PubMed Health⌕ Search

Biomedical subjects

G I Evan

Publications and source records attributed to G I Evan.

At least 19 recordsLinked to original sources
PubMed2001

Caspase-mediated cleavage of the Ca2+/calmodulin-dependent protein kinase-like kinase facilitates neuronal apoptosis.

This study was designed to identify the role of a recently identified Ca(2+)/calmodulin-dependent protein kinase (CaMK)-like kinase (CaMKLK) in neuronal apoptosis. For this purpose, we studied proteolytic cleavage of CaMKLK by caspases in vitro and in neuronal NG108 cells. In addition, we have investigated the effect of overexpression of wild type and mutant CaMKLK proteins on staurosporine- and serum deprivation-induced apoptosis of NG108 cells. We found that CaMKLK is a substrate for caspase-3 and -8, both in vitro and in NG108 cells during staurosporine- and serum withdrawal-induced apoptosis. Substitution of an aspartic acid residue at position 62 in an asparagine residue within a putative caspase cleavage site completely blocked cleavage of CaMKLK, strongly indicating that (59)DEND(62) is the caspase recognition site. Overexpression of an Asp(62) --> Asn CaMKLK mutant protected NG108 cells from staurosporine-induced apoptosis to a similar extent as Bcl-x(L). In contrast, overexpression of wild type CaMKLK did not lead to protection. Moreover, microinjection of Asp(62) --> Asn CaMKLK protected NG108 cells from serum deprivation-induced apoptosis, while overexpression of a caspase-generated noncatalytic N-terminal CaMKLK fragment exacerbated apoptosis. Together, our data suggest that cleavage of CaMKLK and generation of the noncatalytic N-terminal domain of CaMKLK facilitate neuronal apoptosis.

Animals↗
PubMed2001

Proliferation, cell cycle and apoptosis in cancer.

Beneath the complexity and idiopathy of every cancer lies a limited number of 'mission critical' events that have propelled the tumour cell and its progeny into uncontrolled expansion and invasion. One of these is deregulated cell proliferation, which, together with the obligate compensatory suppression of apoptosis needed to support it, provides a minimal 'platform' necessary to support further neoplastic progression. Adroit targeting of these critical events should have potent and specific therapeutic consequences.

Animals↗
PubMed2001

Expression of Mad1 in T cells leads to reduced thymic cellularity and impaired mitogen-induced proliferation.

To investigate Mad1 function in vivo, transgenic mice were generated that express a Mad1 transgene in T lineage cells under the control of the proximal lck promoter. Thymus size in lck-Mad1 transgenic mice is drastically reduced although representation of the various thymocyte sub populations appears normal. To investigate more closely any effects of Mad1 expression on thymocytes, we examined thymic selection using MHC class I-restricted H-Y-TCR transgenic mice. Mad1 expression in vivo reduces the efficiency of positive selection. Furthermore, thymocytes and splenic T cells from lck-Mad1 transgenic mice display a profound proliferative defect in response to activation with either PMA/Ionomycin or immobilized anti-CD3/CD28 antibody. This proliferative defect is not reversed by addition of exogenous IL-2 and is p53-independent. The growth inhibition caused by Mad1 is overcome by expression of active c-Myc.

Animals↗
PubMed2000

Reversible activation of c-Myc in thymocytes enhances positive selection and induces proliferation and apoptosis in vitro.

In order to study the effect of c-Myc activation in T lymphocytes in vivo, we generated transgenic mice that express a 4-hydroxytamoxifen (4-OHT)-dependent switchable c-myc oncoprotein under the control of the proximal lck promoter. Activation of c-MycER causes no obvious alteration in T cell ontogeny. However, using MHC class I restricted H-Y-TCR transgenic mice, we found that c-Myc activation in vivo enhances the efficiency of positive selection. Moreover, splenic T cells derived from lck-c-mycER transgenic mice in which c-Myc had been activated exhibited increased proliferation in vitro in response to activation with anti-CD3/CD28 antibody. Activation of c-MycER also promotes apoptosis in thymocytes in vitro.

Animals↗
PubMed2000

Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson's disease.

Caspase-3 is an effector of apoptosis in experimental models of Parkinson's disease (PD). However, its potential role in the human pathology remains to be demonstrated. Using caspase-3 immunohistochemistry on the postmortem human brain, we observed a positive correlation between the degree of neuronal loss in dopaminergic (DA) cell groups affected in the mesencephalon of PD patients and the percentage of caspase-3-positive neurons in these cell groups in control subjects and a significant decrease of caspase-3-positive pigmented neurons in the substantia nigra pars compacta of PD patients compared with controls that also could be observed in an animal model of PD. This suggests that neurons expressing caspase-3 are more sensitive to the pathological process than those that do not express the protein. In addition, using an antibody raised against activated caspase-3, the percentage of active caspase-3-positive neurons among DA neurons was significantly higher in PD patients than in controls. Finally, electron microscopy analysis in the human brain and in vitro data suggest that caspase-3 activation precedes and is not a consequence of apoptotic cell death in PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

The Drosophila caspase DRONC is regulated by DIAP1.

We have isolated the recently identified Drosophila caspase DRONC through its interaction with the effector caspase drICE. Ectopic expression of DRONC induces cell death in Schizosaccharomyces pombe, mammalian fibroblasts and the developing Drosophila eye. The caspase inhibitor p35 fails to rescue DRONC-induced cell death in vivo and is not cleaved by DRONC in vitro, making DRONC the first identified p35-resistant caspase. The DRONC pro-domain interacts with Drosphila inhibitor of apoptosis protein 1 (DIAP1), and co-expression of DIAP1 in the developing Drosophila eye completely reverts the eye ablation phenotype induced by pro-DRONC expression. In contrast, DIAP1 fails to rescue eye ablation induced by DRONC lacking the pro-domain, indicating that interaction of DIAP1 with the pro-domain of DRONC is required for suppression of DRONC-mediated cell death. Heterozygosity at the diap1 locus enhances the pro-DRONC eye phenotype, consistent with a role for endogenous DIAP1 in suppression of DRONC activation. Both heterozygosity at the dronc locus and expression of dominant-negative DRONC mutants suppress the eye phenotype caused by reaper (RPR) and head involution defective (HID), consistent with the idea that DRONC functions in the RPR and HID pathway.

Animals↗
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

Caspase-8 in apoptosis: the beginning of "the end"?

Caspase-8 is a member of the cysteine proteases, which are implicated in apoptosis and cytokine processing. Like all caspases, caspase-8 is synthesized as an inactive single polypeptide chain zymogen procaspase and is activated by proteolytic cleavage, through either autoactivation after recruitment into a multimeric complex or trans-cleavage by other caspases. Thus, ligand binding-induced trimerization of death receptors results in recruitment of the receptor-specific adapter protein Fas-associated death domain (FADD), which then recruits caspase-8. Activated caspase-8 is known to propagate the apoptotic signal either by directly cleaving and activating downstream caspases or by cleaving the BH3 Bcl2-interacting protein, which leads to the release of cytochrome c from mitochondria, triggering activation of caspase-9 in a complex with dATP and Apaf-1. Activated caspase-9 then activates further "downstream caspases," including caspase-8. Knockout data indicate that caspase-8 is required for killing induced by the death receptors Fas, tumor necrosis factor receptor 1, and death receptor 3. Moreover, caspase-8-/- mice die in utero as a result of defective development of heart muscle and display fewer hematopoietic progenitor cells, suggesting that the FADD/caspase-8 pathway is absolutely required for growth and development of specific cell types.

Adenosine Triphosphate↗
PubMed1999

c-Myc and E1A induced cellular sensitivity to activated NK cells involves cytotoxic granules as death effectors.

The contact of natural killer (NK) cells with foreign cells and with certain virus-infected or tumor cells triggers the cytolytic machinery of NK cells. This triggering leads to exocytosis of the cytotoxic NK cell granules. The oncoproteins c-Myc and E1A render cells vulnerable to NK cell mediated cytolysis yet the mechanisms of sensitization are not well understood. In a model where foreign cells (rat fibroblasts) were cocultured with human IL-2 activated NK cells, we observed that NK cells were capable of efficiently killing their targets only if the cells overexpressed the oncogene c-Myc or E1A. Both the parental and the oncogene expressing fibroblasts similarly triggered phosphoinositide hydrolysis in the bound NK cells, demonstrating that NK cells were cytolytically activated in contact with both resistant parental and oncogene expressing sensitive target fibroblasts. The cell death was independent of wild-type p53 and was not inhibited by an anti-apoptotic protein EIB19K. These results provided evidence that c-Myc and E1A activated the NK cell induced cytolysis at a post-triggering stage of NK cell-target cell interaction. In consistence, the c-Myc and E1A overexpressing fibroblasts were more sensitive to the cytolytic effects of isolated NK cell-derived granules than parental cells. The data indicate that oncogenes activate the cytotoxicity of NK cell granules. This mechanism can have a role in directing the cytolytic action of NK cells towards the virus-infected and cancer cells.

Actins↗
PubMed1999

Caenorhabditis elegans inhibitor of apoptosis protein (IAP) homologue BIR-1 plays a conserved role in cytokinesis.

BACKGROUND: Inhibitor of apoptosis proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals. All IAPs contain at least one copy of the approximately 70 amino-acid baculovirus IAP repeat (BIR), and this domain is essential for the anti-apoptotic activity of the IAPs. Both the marked structural diversity of IAPs and the identification of BIR-containing proteins (BIRPs) in yeast, however, have led to the suggestion that BIRPs might play roles in other, as yet unidentified, cellular processes besides apoptosis. Survivin, a human BIRP, is upregulated 40-fold at G2-M phase and binds to mitotic spindles, although its role at the spindle is still unclear. RESULTS: We have identified and characterised two Caenorhabditis elegans BIRPs,BIR-1 and BIR-2; these proteins are the only BIRPs in C. elegans. The bir-1 gene is highly expressed during embryogenesis with detectable expression throughout other stages of development; bir-2 expression is detectable only in adults and embryos. Overexpression of bir-1 was unable to inhibit developmentally occurring cell death in C. elegans and inhibition of bir-1 expression did not increase cell death. Instead, embryos lacking bir-1 were unable to complete cytokinesis and they became multinucleate. This cytokinesis defect could be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis. CONCLUSIONS: BIR-1, a C. elegans BIRP, is probably not involved in the general regulation of apoptosis but is required for embryonic cytokinesis. We suggest that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis.

Amino Acid Sequence↗
PubMed1998

The opposing roles of the Akt and c-Myc signalling pathways in survival from CD95-mediated apoptosis.

Expression of the proto-oncogene c-myc stimulates cell proliferation in the presence of the appropriate survival factors and triggers apoptosis in their absence; this dual capacity ensures that cell growth is restricted to the correct paracrine environment and is thereby strictly controlled. Recently our laboratory demonstrated that c-Myc-induced apoptosis requires the CD95 death receptor pathway and that insulin-like growth factor (IGF-1) signalling suppresses this killing. To investigate further the links between c-Myc and IGF-1 pathways in CD95-induced apoptosis, we examined the effects of c-Myc and a downstream IGF-1 survival kinase, Akt, on killing mediated by CD95 and its recruited effector proteins (FADD and caspase-8). Here, we show that c-Myc activation does not exacerbate killing induced by FADD or pro-caspase-8, which narrows the point at which c-Myc exerts its action downstream of the interaction of CD95 with its ligand and upstream of FADD. We show further that activated Akt suppresses CD95-induced apoptosis and that Akt exerts its activity at a point downstream of FADD but upstream of caspase-8. These results restrict the possible mechanisms by which CD95-induced apoptosis is modulated by death signals and survival factors.

3T3 Cells↗
PubMed1998

Increased levels of TrkA in the regenerating retinal ganglion cells of fish.

Retinal ganglion cells of the fish have the spontaneous capacity to regenerate after nerve crush, a phenomenon known to be facilitated by nerve growth factor (NGF). We have studied the high-affinity NGF receptor TrkA, during the regeneration of the tench (Tinca tinca L.) optic nerve, using immunocytochemical techniques. TrkA-like immunoreactivity increased during the regeneration of the retinal ganglion cells. The increase is followed by a change in the subcellular distribution from perinuclear in control cells to cytoplasmic and perinuclear in regenerating ones. This increase was observed when antibodies against the extracellular domain of TrkA were used; no changes in TrkA-like immunoreactivity were observed with antibodies against the intracellular domain of TrkA. We thus conclude that modulation of TrkA is involved in the regeneration of fish retinal ganglion cells.

3T3 Cells↗
PubMed1998

Cell cycle phase-specific surface expression of nerve growth factor receptors TrkA and p75(NTR).

Expression of the nerve growth factor (NGF) receptors TrkA and p75(NTR) was found to vary at the surface of PC12 cells in a cell cycle phase-specific manner. This was evidenced by using flow cytometric and microscopic analysis of cell populations labeled with antibodies to the extracellular domains of both receptors. Differential expression of these receptors also was evidenced by biotinylation of surface proteins and Western analysis, using antibodies specific for the extracellular domains of TrkA and p75(NTR). TrkA is expressed most strongly at the cell surface in M and early G1 phases, whereas p75(NTR) is expressed mainly in late G1, S, and G2 phases. This expression reflects the molecular and cellular responses to NGF in specific phases of the cell cycle; in the G1 phase NGF elicits both the anti-mitogenic effect, i.e., inhibition of the G1 to S transition, and the differentiation response whereas a survival effect is provoked elsewhere in the cell cycle. A model is proposed relating these responses to the surface expression of the two receptors. These observations open the way for novel approaches to the investigation of the mechanism of NGF signal transduction.

Animals↗
PubMed1998

Traps to catch unwary oncogenes.

The MYC proto-oncogene has long been implicated in the control of normal cell growth and its deregulation is associated with the development of neoplasia. The MYC protein has a well-established role as a component of signal-transduction pathways promoting both proliferation and apoptosis. Because signalling pathways that drive cell death and cell proliferation are so tightly coupled, a synergy between genetic lesions leading to suppression of cell death and those promoting cell proliferation is observed during carcinogenesis. We discuss such synergy with respect to the cooperating oncogenes MYC, RAS and BCL2.

Animals↗
PubMed1997

Induction of TNF-sensitive cellular phenotype by c-Myc involves p53 and impaired NF-kappaB activation.

Normal fibroblasts are resistant to the cytotoxic action of tumor necrosis factor (TNF), but are rendered TNF-sensitive upon deregulation of c-Myc. To assess if oncoproteins induce the cytotoxic TNF activity by modulating TNF signaling, we investigated the TNF-elicited signaling responses in fibroblasts containing a conditionally active c-Myc protein. In association with cell death, c-Myc impaired TNF-induced activation of phospholipase A2, JNK protein kinase and cell survival-signaling-associated NF-kappaB transcription factor complex. The TNF-induced death of mouse primary fibroblasts expressing deregulated c-Myc was inhibited by transient overexpression of the p65 subunit of NF-kappaB, which increased NF-kappaB activity in the cells. Unlike other TNF-induced signals, TNF-induced accumulation of the wild-type p53 mRNA and protein was not inhibited by c-Myc. TNF, with c-Myc, induced apoptosis in mouse primary fibroblasts but only weakly in p53-deficient primary fibroblasts. The C-terminal domain of p53, which is a transacting dominant inhibitor of wild-type p53, failed to inhibit apoptosis by c-Myc and TNF, suggesting that the cell death was not dependent on the transcription-activating function of p53. Taken together, the present findings show that the cytotoxic activity of TNF towards oncoprotein-expressing cells involves p53 and an impaired signaling for survival in such cells.

3T3 Cells↗