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D R Green

Publications and source records attributed to D R Green.

At least 55 records · Page 3Linked to original sources
PubMed2000

Nitric oxide inhibits apoptosis via AP-1-dependent CD95L transactivation.

Several inducers of cytotoxic stress promote apoptotic cell death, which, at least in some cases, involves the CD95/CD95 ligand (CD95L) pathway. The induction of the CD95/CD95L pathway can be activated by the activator protein-1 (AP-1)-mediated up-regulation of the CD95L promoter, which is responsible for the induction of apoptosis elicited by stimuli such as etoposide. We show that nitric oxide (NO) represents a regulatory element able to block apoptosis by interfering with this loop. Etoposide- and C6-ceramide-induced apoptosis in Jurkat T cells with different kinetics. Cell death was accompanied by an increase in DNA-binding activity of the transcription factor AP-1, transactivation of the AP-1 site-containing CD95L promoter, and caspase 3-like protease activation. Using different NO-releasing compounds, we found that apoptosis was prevented in a dose-dependent manner. Furthermore, in both models of apoptosis, NO-releasing compounds dose-dependently reduced: (a) the number of the titratable thiol groups (cysteine residues) of c-Jun; (b) induction of AP-1 DNA-binding activity; (c) AP-1-driven transactivation of the CD95L promoter; and (d) caspase activation. In conclusion, our data demonstrate that NO can modulate cell death at an upstream level, by interfering with the ability of AP-1 to induce CD95L expression.

Amino Acid Sequence↗
PubMed2000

Regulation of FasL by NF-kappaB and AP-1 in Fas-dependent thymineless death of human colon carcinoma cells.

Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS(-)) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS(-) cells by anti-FasL and in enhanced survival of TS(-) clones selected for resistance to Fas-mediated apoptosis, following dThd deprivation. During thymineless stress in TS(-) cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-kappaB or AP-1 and by preventing NF-kappaB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IkappaBalphaM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-kappaB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells.

Apoptosis↗
PubMed2000

Expression of Fas ligand in activated T cells is regulated by c-Myc.

The transcription factor c-Myc is important for the control of cell cycle progression, neoplasia, and apoptotic cell death. c-Myc dimerizes with its partner Max to form an active transcription factor complex. Little is known, however, about the transcriptional targets of c-Myc and their roles in c-Myc-induced cell death. Here we demonstrate that T cell activation-induced expression of Fas ligand (FasL, CD95-L, APO-1-L), which can induce apoptotic cell death in many different cell types, is regulated by c-Myc. Down-modulation of c-Myc protein via antisense oligonucleotides blocked activation-induced FasL mRNA and protein expression and functional FasL expression in activated T cells and T cell lines. Further, FasL promoter activity in T cells is driven by overexpression of c-Myc and inhibited by expression of dominant-negative mutants of c-Myc and Max. Our findings indicate that c-Myc controls apoptotic cell death in T cells through regulation of FasL expression.

Base Sequence↗
PubMed2000

Role of acidic sphingomyelinase in Fas/CD95-mediated cell death.

Engagement of the Fas receptor has been reported to induce ceramide generation via activation of acidic sphingomyelinase (aSMase). However, the role of aSMase in Fas-mediated cell death is controversial. Using genetically engineered mice deficient in the aSMase gene (aSMase(-/-)), we found that thymocytes, concanavalin A-activated T cells, and lipopolysaccharide-activated B cells derived from both aSMase(-/-) and aSMase(+/+) mice were equally sensitive to Fas-mediated cell death, triggered by either anti-Fas antibody or Fas ligand in vitro. Similarly, activation-induced apoptosis of T lymphocytes was unaffected by the status of aSMase, and aSMase(-/-) mice failed to show immunological symptoms seen in animals with defects in Fas function. In vivo, intravenous injection of 3 microg/25 g mouse body weight of anti-Fas Jo2 antibody into aSMase(-/-) mice failed to affect hepatocyte apoptosis or mortality, whereas massive hepatocyte apoptosis and animal death occurred in wild type littermates. Animals heterozygous for aSMase deficiency were also significantly protected. Susceptibility of aSMase(-/-) mice to anti-Fas antibody was demonstrated with higher antibody doses (>/=4 microg/25 g mouse). These data indicate a role for aSMase in Fas-mediated cell death in some but not all tissues.

Animals↗
PubMed2000

p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release.

The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 death signals lead to caspase activation; however, the mechanism of caspase activation by p53 still is unclear. Expressing wild type p53 by means of an adenoviral expression vector, we were able to induce apoptotic cell death, as characterized by morphological changes, phosphatidylserine externalization, and internucleosomal DNA fragmentation, in p53(null) Saos-2 cells. This cell death was accompanied by caspase activation as well as by cleavage of caspase substrates and was preceded by mitochondrial cytochrome c release. The addition of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transduction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcysteine, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduced with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of zVAD-fmk. When Bax was immunodepleted from the cytosolic extracts of p53-expressing cells before incubation with isolated mitochondria, the in vitro cytochrome c release was abolished. Thus, we could demonstrate in cells and in vitro that p53 activates the apoptotic machinery through induction of the release of cytochrome c from the mitochondrial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bax for this cytochrome c-releasing activity of p53 in Saos-2 cells.

Acetylcysteine↗
PubMed2000

Cloak and dagger in the avoidance of immune surveillance.

CD95 and CD95-ligand (CD95L) are physiological mediators of apoptosis required for the control of cell numbers in the human immune system. Discoveries in CD95-dependent mechanisms of immune evasion by tumours suggest regulation by oncogene expression. Clonal contraction of lymphocytes by a CD95/CD95L-independent mechanism has been reported and new evidence supports a role for CD95-dependent peripheral lymphocyte deletion by non-lymphoid tissue. Additionally, factors affecting the pro- and anti-inflammatory effects of CD95L point to a balance of cytokines and growth factors.

Apoptosis↗
PubMed2000

The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant.

Release of cytochrome c from mitochondria triggers activation of caspase proteases and death of a cell by apoptosis. However, the mechanism and kinetics of cytochrome c release remain unknown. Here we study this event by using green fluorescent protein (GFP)-tagged cytochrome c, and find that the release of cytochrome-c-GFP always precedes exposure of phosphatidylserine and the loss of plasma-membrane integrity - characteristics of apoptotic cells. Once initiated, the release of cytochrome- c-GFP continues until all of the protein is released from all mitochondria in individual cells, within about 5 minutes, regardless of the type or strength of stimulus or the time elapsed since the stimulus was applied. Temperatures ranging from 24 degrees C to 37 degrees C do not change the duration of release, and nor does the addition of caspase inhibitors. Further, we find that the electron-transport chain can maintain the mitochondrial transmembrane potential even after cytochrome c has been released.

Adenosine Triphosphate↗
PubMed2000

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

Apoptosis↗
PubMed2000

Murder by proxy.

Explore the source record for details and available documents.

Apoptosis↗
PubMed2000

Bcl-xL does not inhibit the function of Apaf-1.

Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1-5 microM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9.

Amino Acid Sequence↗
PubMed2000

The 'harmless' release of cytochrome c.

Release of cytochrome c from the mitochondria plays an integral role in apoptosis; however, the mechanism by which cytochrome c is released remains one of the conundrums that has occupied the field. Recently, evidence has emerged that the commitment to death may be regulated downstream of cytochrome c release; therefore the mechanism of release must be subtle enough for the cell to recover from this event. In this review, we discuss the evidence that cytochrome c release is mediated by Bcl-2 family proteins in a process that involves only outer membrane permeability but leaves inner membrane energization, protein import function and the ultrastructure of mitochondria intact. Cell Death and Differentiation (2000) 7, 1192 - 1199.

Animals↗
PubMed2000

A single cell analysis of apoptosis. Ordering the apoptotic phenotype.

Microcinematography was applied to the analysis of the kinetics of apoptotic cell death. Apoptosis was found to be a process that proceeds in different cells at different times after an initial stress, and therefore kinetic studies of apoptotic events in bulk cultures can be problematic. Using single cell analysis we found that stronger apoptotic stimuli induce an earlier onset of apoptosis, but that there is no relationship between time of onset and duration of the apoptotic process. That is, cells that initiate apoptosis shortly after induction do not proceed more rapidly through the process. This suggests an all-or-non-mechanism that is supported by some models of the biochemical pathways of apoptosis.

Apoptosis↗
PubMed2000

Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid.

Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.

Apoptosis↗
PubMed2000

Fas and Fas ligand in gut and liver.

Apoptosis (programmed cell death) has been shown to play a major role in development and in the pathogenesis of numerous diseases. A principal mechanism of apoptosis is molecular interaction between surface molecules known as the "death receptors" and their ligands. Perhaps the best-studied death receptor and ligand system is the Fas/Fas ligand (FasL) system, in which FasL, a member of the tumor necrosis factor (TNF) family of death-inducing ligands, signals death through the death receptor Fas, thereby resulting in the apoptotic death of the cell. Numerous cells in the liver and gastrointestinal tract have been shown to express Fas/FasL, and there is a growing body of evidence that the Fas/FasL system plays a major role in the pathogenesis of many liver and gastrointestinal diseases, such as inflammatory bowel disease, graft vs. host disease, and hepatitis. Here we review the Fas/FasL system and the evidence that it is involved in the pathogenesis of liver and gastrointestinal diseases.

Animals↗