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H O Fearnhead

Publications and source records attributed to H O Fearnhead.

11 recordsLinked to original sources
PubMed2005

Small molecule inhibitors of Apaf-1-related caspase- 3/-9 activation that control mitochondrial-dependent apoptosis.

Apoptosis is a biological process relevant to human disease states that is strongly regulated through protein-protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic protease-activating factor), dATP and procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversity-oriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis.

Apoptosis↗
PubMed2004

Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2.

An assay for the autoubiquitination activity of the E3 ligase HDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used to measure the activity of other E3s and may be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.

Animals↗
PubMed2001

Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family.

Inhibitor of apoptosis protein (IAP)-like protein-1 (ILP-1) (also known as X-linked IAP [XIAP] and mammalian IAP homolog A [MIHA]) is a potent inhibitor of apoptosis and exerts its effects, at least in part, by the direct association with and inhibition of specific caspases. Here, we describe the molecular cloning and characterization of a human gene related to ILP-1, termed ILP-2. Despite high homology to ILP-1, ILP-2 is encoded by a distinct gene, which in normal tissues is expressed solely in testis. In contrast to ILP-1, overexpression of ILP-2 had no protective effect on apoptosis mediated by Fas (also known as CD95) or tumor necrosis factor. However, ILP-2 potently inhibited apoptosis induced by overexpression of Bax or by coexpression of caspase 9 with Apaf-1, and preincubation of cytosolic extracts with ILP-2 abrogated caspase activation in vitro. A processed form of caspase 9 could be coprecipitated with ILP-2 from cells, suggesting a physical interaction between ILP-2 and caspase 9. Thus, ILP-2 is a novel IAP family member with restricted specificity for caspase 9.

Amino Acid Sequence↗
PubMed1998

Oncogene-dependent apoptosis is mediated by caspase-9.

Understanding how oncogenic transformation sensitizes cells to apoptosis may provide a strategy to kill tumor cells selectively. We previously developed a cell-free system that recapitulates oncogene dependent apoptosis as reflected by activation of caspases, the core of the apoptotic machinery. Here, we show that this activation requires a previously identified apoptosis-promoting complex consisting of caspase-9, APAF-1, and cytochrome c. As predicted by the in vitro system, preventing caspase-9 activation blocked drug-induced apoptosis in cells sensitized by E1A, an adenoviral oncogene. Oncogenes, such as E1A, appear to facilitate caspase-9 activation by several mechanisms, including the control of cytochrome c release from the mitochondria.

Adenovirus E1A Proteins↗
PubMed1997

Oncogene-dependent apoptosis in extracts from drug-resistant cells.

Many genotoxic agents kill tumor cells by inducing apoptosis; hence, mutations that suppress apoptosis produce resistance to chemotherapy. Although directly activating the apoptotic machinery may bypass these mutations, how to achieve this activation in cancer cells selectively is not clear. In this study, we show that the drug-resistant 293 cell line is unable to activate components of the apoptotic machinery-the ICE-like proteases (caspases)-following treatment with an anticancer drug. Remarkably, extracts from untreated cells spontaneously activate caspases and induce apoptosis in a cell-free system, indicating that drug-resistant cells have not only the apoptotic machinery but also its activator. Comparing extracts from cells with defined genetic differences, we show that this activator is generated by the adenovirus E1A oncogene and is absent from normal cells. We provide preliminary characterization of this oncogene generated activity (OGA) and show that partially purified OGA activates caspases when added to extracts from untransformed cells. We suggest that agents that link OGA to caspases in cells would kill tumor cells otherwise resistant to conventional cancer therapy. As this killing relies on an activity generated by an oncogene, the effect of these agents should be selective for transformed cells.

Adenosine Triphosphate↗
PubMed1995

An interleukin-1 beta-converting enzyme-like protease is a common mediator of apoptosis in thymocytes.

Apoptosis was induced in thymocytes using diverse stimuli in order to identify events within a common apoptotic pathway. Benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone (Z-VAD.FMK), an interleukin-1 beta-converting enzyme (ICE)-like protease inhibitor, inhibited apoptosis assessed by flow cytometry, proteolysis of poly (ADP)-ribose polymerase of DNA to both large kilobase pair fragments (30-50 and 200-300 kbp) and to nucleosomal fragments. Z-VAD.FMK also blocked all the classical ultrastructural features of apoptosis including chromatin condensation to one pole of the nucleus, nucleolar disintegration and cytoplasmic vacuolation. These results suggest the involvement of an ICE-like protease as a common mediator of apoptosis in thymocytes.

Amino Acid Chloromethyl Ketones↗
PubMed1995

An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells.

Apoptosis was induced in THP.1 cells, a human monocytic tumour cell line, by diverse stimuli including cycloheximide, thapsigargin, etoposide and staurosporine. Induction of apoptosis by all these stimuli, except etoposide, was enhanced in the presence of the trypsin-like protease inhibitor, N alpha-tosyl-L-lysinyl chloromethyl ketone (TLCK). Induction of apoptosis, assessed by morphological, flow cytometric and biochemical criteria, including proteolysis of poly(ADP-ribose) polymerase and cleavage of DNA to large kilobasepair fragments, was completely abrogated when cells were pretreated with an ICE-like protease inhibitor, Z-Val-Ala-Asp.fluoromethylketone. This suggested that an ICE homologue was a common mediator of apoptosis in THP.1 cells.

Amino Acid Chloromethyl Ketones↗
PubMed1995

A pre-existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli.

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the inhibitors N-tosyl-L-lysyl chloromethylketone (TLCK) and N-tosyl-L-phenylalanyl chloromethylketone (TPCK) have very different effects on apoptosis. TLCK inhibits apoptosis induced by diverse stimuli at an early stage prior to both DNA fragmentation and cytoplasmic changes. We show that the TLCK-sensitive target is pre-existing and not synthesized in response to apoptotic stimuli. The contrasting effects of TLCK and TPCK support the hypothesis that the TLCK target is a trypsin-like protease which is a common effector of thymocyte apoptosis.

Animals↗
PubMed1995

DNA degradation and proteolysis in thymocyte apoptosis.

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the protease inhibitors N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) and benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethylketone (Z-VAD.FMK) inhibit apoptosis. N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) has a very different effect, inducing the early morphological and biochemical changes associated with apoptosis. TLCK inhibits trypsin-like proteases whilst Z-VAD.FMK inhibits interleukin-1 beta-converting enzyme (ICE)-like proteases; this and the contrasting effects of TPCK support the hypothesis that thymocyte apoptosis involves a hierarchy of proteases which act at different stages of the process.

Animals↗
PubMed1994

Dexamethasone and etoposide induce apoptosis in rat thymocytes from different phases of the cell cycle.

Dexamethasone and etoposide both induce apoptosis in immature rat thymocytes. We investigated the dependence of apoptosis on the phase of the cell cycle after incubation with these drugs. Cell cycle progression was followed by a combination of pulse labelling with 5-bromo-2'-deoxyuridine (BrdU), labelling fixed cells with an anti-BrdU antibody and flow cytometry. Dexamethasone had little effect on the cell cycle progression of proliferating thymocytes, while etoposide caused cell cycle arrest. Normal and apoptotic thymocytes were separated by centrifugation on discontinuous Percoll gradients into four fractions (F1-F4). It was found that both dexamethasone and etoposide induced apoptosis in cells in G0/G1 and G2/M of the cell cycle, whereas only etoposide induced apoptosis of cells in S phase. These results demonstrated that dexamethasone induced apoptosis in quiescent cells while only etoposide could induce apoptosis in cells from the proliferative compartment. Following treatment of thymocytes with etoposide, some of the proliferating thymocytes (F1) were converted to cells with intermediate size and density (F3). We have recently identified these cells as a population of preapoptotic thymocytes, at an early stage of apoptosis. These cells then further progressed to fully apoptotic cells (F4). These data support the hypothesis that normal thymocytes (F1) became apoptotic (F4) via an intermediate population (F3).

Animals↗