Inhibition of caspase activity prevents CD95-mediated hepatic microvascular perfusion failure and restores Kupffer cell clearance capacity. (49/1161)

Using a murine model, we studied the effect of agonistic anti-CD95 antibodies (aCD95) on sinusoidal lining cells and a potential protection by caspase inhibition. C3H/HeN mice were intravenously administered aCD95 (10 microgram/mouse) or unspecific IgG (control) in the presence or absence of the caspase inhibitor z-VAD-fmk. Analysis of hepatic microcirculation using intravital fluorescence microscopy revealed severe (P<0.01) sinusoidal perfusion failure and reduced (P<0.05) phagocytic activity of Kupffer cells (KC) within 2 h. Transmission electron micrographs demonstrated loss of integrity of sinusoidal endothelial cells as early as 1 h after aCD95 application, whereas histological manifestation of hepatocellular apoptosis and hemorrhagic necrosis was most pronounced at 6 h. Blocking of caspase activity attenuated (P<0.01) both hepatic microvascular perfusion failure and KC dysfunction. Accordingly, full protection of the liver from apoptotic damage and intact microarchitecture was observed in histological sections after z-VAD-fmk treatment. Mortality rate was 40% 6 h after aCD95 administration, whereas all animals survived in the z-VAD-fmk group (P<0.05). The activation of caspases through CD95 may primarily lead to damage of sinusoidal endothelial cells and hepatic microvascular perfusion failure. Moreover, reduced phagocytic capacity of KC may contribute to accumulation of toxic metabolites released by dying cells at the local site of inflammation, further aggravating liver injury.  (+info)

Increase of ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)- retinamide in neuroblastoma cell lines. (50/1161)

BACKGROUND: The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR or fenretinide) is toxic to myeloid leukemia and cervical carcinoma cell lines, probably in part due to its ability to increase levels of reactive oxygen species (ROS). We have studied the effects of 4-HPR on neuroblastoma cell lines. Since neuroblastomas commonly relapse in bone marrow, a hypoxic tissue compartment, and many chemotherapeutic agents are antagonized by hypoxia, our purpose was to study in these cell lines several factors influencing 4-HPR-induced cytotoxicity, including induced levels of ROS, effects of physiologic hypoxia and antioxidants, levels of ceramide, and the mechanism of cell death. METHODS: ROS generation was measured by carboxydichlorofluorescein diacetate fluoresence. Ceramide was quantified by radiolabeling and thin-layer chromatography. Immunoblotting was used to assess p53 protein levels. Apoptosis (programmed cell death) and necrosis were analyzed by nuclear morphology and internucleosomal DNA fragmentation patterns. Cytotoxicity was measured by a fluorescence-based assay employing digital imaging microscopy in the presence or absence of the pancaspase enzyme inhibitor BOC-d-fmk. Statistical tests were two-sided. RESULTS/CONCLUSIONS: In addition to increasing ROS, 4-HPR (2.5-10 microM) statistically significantly increased the level of intracellular ceramide (up to approximately 10-fold; P<.001) in a dose-dependent manner in two neuroblastoma cell lines, one of which is highly resistant to alkylating agents and to etoposide. Cell death induced by 4-HPR was reduced but not abrogated by hypoxia in the presence or absence of an antioxidant, N-acetyl-L-cysteine. Expression of p53 protein was not affected by 4-HPR. Furthermore, the pan-caspase enzyme inhibitor BOC-d-fmk prevented apoptosis, but not necrosis, and only partially decreased cytotoxicity induced by 4-HPR, indicating that 4-HPR induced both apoptosis and necrosis in neuroblastoma cells. IMPLICATIONS: 4-HPR may form the basis for a novel, p53-independent chemotherapy that operates through increased intracellular levels of ceramide and that retains cytotoxicity under reduced oxygen conditions.  (+info)

Tissue plasminogen activator binds to human vascular smooth muscle cells by a novel mechanism. Evidence for a reciprocal linkage between inhibition of catalytic activity and cellular binding. (51/1161)

Human vascular smooth muscle cells (VSMC) bind tissue plasminogen activator (tPA) specifically, saturably, and with relatively high affinity (K(d) 25 nM), and this binding potentiates the activation of cell-associated plasminogen (Ellis, V., and Whawell, S. A. (1997) Blood 90, 2312-2322). We have observed that this binding can be efficiently competed by DFP-inactivated tPA and S478A-tPA but not by tPA inactivated with H-D-Phe-Pro-Arg-chloromethyl ketone (PPACK). VSMC-bound tPA also exhibited a markedly reduced inhibition by PPACK, displaying biphasic kinetics with second-order rate constants of 7. 5 x 10(3) M(-1) s(-1) and 0.48 x 10(3) M(-1) s(-1), compared with 7. 2 x 10(3) M(-1) s(-1) in the solution phase. By contrast, tPA binding to fibrin was competed equally well by all forms of tPA, and its inhibition was unaltered. These effects were shown to extend to the physiological tPA inhibitor, plasminogen activator inhibitor 1. tPA.plasminogen activator inhibitor 1 complex did not compete tPA binding to VSMC, and the inhibition of bound tPA was reduced by 30-fold. The behavior of the various forms of tPA bound to VSMC correlated with conformational changes in tPA detected by CD spectroscopy. These data suggest that tPA binds to its specific high affinity site on VSMC by a novel mechanism involving the serine protease domain of tPA and distinct from its binding to fibrin. Furthermore, reciprocally linked conformational changes in tPA appear to have functionally significant effects on both the interaction of tPA with its VSMC binding site and the susceptibility of bound tPA to inhibition.  (+info)

Regulation of apoptosis in myeloid cells by interferon consensus sequence-binding protein. (52/1161)

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-X(L). These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.  (+info)

Redistribution of cytochrome c precedes the caspase-dependent formation of ultracondensed mitochondria, with a reduced inner membrane potential, in apoptotic monocytes. (53/1161)

Apoptosis was induced in human monocytic THP.1 cells by the use of chemicals with disparate mechanisms of action. Apoptotic cells were characterized by a reduced inner mitochondrial membrane potential, increased cytosolic cytochrome c, ultracondensed mitochondria, condensed chromatin, cytoplasmic inclusions of beta-actin, and fragmentation of the Golgi apparatus. All of these changes, except the release of cytochrome c, were prevented by caspase inhibition. Cells were separated into two populations, with either normal or low inner mitochondrial membrane potential, using fluorescence-activated cell sorting. Ultracondensed mitochondria were observed only in the cells with low inner mitochondrial membrane potential, whereas noncondensed mitochondria were found in the cells with a normal inner mitochondrial membrane potential. We have demonstrated a sequence of related biochemical and ultrastructural changes, commencing with the release of mitochondrial cytochrome c, followed by activation of caspases and a reduction of inner mitochondrial membrane potential. These changes involved the formation of ultracondensed but not swollen mitochondria. Thus the release of mitochondrial cytochrome c was not the result of the mitochondrial permeability transition, reduction of inner mitochondrial membrane potential, or rupture of the outer mitochondrial membrane. Discontinuities in the outer membrane of ultracondensed mitochondria may, however, facilitate the further release of caspase-activating proteins, thereby amplifying the apoptotic process.  (+info)

The zinc finger protein A20 interacts with a novel anti-apoptotic protein which is cleaved by specific caspases. (54/1161)

A20 is a Cys2/Cys2 zinc finger protein which is induced by a variety of inflammatory stimuli and which has been characterized as an inhibitor of cell death by a yet unknown mechanism. In order to clarify its molecular mechanism of action, we used the yeast two-hybrid system to screen for proteins that interact with A20. A cDNA fragment was isolated which encoded a portion of a novel protein (TXBP151), which was recently found to be a human T-cell leukemia virus type-I (HTLV-I) Tax-binding protein. The full-length 2386 bp TXBP151 mRNA encodes a protein of 86 kDa. Like A20, overexpression of TXBP151 could inhibit apoptosis induced by tumour necrosis factor (TNF) in NIH3T3 cells. Moreover, transfection of antisense TXBP151 partially abolished the anti-apoptotic effect of A20. Furthermore, apoptosis induced by TNF or CD95 (Fas/APO-1) was associated with proteolysis of TXBP151. This degradation could be inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of the cowpox virus-derived inhibitor CrmA, suggesting that TXBP151 is a novel substrate for caspase family members. TXBP151 was indeed found to be specifically cleaved in vitro by members of the caspase-3-like subfamily, viz. caspase-3, caspase-6 and caspase-7. Thus TXBP151 appears to be a novel A20-binding protein which might mediate the anti-apoptotic activity of A20, and which can be processed by specific caspases.  (+info)

Implication of c-Myc in apoptosis induced by the retinoid CD437 in human lung carcinoma cells. (55/1161)

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been recently identified to be a potent inducer of apoptosis in human non-small cell lung carcinoma (NSCLC) cells through a nuclear retinoic acid receptor independent mechanism. To approach the mechanism by which CD437 induces apoptosis in NSCLC cells, we investigated the involvement of c-Myc in CD437-induced apoptosis. CD437 (1 microM) up-regulated the expression of c-Myc and of its downstream target genes ornithine decarboxylase (ODC) and cdc25A in all three NSCLC cell lines (i.e., H460, SK-MES-1 and H1792) used. These effects were correlated with cellular susceptibilities to induction of apoptosis by CD437. Furthermore, CD437-induced apoptosis could be blocked by the ODC inhibitor difluoromethylornithine, the caspase inhibitors Z-VAD FMK and Z-DEVD FMK, and c-Myc antisense oligodeoxynucleotide, respectively. These data indicate that c-Myc gene plays an important role in mediating CD437-induced apoptosis in human NSCLC cells.  (+info)

Caspase inhibition in camptothecin-treated U-937 cells is coupled with a shift from apoptosis to transient G1 arrest followed by necrotic cell death. (56/1161)

Leukemia U-937 cells rapidly undergo characteristic morphological changes, caspase activation, and DNA fragmentation typical of apoptosis on treatment with the DNA topoisomerase I inhibitor camptothecin (CPT). In a previous report (Sane, A. T., and Bertrand, R., Cancer Res., 58: 3066-3072, 1998), we showed that, after CPT treatment, caspase inhibition by the tripeptide derivative benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethyl ketone (zVAD-fmk) blocked apoptosis and slowed passage of the cells through S-G2 and caused a transient accumulation of these cells at the G1 phase of the cell cycle. Accumulation of these cells at G1 is not associated with major changes in expression level of cyclin-dependent kinase (cdk)2, cdk4, and cdk6; cyclin D1 and cyclin E; or p16, p21, p27, and p57 after CPT treatment. Furthermore, cdk2, cdk4, and cdk6 kinase activities remain unaffected after CPT treatment. These results indicate that the G1 arrest of these cells does not correlate with a classical driven cell cycle checkpoint but with the known effect of CPT in mediating inhibition of DNA replication and RNA transcription after stabilization of topoisomerase I-linked DNA strand breaks. However, persistent caspase inhibition after CPT treatment also results in cells falling into necrosis after the transient G1 arrest. These results indicate that the enforced inhibition of caspase activities does not confer a survival advantage upon CPT-treated cells but is coupled with a shift from apoptosis to transient G1 arrest followed by massive necrosis.  (+info)