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Biomedical subjects

D R Green

Publications and source records attributed to D R Green.

At least 19 recordsLinked to original sources
PubMed2006

Mitochondrial outer membrane permeabilization during apoptosis: the innocent bystander scenario.

Mitochondrial outer membrane permeabilization (MOMP) is considered the 'point of no return' as this event is responsible for engaging the apoptotic cascade in numerous cell death pathways. MOMP is directly governed by a subset of the BCL-2 family of proapoptotic proteins, which induce disruptions in the outer mitochondrial membrane (OMM) and subsequent release of death-promoting proteins like cytochrome c. The proposal here is centered on our hypothesis that MOMP is dictated by an interaction between the cytosol and the OMM, and although proteins of the OMM may be important in the process, the 'decision' to undergo apoptosis originates within the cytosol with no participation (in terms of yes, no and when) by mitochondria.

Animals↗
PubMed2006

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases.

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.

Animals↗
PubMed2006

Functional dissociation of DeltaPsim and cytochrome c release defines the contribution of mitochondria upstream of caspase activation during granzyme B-induced apoptosis.

Loss of Bid confers clonogenic survival to granzyme B-treated cells, however the exact role of Bid-induced mitochondrial damage--upstream or downstream of caspases--remains controversial. Here we show that direct cleavage of Bid by granzyme B, but not caspases, was required for granzyme B-induced apoptosis. Release of cytochrome c and SMAC, but not AIF or endonuclease G, occurred in the absence of caspase activity and correlated with the onset of apoptosis and loss of clonogenic potential. Loss of mitochondrial trans-membrane potential (DeltaPsim) was also caspase independent, however if caspase activity was blocked the mitochondria regenerated their DeltaPsim. Loss of DeltaPsim was not required for rapid granzyme B-induced apoptosis and regeneration of DeltaPsim following cytochrome c release did not confer clonogenic survival. This functional dissociation of cytochrome c and SMAC release from loss of DeltaPsim demonstrates the essential contribution of Bid upstream of caspase activation during granzyme B-induced apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Dissecting p53-dependent apoptosis.

The complexity of the p53 protein, coupled with the vast cellular responses to p53, is simply astonishing. As new isoforms, functional domains and protein-protein interactions are described; each morsel of information forces us to think (and re-think) about how it 'fits' into the current p53 paradigm. One aspect of p53 signaling that is under refinement is the mechanism(s) leading to apoptosis. Here we discuss what is known about p53-induced apoptosis, what proteins and protein-protein interactions are responsible for regulating apoptosis, how can this cascade be genetically dissected, and what pharmacological tools are available to modulate p53-dependent apoptosis. While everything may not comfortably fit into our understanding of p53, all of these data will certainly broaden our viewpoint on the complexity and significance of the p53-induced apoptotic pathway. Here, our discussion is primarily focused on the works presented at the 12th International p53 Workshop, except where appropriate background is required.

Animals↗
PubMed2005

Cytochrome c is released in a single step during apoptosis.

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).

Apoptosis↗
PubMed2003

p53 triggers apoptosis in oncogene-expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation.

The mechanism of p53-dependent apoptosis is still only partly defined. Using early-passage embryonic fibroblasts (MEF) from wild-type (wt), p53(-/-) and bax(-/-) mice, we observe a p53-dependent translocation of Bax to the mitochondria and a release of mitochondrial Cytochrome c during stress-induced apoptosis. These events proceed independent of zVAD-inhibitable caspase activation, are not prevented by dominant negative FADD (DN-FADD), but are negatively regulated by Mdm-2. Bcl-x(L) expression prevents the release of mitochondrial Cytochrome c and apoptosis, but not Bax translocation. At a single-cell level, enforced expression of p53 is sufficient to induce Bax translocation and Cytochrome c release. Real-time RT-PCR analysis reveals a significant induction of RNA expression of Noxa and Bax in p53(+/+), but not in p53(-/-) MEF. Noxa protein expression becomes detectable prior to Bax translocation, and downregulation of endogenous Noxa by RNA interference protects wt MEF against p53-dependent apoptosis. Hence, in oncogene-expressing MEF p53 induces apoptosis by BH3 protein-dependent caspase activation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Bcr-Abl-mediated resistance to apoptosis is independent of constant tyrosine-kinase activity.

Bcr-Abl is one of the most potent antiapoptotic molecules and is the tyrosine-kinase implicated in Philadelphia (Ph) chromosome-positive leukemia. It is still obscure how Bcr-Abl provides the leukemic cell a strong resistance to chemotherapeutic drugs. A rational drug development produced a specific inhibitor of the catalytic activity of Bcr-Abl called STI571. This drug was shown to eliminate Bcr-Abl-positive cells both in vitro and in vivo, although resistant cells may appear in culture and relapse occurs in some patients. In the study described here, Bcr-Abl-positive cells treated with tyrosine-kinase inhibitors such as herbimycin A, genistein or STI571 lost their phosphotyrosine-containing proteins, but were still extremely resistant to apoptosis. Therefore, in the absence of tyrosine-kinase activity, Bcr-Abl-positive cells continue to signal biochemically to prevent apoptosis induced by chemotherapeutic drugs. We propose that secondary antiapoptotic signals are entirely responsible for the resistance of Bcr-Abl-positive cells. Precise determination of such signals and rational drug development against them should improve the means to combat Ph chromosome-positive leukemia.

Antineoplastic Agents↗
PubMed2001

Accumulation and activation-induced release of preformed Fas (CD95) ligand during the pathogenesis of experimental graft-versus-host disease.

Fas (CD95/APO-1) ligand (FasL)-mediated cytotoxicity has been implicated in tissue destruction in a variety of diseases, including acute graft-vs-host disease (GVHD). In this study, we have analyzed FasL expression and regulation during the course of experimental murine acute GVHD. Although activation-induced FasL-mediated cytotoxicity in control T cells was sensitive to the immunosuppressant cyclosporin A, we observed that functional FasL expression of GVHD T cells became increasingly cyclosporin A unresponsive. This was found to be the result of a massive in vivo accumulation and intracellular storage of FasL protein and its release in a transcription- and protein synthesis-independent manner. Immunohistochemistry analysis of FasL expression in situ revealed accumulation of FasL-expressing cells in the spleen, the liver, and small intestine, with a typical cytoplasmic and granular expression pattern. Thus, we conclude that the release of preformed FasL by infiltrating donor T cells may contribute to recipient tissue damage during the pathogenesis of acute GVHD.

Acute Disease↗
PubMed2001

Defective cytochrome c-dependent caspase activation in ovarian cancer cell lines due to diminished or absent apoptotic protease activating factor-1 activity.

Apoptosis via the mitochondrial pathway requires release of cytochrome c into the cytosol to initiate formation of an oligomeric apoptotic protease-activating factor-1 (APAF-1) apoptosome. The apoptosome recruits and activates caspase-9, which in turn activates caspase-3 and -7, which then kill the cell by proteolysis. Because inactivation of this pathway may promote oncogenesis, we examined 10 ovarian cancer cell lines for resistance to cytochrome c-dependent caspase activation using a cell-free system. Strikingly, we found that cytosolic extracts from all cell lines had diminished cytochrome c-dependent caspase activation compared with normal ovarian epithelium extracts. The resistant cell lines expressed APAF-1 and caspase-9, -3, and -7; however, each demonstrated diminished APAF-1 activity relative to the normal ovarian epithelium cell lines. A competitive APAF-1 inhibitor may account for the diminished APAF-1 activity because we did not detect dominant APAF-1 inhibitors, altered APAF-1 isoform expression, or APAF-1 deletion, degradation, or mutation. Lack of APAF-1 activity correlated in some but not all cell lines with resistance to apoptosis. These data suggest that regulation of APAF-1 activity may be important for apoptosis regulation in some ovarian cancers.

Apoptosis↗
PubMed2001

Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process.

During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

Amino Acid Chloromethyl Ketones↗
PubMed2001

p53-dependent radiation-induced crypt intestinal epithelial cells apoptosis is mediated in part through TNF-TNFR1 system.

Radiation induces apoptosis of crypt intestinal epithelial cells (IEC) through a pathway that is largely dependent on p53. However, exactly how p53 mediates IEC apoptosis is unclear. Studies in vitro suggest that one mechanism by which p53 mediates apoptosis is through its ability to transactivate members of the TNF receptor family of 'Death Receptors'. Here, we examined the role of one of its member, TNF receptor type 1 (TNFR1), in an in vivo model of p53-dependent radiation-induced IEC apoptosis. We demonstrate that mice genetically engineered to be deficient in TNF receptor type 1 (TNFR1(-/-)) and mice injected with TNFR1-fusion chimeric protein (TNFR1-Fc; a competitive inhibitor of TNFR1) were partially protected (30-40%) from p53-dependent radiation-induced IEC apoptosis. However, we found no evidence to support the possibility p53 transcriptionally regulates the expression of TNFR1 nor increases the susceptibility of IEC to TNF-mediated apoptosis. Interestingly, we found that injection of TNF readily induced IEC apoptosis and that radiation induced a p53-dependent increase in the intestinal level of TNF. Furthermore, injection of a neutralizing anti-TNF mAb reduced p53-dependent radiation-induced IEC apoptosis by approximately 60%. Overall, these results suggest that p53-dependent radiation-induced IEC apoptosis is mediated in part through ability of p53 to regulate TNF, which subsequently induces IEC apoptosis through TNFR1.

Animals↗
PubMed2001

Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway.

Cytotoxic T lymphocytes kill virus-infected and tumor cell targets through the concerted action of proteins contained in cytolytic granules, primarily granzyme B and perforin. Granzyme B, a serine proteinase with substrate specificity similar to the caspase family of apoptotic cysteine proteinases, is capable of cleaving and activating a number of death proteins in target cells. Despite the ability to engage the death pathway at multiple entry points, the preferred mechanism for rapid induction of apoptosis by granzyme B has yet to be clearly established. Here we use time lapse confocal microscopy to demonstrate that mitochondrial cytochrome c release is the primary mode of granzyme B-induced apoptosis and that Bcl-2 is a potent inhibitor of this pivotal event. Caspase activation is not required for cytochrome c release, an activity that correlates with cleavage and activation of Bid, which we have found to be cleaved more readily by granzyme B than either caspase-3 or caspase-8. Bcl-2 blocks the rapid destruction of targets by granzyme B by blocking mitochondrial involvement in the process.

Amino Acid Sequence↗
PubMed2001

Role of CD95-activated caspase-1 processing of IL-1beta in TCR-mediated proliferation of HIV-infected CD4(+) T cells.

CD95 plays a critical role in the homeostasis of the immune system, and has been reported to participate in T cell death during HIV infection. Here we report that the response to CD3-TCR stimulation of CD4(+) T cells from HIV-infected individuals and CD4(+) T cells from healthy donors incubated in vitro with HIV-1(Lai) depends on the manner the CD3-TCR complex is engaged. While stimulation by anti-CD3 antibodies in solution induced CD4 T cell apoptosis both in the absence or presence of anti-CD95 antibodies, stimulation by immobilized anti-CD3 antibodies rendered CD4(+) T cells resistant to CD95-mediated death and led to increased CD4 T cell proliferation in response to CD95 ligation. CD95 ligation of CD4(+) T cells led to the activation of caspases, while costimulation induced by anti-CD3 and anti-CD95 mAb prevented the full processing of caspase-3 and caspase-8. Proliferation of CD4(+) T cells induced by CD3-TCR and CD95 costimulation was decreased by treatments with a caspase-1 inhibitor or with neutralizing antibodies to IL-1ss, indicating a requirement for caspase-1-mediated IL-1beta processing and secretion. Our findings suggest a novel mechanism whereby in addition to its role in inducing T cell apoptosis, CD95 signaling during HIV infection may also provide a costimulatory signal leading to an enhancement of CD4 T cell proliferation in response to CD3-TCR complex engagement.

Acquired Immunodeficiency Syndrome↗