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W C Earnshaw

Publications and source records attributed to W C Earnshaw.

At least 19 recordsLinked to original sources
PubMed1999

Characterization of caspase processing and activation in HL-60 cell cytosol under cell-free conditions. Nucleotide requirement and inhibitor profile.

The present studies compared caspase activation under cell-free conditions in vitro and in etoposide-treated HL-60 leukemia cells in situ. Immunoblotting revealed that incubation of HL-60 cytosol at 30 degrees C in the presence of cytochrome c and ATP (or dATP) resulted in activation of procaspases-3, -6, and -7 but not -2 and -8. Although similar selectivity was observed in intact cells, affinity labeling revealed that the active caspase species generated in vitro and in situ differed in charge and abundance. ATP and dATP levels in intact HL-60 cells were higher than required for caspase activation in vitro and did not change before caspase activation in situ. Replacement of ATP with the poorly hydrolyzable analogs 5'-adenylyl methylenediphosphate, 5'-adenylyl imidodiphosphate, or 5'-adenylyl-O-(3-thiotriphos-phate) slowed caspase activation in vitro, suggesting that ATP hydrolysis is required. Caspase activation in vitro was insensitive to phosphatase and kinase inhibitors (okadaic acid, staurosporine, and genistein) but was inhibited by Zn(2+), aurintricarboxylic acid, and various protease inhibitors, including 3,4-dichloroisocoumarin, N(alpha)-p-tosyl-L-phenylalanine chloromethyl ketone, N(alpha)-p-tosyl-L-lysine chloromethyl ketone, and N-(N(alpha)-benzyloxycarbonylphenylalanyl)alanine fluoromethyl ketone, each of which inhibited recombinant caspases-3, -6, -7, and -9. Experiments with anti-neoepitope antiserum confirmed that these agents inhibited caspase-9 activation. Collectively, these results suggest that caspase-9 activation requires nucleotide hydrolysis and is inhibited by agents previously thought to affect apoptosis by other means.

Adenosine Triphosphate↗
PubMed1999

Comparison of paclitaxel-, 5-fluoro-2'-deoxyuridine-, and epidermal growth factor (EGF)-induced apoptosis. Evidence for EGF-induced anoikis.

Epidermal growth factor (EGF), a hormone that stimulates proliferation of many cell types, induces apoptosis in some cell lines that overexpress the EGF receptor. To evaluate the mechanism of EGF-induced apoptosis, MDA-MB-468 breast cancer cells were examined by microscopy, flow cytometry, immunoblotting, enzyme assays, and affinity labeling after treatment with EGF, paclitaxel, or 5-fluoro-2'-deoxyuridine (5FUdR). Apoptosis induced by all three agents was accompanied by activation of caspases-3, -6, and -7, as indicated by disappearance of the corresponding zymogens from immunoblots, cleavage of substrate polypeptides in situ, and detection of active forms of these caspases in cytosol and nuclei using fluorogenic assays and affinity labeling. Further analysis indicated involvement of the cytochrome c/Apaf-1/caspase-9 pathway of caspase activation, but not the Fas/Fas ligand pathway. Interestingly, caspase activation was consistently lower after EGF treatment than after paclitaxel or 5FUdR treatment. Additional experiments revealed that the majority of cells detaching from the substratum after EGF (but not paclitaxel or 5FUdR) were morphologically normal and retained the capacity to readhere, suggesting that EGF-induced apoptosis involves cell detachment followed by anoikis. These observations not only indicate that EGF- and chemotherapy-induced apoptosis in this cell line involve the same downstream pathways but also suggest that detachment-induced apoptosis is responsible for the paradoxical antiproliferative effects of EGF.

Affinity Labels↗
PubMed1999

Specific destruction of kinetochore protein CENP-C and disruption of cell division by herpes simplex virus immediate-early protein Vmw110.

Examination of cells at the early stages of herpes simplex virus type 1 infection revealed that the viral immediate-early protein Vmw110 (also known as ICP0) formed discrete punctate accumulations associated with centromeres in both mitotic and interphase cells. The RING finger domain of Vmw110 (but not the C-terminal region) was essential for its localization at centromeres, thus distinguishing the Vmw110 sequences required for centromere association from those required for its localization at other discrete nuclear structures known as ND10, promyelocytic leukaemia (PML) bodies or PODs. We have shown recently that Vmw110 can induce the proteasome-dependent loss of several cellular proteins, including a number of probable SUMO-1-conjugated isoforms of PML, and this results in the disruption of ND10. In this study, we found some striking similarities between the interactions of Vmw110 with ND10 and centromeres. Specifically, centromeric protein CENP-C was lost from centromeres during virus infection in a Vmw110- and proteasome-dependent manner, causing substantial ultrastructural changes in the kinetochore. In consequence, dividing cells either became stalled in mitosis or underwent an unusual cytokinesis resulting in daughter cells with many micronuclei. These results emphasize the importance of CENP-C for mitotic progression and suggest that Vmw110 may be interfering with biochemical mechanisms which are relevant to both centromeres and ND10.

Animals↗
PubMed1999

Caspase-mediated cleavage of DNA topoisomerase I at unconventional sites during apoptosis.

Previous studies have demonstrated that topoisomerase I is cleaved late during apoptosis, but have not identified the proteases responsible or examined the functional consequences of this cleavage. Here, we have shown that treatment of purified topoisomerase I with caspase-3 resulted in cleavage at DDVD146 downward arrowY and EEED170 downward arrowG, whereas treatment with caspase-6 resulted in cleavage at PEDD123 downward arrowG and EEED170 downward arrowG. After treatment of Jurkat T lymphocytic leukemia cells with anti-Fas antibody or A549 lung cancer cells with topotecan, etoposide, or paclitaxel, the topoisomerase I fragment comigrated with the product that resulted from caspase-3 cleavage at DDVD146 downward arrowY. In contrast, two discrete topoisomerase I fragments that appeared to result from cleavage at DDVD146 downward arrowY and EEED170 downward arrowG were observed after treatment of MDA-MB-468 breast cancer cells with paclitaxel. Topoisomerase I cleavage did not occur in apoptotic MCF-7 cells, which lack caspase-3. Cell fractionation and band depletion studies with the topoisomerase I poison topotecan revealed that the topoisomerase I fragment remains in proximity to the chromatin and retains the ability to bind to and cleave DNA. These observations indicate that topoisomerase I is a substrate of caspase-3 and possibly caspase-6, but is cleaved at sequences that differ from those ordinarily preferred by these enzymes, thereby providing a potential explanation why topoisomerase I cleavage lags behind that of classical caspase substrates such as poly(ADP-ribose) polymerase and lamin B1.

Animals↗
PubMed1999

Mammalian centromeres: DNA sequence, protein composition, and role in cell cycle progression.

The centromere is a specialized region of the eukaryotic chromosome that is responsible for directing chromosome movements in mitosis and for coordinating the progression of mitotic events at the crucial transition between metaphase and anaphase. In this review, we will focus on recent advances in the understanding of centromere composition at the protein and DNA level and of the role of centromeres in sister-chromatid cohesion and mitotic checkpoint control.

Animals↗
PubMed1999

The structure and dynamics of ring chromosomes in human neoplastic and non-neoplastic cells.

Acquired ring chromosomes have been found in most types of human neoplasia, with a frequency approaching 10% in malignant mesenchymal tumours. In this study, the composition and dynamics of ring chromosomes were analysed in eight cases of acute myelogenous leukaemia, 17 solid tumours, and five cases with constitutional rings. Chromosomal banding and fluorescence in situ hybridisation were performed to determine the content and the structural heterogeneity of the rings. Telomeric repeats were detected using peptide nucleic acid probes or primed in situ labelling, whereas centromeric activity was evaluated by detection of kinetochore proteins. Mitotic instability was assessed by the frequency of anaphase bridges. The results suggest that human ring chromosomes can be structurally and functionally divided into two categories. In the first of these, size variation is minimal and rearrangement at cell division is uncommon. The majority of such rings contain subtelomeric sequences. Constitutional ring chromosomes and most rings in leukaemias belong to this group, whereas only a few mesenchymal tumours exhibit rings of this type. The second category consists of rings with amplified sequences, primarily from chromosome 12, characteristically occurring in atypical lipomatous tumours and other subtypes of low or borderline malignant mesenchymal neoplasms. Variation in size and number is extensive, and breakage-fusion-bridge events occur at a high frequency. Abnormalities in pericentromeric sequences are common and, in some cases, kinetochores assemble in the absence of alphoid DNA. We conclude that it is not only the ring structure per se or the neoplastic nature of the host cell that determines ring instability, but probably also the functional role of the genes carried in the ring.

Abnormalities, Multiple↗
PubMed1999

Transmission of a fully functional human neocentromere through three generations.

An unusual Y chromosome with a primary constriction inside the long-arm heterochromatin was found in the amniocytes of a 38-year-old woman. The same Y chromosome was found in her husband and brother-in-law, thus proving that it was already present in the father. FISH with alphoid DNA showed hybridization signals at the usual position of the Y centromere but not at the primary constriction. Centromere proteins (CENP)-A, CENP-C, and CENP-E could not be detected at the site of the canonic centromere but were present at the new constriction, whereas CENP-B was not detected on this Y chromosome. Experiments with 82 Y-specific loci distributed throughout the chromosome confirmed that no gross deletion or rearrangement had taken place, and that the Y chromosome belonged to a haplogroup whose members have a mean alphoid array of 770 kb (range 430-1,600 kb), whereas that of this case was approximately 250 kb. Thus, this Y chromosome appeared to be deleted for part of the alphoid DNA. It seems likely that this deletion was responsible for the silencing of the normal centromere and that the activation of the neocentromere prevented the loss of this chromosome. Alternatively, neocentromere activation could have occurred first and stimulated inactivation of the normal centromere by partial deletion. Whatever the mechanism, the presence of this chromosome in three generations demonstrates that it functions sufficiently well in mitosis for male sex determination and fertility and that neocentromeres can be transmitted normally at meiosis.

Adult↗
PubMed1999

Cleavage furrows formed between centrosomes lacking an intervening spindle and chromosomes contain microtubule bundles, INCENP, and CHO1 but not CENP-E.

PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundles as well as INCENP and CHO1. Together these data suggest that the mechanism defining the site of furrow formation during mitosis in vertebrates does not depend on the presence of underlying microtubule bundles and chromosomes or on the stable association of INCENP or CHO1. The data also suggest that the completion of cytokinesis requires the presence of microtubule bundles and specific proteins (e.g., INCENP, CHO1, etc.) that do not include CENP-E.

Animals↗
PubMed1999

A dynamic connection between centromeres and ND10 proteins.

ND10, otherwise known as nuclear dots, PML nuclear bodies or PODs, are punctate foci in interphase nuclei that contain several cellular proteins. The functions of ND10 have not been well defined, but they are sensitive to external stimuli such as stress and virus infection, and they are disrupted in malignant promyelocytic leukaemia cells. Herpes simplex virus type 1 regulatory protein Vmw110 induces the proteasome-dependent degradation of ND10 component proteins PML and Sp100, particularly the species of these proteins which are covalently conjugated to the ubiquitin-like protein SUMO-1. We have recently reported that Vmw110 also induces the degradation of centromere protein CENP-C with consequent disruption of centromere structure. These observations led us to examine whether there were hitherto undetected connections between ND10 and centromeres. In this paper we report that hDaxx and HP1 (which have been shown to interact with CENP-C and Sp100, respectively) are present in a proportion of both ND10 and interphase centromeres. Furthermore, the proteasome inhibitor MG132 induced an association between centromeres and ND10 proteins PML and Sp100 in a significant number of cells in the G(2) phase of the cell cycle. These results imply that there is a dynamic, cell cycle regulated connection between centromeres and ND10 proteins which can be stabilised by inhibition of proteasome-mediated proteolysis.

Adaptor Proteins, Signal Transducing↗
PubMed1998

INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1.

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13-amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11-amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1(Hsalpha). Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.

Anaphase↗
PubMed1998

Phosphorylated forms of activated caspases are present in cytosol from HL-60 cells during etoposide-induced apoptosis.

Treatment of HL-60 human leukemia cells with etoposide induces apoptotic cell death and activation of at least 18 electrophoretically distinct cysteine-dependent aspartate-directed protease (caspase) isoforms, several of which differ only in their isoelectric points. The purpose of the present study was to determine whether activated caspases are phosphorylated. Phosphatase treatment of cytosolic extracts containing active caspases followed by affinity labeling with N-(N-benzyloxycarbonylglutamyl-N-biotinyllysyl)aspartic acid [(2, 6-dimethylbenzoyl)oxy] methyl ketone (Z-EK(bio)D-aomk) showed a mobility shift in several of the labeled species, suggesting that phosphorylated forms of these enzymes are present in the extracts. Metabolic labeling with 32P followed by etoposide treatment and subsequent affinity purification of affinity-labeled caspases confirmed that at least three caspase species were phosphorylated. To detect effects of the phosphorylation on enzymatic activity, caspase-mediated cleavage of aspartylglutamylvalinylaspartyl-7-amino-4-trifluoromethylcoumarin (DEVD-AFC) and poly(ADP-ribose) polymerase (PARP) by phosphorylated and dephosphorylated extracts was measured. No significant changes in Km or vmax were detected using DEVD-AFC. In contrast, a slight, but significant enhancement of PARP cleavage was observed in dephosphorylated extracts, suggesting that phosphorylation of active caspases could have an inhibitory effect on enzyme activity. These observations, which provide the first evidence that caspases are phosphoproteins, suggest that caspases may be targets for some of the growing list of protein kinases that are involved in apoptotic events.

Apoptosis↗
PubMed1998

Transition from caspase-dependent to caspase-independent mechanisms at the onset of apoptotic execution.

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal "committed stage" cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the "execution phase" extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.

Animals↗
PubMed1998

ICAD/DFF regulator of apoptotic nuclease is nuclear.

In nonapoptotic cells, the caspase-activated DNase CAD/CPAN is associated with a regulatory subunit, ICAD/DFF, that binds to it and blocks its enzymatic activity. It has been proposed that a major function of ICAD is to restrain CAD in the cytoplasm in healthy cells. The experiments presented here demonstrate that rather than being cytoplasmic, a GFP-ICAD fusion protein is nuclear in healthy human, pig, and chicken cells. Furthermore, immunoblots using antibodies to murine ICAD confirm the presence of endogenous ICAD and the marker protein DNA topoisomerase I in human nuclei, while tubulin was found solely in the cytosolic fraction. Since ICAD is located in cell nuclei, it is unlikely that the protein functions primarily in the cytoplasm either as an anchor for CAD/CPAN or as a factor that enters the nucleus following caspase cleavage in order to activate resident endonucleases.

Animals↗
PubMed1998

Apparent caspase independence of programmed cell death in Dictyostelium.

During normal development, cell elimination [1,2] occurs by programmed cell death (PCD) [3], of which apoptosis [4] is the best known morphological type. Activation of cysteine proteases termed caspases [5] is required in many instances of animal PCD [6-9], but its role outside the animal kingdom is as yet unknown. PCD occurs during developmental stages in the slime mold Dictyostelium discoideum [10,11]. Under favorable conditions, Dictyostelium multiplies as a unicellular organism. Upon starvation, a pathway involving aggregation, differentiation and morphogenesis induces the formation of a multicellular fungus-like structure called a sorocarp [12], consisting mainly of spores and stalk cells, the latter being a result of cell death. Dictyostelium cell death is similar to classical apoptosis in that some cytoplasmic and chromatin condensation occurs but differs from apoptosis because it involves massive vacuolisation and, interestingly, lacks DNA fragmentation [11]. We examined whether caspase activity is required for Dictyostelium cell death. We found that caspase inhibitors did not affect cell death, although some caspase inhibitors that did not inhibit cell death impaired other stages in development and could block affinity-labelling of soluble extracts of Dictyostelium cells with an activated caspase-specific reagent. The simplest interpretation of these results is that in Dictyostelium, whether or not caspase-like molecules exist and are required for some developmental steps, caspase activation is not required for cell death itself.

Animals↗
PubMed1998

Interphase-specific association of intrinsic centromere protein CENP-C with HDaxx, a death domain-binding protein implicated in Fas-mediated cell death.

CENP-C, one of the few known intrinsic proteins of the human centromere, is thought to play structural as well as regulatory roles crucial to proper chromosome segregation and mitotic progression. To further define the functions of CENP-C throughout the cell cycle we have used the yeast interaction trap to identify proteins with which it interacts. One specific CENP-C interactor, which we have named HDaxx, was characterized in detail and found to be homologous to murine Daxx, a protein identified through its ability to bind the death domain of Fas (CD95). The interaction between CENP-C and HDaxx is mediated by the amino-terminal 315 amino acids of CENP-C and the carboxyl-terminal 104 amino acids of HDaxx. This region of Daxx is responsible for binding to death domains of several apoptosis signalling proteins. The biological significance of the interaction between CENP-C and HDaxx was confirmed by immunofluorescence colocalization of these two proteins at discrete spots in the nuclei of some interphase HeLa cells. We discuss the functional implications of the interphase-restricted association of HDaxx with centromeres.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Structure and function in the nucleus.

Current evidence suggests that the nucleus has a distinct substructure, albeit one that is dynamic rather than a rigid framework. Viral infection, oncogene expression, and inherited human disorders can each cause profound and specific changes in nuclear organization. This review summarizes recent progress in understanding nuclear organization, highlighting in particular the dynamic aspects of nuclear structure.

Animals↗
PubMed1998

A dominant mutant of inner centromere protein (INCENP), a chromosomal protein, disrupts prometaphase congression and cytokinesis.

INCENP is a tightly bound chromosomal protein that transfers to the spindle midzone at the metaphase/anaphase transition. Here, we show that an INCENP truncation mutant (INCENP382-839) associates with microtubules but does not bind to chromosomes, and coats the entire spindle throughout mitosis. Furthermore, an INCENP truncation mutant (INCENP43-839) previously shown not to transfer to the spindle at anaphase (Mackay, A.M., D.M. Eckley, C. Chue, and W.C. Earnshaw. 1993. J. Cell Biol. 123:373-385), is shown here to bind chromosomes, but is unable to target to the centromere. Thus, association with the chromosomes, and specifically with centromeres, appears to be essential for INCENP targeting to the correct spindle subdomain at anaphase. An INCENP truncation mutant (INCENP1-405) that targets to centromeres but lacks the microtubule association region acquires strong dominant-negative characteristics. INCENP1-405 interferes with both prometaphase chromosome alignment and the completion of cytokinesis. INCENP1-405 apparently exerts its effect by displacing the endogenous protein from centromeres. These experiments provide evidence of an unexpected link between this chromosomal protein and cytokinesis, and suggest that one function of INCENP may be to integrate the chromosomal and cytoskeletal events of mitosis.

Animals↗