzymes , Humans , Jurkat Cells , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/metabolism , Substrate Specificity , Tumor Cells, Cultured
14.
J Neurosci ; 17(16): 6165-78, 1997 Aug 15.
Article in English | MEDLINE | ID: mdl-9236228

ABSTRACT

Apoptosis is a fundamental process required for normal development of the nervous system and is triggered during neurodegenerative disease. To dissect the molecular events leading to neuronal cell death, we have developed a cell-free model of neuronal apoptosis. The model faithfully reproduces key elements of apoptosis, including chromatin condensation, DNA fragmentation, caspase activation/processing, and selective substrate cleavage. We report that cell-free apoptosis is activated in premitochondrial, mitochondrial, and postmitochondrial phases by tamoxifen, mastoparan, and cytochrome c, respectively, allowing a functional ordering of these proapoptotic modulators. Furthermore, this is the first report of mitochondrial-mediated activation of cell-free apoptosis in a cell extract. Although Bcl-2 blocks activation at the premitochondrial and mitochondrial levels, it does not affect the postmitochondrial level. The cell-free system described here provides a valuable tool to elucidate the molecular events leading to neuronal cell death.


Subject(s)
Apoptosis/physiology , Caspases , Cerebellum/cytology , Mitochondria/enzymology , Neurons/cytology , Adenosine Triphosphate/pharmacology , Animals , Apoptosis/drug effects , Atractyloside/pharmacology , Caspase 3 , Cell Extracts/pharmacology , Cell-Free System , Cells, Cultured , Cysteine Endopeptidases/metabolism , Cytochrome c Group/pharmacology , Enzyme Inhibitors/pharmacology , Enzyme Precursors/metabolism , Estrogen Antagonists/pharmacology , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Neurons/enzymology , Neurons/ultrastructure , Peptides , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Rats , Rats, Sprague-Dawley , Sequence Homology, Amino Acid , Staurosporine/pharmacology , Tamoxifen/pharmacology , Wasp Venoms/pharmacology
15.
J Immunol ; 158(7): 3521-8, 1997 Apr 01.
Article in English | MEDLINE | ID: mdl-9120314

ABSTRACT

The heavy metal mercury elicits a genetically restricted, anti-nucleolar autoantibody response that targets fibrillarin, a 34-kDa protein component of many small nucleolar ribonucleoprotein particles. The mechanisms by which a toxin such as mercury elicits an autoantibody response that predominantly targets a single intracellular protein autoantigen remain uncertain, but may be prefaced by mercury gaining access to the intracellular environment. Mercury-induced cell death was associated with loss of fibrillarin antigenicity and modification of the molecular properties of fibrillarin as revealed by aberrant migration under nonreducing conditions in SDS-PAGE. Addition of mercury to isolated nuclei also resulted in aberrant migration of fibrillarin, but not other nuclear autoantigens. The sensitivity of the HgCl2-induced modification of fibrillarin to 2-ME, iodoacetamide, and hydrogen peroxide suggested interaction of mercury with the two cysteines in the fibrillarin sequence. This was confirmed by mutation of the cysteines to alanines, which abolished the aberrant migration of fibrillarin in the presence of HgCl2. The modification of the molecular structure of fibrillarin by mercury reduced immunoprecipitation by anti-fibrillarin autoantibodies, pointing to unmodified fibrillarin as the B cell Ag and implicating mercury-modified fibrillarin as the source of T cell antigenicity. These observations demonstrate for the first time that an environmental toxin can alter the physicochemical properties of an autoantigen and may help to explain the antigenic specificity of mercury-induced murine autoimmunity.


Subject(s)
Autoantigens/drug effects , Autoantigens/immunology , Chromosomal Proteins, Non-Histone/immunology , Chromosomal Proteins, Non-Histone/pharmacology , Mercuric Chloride/immunology , Mercuric Chloride/pharmacology , Xenobiotics/immunology , Xenobiotics/pharmacology , Antibodies, Monoclonal/chemistry , Autoantibodies/metabolism , Binding Sites, Antibody , Cell Death/drug effects , Cell Death/immunology , Cell Nucleus/drug effects , Cell Nucleus/immunology , Chromosomal Proteins, Non-Histone/drug effects , Cysteine/physiology , Disulfides/chemistry , Electrophoresis, Polyacrylamide Gel , Epitopes/chemistry , Humans , Subcellular Fractions/drug effects , Subcellular Fractions/immunology
16.
Int Arch Allergy Immunol ; 111(4): 308-13, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8957101

ABSTRACT

Studies of antinuclear autoantibodies (ANAs) associated with systemic autoimmune diseases and their target autoantigens have revealed several key features of the nature of the ANA response. First, each systemic autoimmune disease has a characteristic ANA spectrum, suggesting that specific inciting antigens must be associated with each disease. Second, ANAs are directed against components of functionally important subcellular particles. Third, ANAs recognize highly conserved, conformation-dependent epitopes associated with active regions of the targeted subcellular particle. Fourth, ANAs often target autoantigens associated with active cell division or proliferation. These features support the hypothesis that ANAs are driven by subcellular particles such as organelles or macromolecular complexes which might be in an activated or functional state. This hypothesis leads to the central question of how endogenous subcellular particles that are normally sequestered can be released from cells and exposed to the immune system in a manner that renders them capable of driving a sustained ANA response. An emerging view is that apoptosis could be a mechanism by which potentially immunostimulatory self-antigens might be released from cells. Unregulated cell death or aberrant phagocytic clearance and presentation of debris from dying cells might facilitate the exposure to the immune system of excessive amounts of intracellular material which could potentially induce and maintain, by repeated stimulation, an ANA response.


Subject(s)
Antibodies, Antinuclear/immunology , Autoimmune Diseases/immunology , Apoptosis , Autoantigens/immunology , Autoimmunity , Humans , Subcellular Fractions/immunology
17.
Cytometry ; 25(1): 90-8, 1996 Sep 01.
Article in English | MEDLINE | ID: mdl-8875058

ABSTRACT

p330d, also known as CENP-F, is a newly characterized cell cycle specific nuclear autoantigen which is associated both with the centromeres and the nuclear matrix. It is expressed in low amounts in G0/G1-cells and accumulates in the nuclear matrix during S-phase with a maximum expression in G2/M-cells. In the present study we have investigated if p330d/CENP-F could be used as a marker for proliferation in different human malignancies. A flow cytometric method was developed by which p330d/CENP-F expression and DNA-content could be assessed on hematopoietic and solid tumors. Twenty-four different human hematopoietic malignancies, 12 breast cancers, and several cell lines were analyzed and the number of p330d/CENP-F positive cells and the S-phase fraction were determined. The percentage of p330d/CENP-F positive cells correlated with the fraction of S-phase cells in all human malignancies tested. Various cell lines revealed a similar cell cycle specific distribution. The association of p330d/CENP-F with the nuclear matrix facilitated the flow cytometric analysis of this protein due to its resistance to different preparation and fixation procedures. In summary, p330d/CENP-F seems to be a potentially valuable proliferation marker which can be applied to different tumors.


Subject(s)
Antigens, Neoplasm/analysis , Autoantigens/analysis , Biomarkers, Tumor/analysis , Chromosomal Proteins, Non-Histone/analysis , Flow Cytometry/methods , Nuclear Matrix/chemistry , Breast Neoplasms/immunology , Cell Division , DNA, Neoplasm/analysis , Female , Hematologic Neoplasms/immunology , Humans , Interphase , Male , Microfilament Proteins , S Phase , Tissue Fixation , Tumor Cells, Cultured
18.
J Exp Med ; 184(2): 765-70, 1996 Aug 01.
Article in English | MEDLINE | ID: mdl-8760832

ABSTRACT

Intracellular proteases appear to be important mediators of apoptosis. Substrates cleaved by proteases during apoptosis include nuclear autoantigens targeted in systemic autoimmune diseases. Using human autoantibodies as probes, we demonstrate here that T cell apoptosis mediated by CD95 (Fas/APO-1) is associated with substantial cleavage of a subset of nuclear autoantigens (7 of 33 examined). This subset included poly (ADP-ribose) polymerase, the 70-kD protein of the U1 small nuclear ribonucleoprotein particle, lamin B, the nuclear mitotic apparatus protein NuMA, DNA topoisomerases I and II, and the RNA polymerase I upstream binding factor UBF. Several of the cleaved autoantigens are involved in ensuring the integrity and proper conformation of DNA in the nucleus through interactions with the nuclear matrix, suggesting the possibility that their cleavage may contribute to the collapse of nuclear structure during apoptosis. The relative cleavage kinetics indicated that the autoantigens were targeted at various times after induction of apoptosis, suggesting either differential accessibility or activation of distinct proteases during the cell death process. These data reinforce the hypothesis that apoptosis is accompanied by selective cleavage of key substrates and not by a generalized degradation of intracellular material.


Subject(s)
Apoptosis , Autoantigens/metabolism , Nuclear Proteins/metabolism , Pol1 Transcription Initiation Complex Proteins , fas Receptor/physiology , Cell Line , Cell Nucleus/metabolism , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/metabolism , Endopeptidases/metabolism , Humans , Molecular Weight , Peptide Fragments/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Transcription Factors/metabolism
19.
EMBO J ; 15(10): 2407-16, 1996 May 15.
Article in English | MEDLINE | ID: mdl-8665848

ABSTRACT

The major mechanism of cytotoxic lymphocyte killing involves the directed release of granules containing perforin and a number of proteases onto the target cell membrane. One of these proteases, granzyme B, has an unusual substrate site preference for Asp residues, a property that it shares with members of the emerging interleukin-1beta-converting enzyme (ICE)/CED-3 family of proteases. Here we show that granzyme B is sufficient to reproduce rapidly all of the key features of apoptosis, including the degradation of several protein substrates, when introduced into Jurkat cell-free extracts. Granzyme B-induced apoptosis was neutralized by a tetrapeptide inhibitor of the ICE/CED-3 family protease, CPP32, whereas a similar inhibitor of ICE had no effect. Granzyme B was found to convert CPP32, but not ICE, to its active form by cleaving between the large and small subunits of the CPP32 proenzyme, resulting in removal of the prodomain via an autocatalytic step. The cowpox virus protein CrmA, a known inhibitor of ICE family proteases as well as granzyme B, inhibited granzyme B-mediated CPP32 processing and apoptosis. These data demonstrate that CPP32 activation is a key event during apoptosis initiated by granzyme B.


Subject(s)
Apoptosis/physiology , Caspases , Cysteine Endopeptidases/metabolism , Serine Endopeptidases/physiology , Viral Proteins , Caspase 1 , Caspase 3 , Cell-Free System , Cysteine Endopeptidases/classification , Enzyme Activation/drug effects , Granzymes , Leukemia-Lymphoma, Adult T-Cell/pathology , Models, Biological , Neoplasm Proteins/metabolism , Protease Inhibitors/pharmacology , Serpins/pharmacology , Substrate Specificity , Tumor Cells, Cultured
20.
Cell Prolif ; 29(4): 183-96, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8695747

ABSTRACT

p330d/CENP-F is a recently described nuclear autoantigen that was detected in PHA-stimulated but not in resting peripheral lymphocytes. This protein accumulates in the nucleus during S-phase and reaches maximum levels during the G2 and M phases of the cell cycles. We compared the expression of p330d/CENP-F and proliferating cell nuclear antigen (PCNA) during the induction of terminal myeloid differentiation of HL-60 tumour cells. HL-60 cells were induced to differentiate with retinoic acid (RA), dimethyl sulfoxide (DMSO), and 3-nitrobenzothiazolo [3,2-]quinolinium (NBQ), and collected at different intervals. Control and treated cells were analyzed by two-parameter flow cytometry using propidium iodide and antibodies to p330d/CENP-F and PCNA. The percentage of p330d/CENP-F and PCNA positive cells was found to be proportional to the percentage of proliferating cells. After two cell cycles (65 h), the percentage of p330d/CENP-F and PCNA positive cells was reduced proportionately to the number of cells that had differentiated. Reduction in the expression of both antigens was completed after 120 h when 80% to 85% of the cells were arrested in G1 and displayed the mature phenotype. The expression of p330d/CENP-F and PCNA was also assessed in the growth inhibition of HT-29 cells induced by various concentrations of camptothecin (CPT), etoposide (VP-16), and aphidicolin (APH). There was a dose-dependent displacement of cells to late S-phase by CPT while VP-16 induced cells to accumulate in G2+M, and as expected these effects caused a strong increase in the cellular levels of both antigens. The arrest of cells in G1 by APH led to a significant decrease in their expression. The dramatic reduction in p330d/CENP-F levels during differentiation, and the correlation of its expression with the cell cycle effects of the cytotoxic drugs are consistent with the behaviour expected for a proliferation marker.


Subject(s)
Autoantigens/metabolism , Cell Cycle , Chromosomal Proteins, Non-Histone/metabolism , Growth Inhibitors/pharmacology , Proliferating Cell Nuclear Antigen/metabolism , Aphidicolin/pharmacology , Camptothecin/pharmacology , Cell Differentiation , Centromere/metabolism , DNA Replication , Etoposide/pharmacology , Flow Cytometry , Fluorescent Antibody Technique, Indirect , HL-60 Cells , Humans , Microfilament Proteins , Tumor Cells, Cultured
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