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Biomedical subjects

M MacFarlane

Publications and source records attributed to M MacFarlane.

At least 19 recordsLinked to original sources
PubMed2005

Increased sensitivity to TRAIL-induced apoptosis occurs during the adenoma to carcinoma transition of colorectal carcinogenesis.

The death ligand TRAIL (Apo2L) has potential for cancer therapy, since tumour cells are thought to be more sensitive than normal cells. We investigated whether sensitivity to TRAIL increases during the adenoma to carcinoma transition of colorectal carcinogenesis. Under the same culture conditions, we compared the extent of TRAIL-induced apoptosis in four premalignant adenoma and three carcinoma cell lines. Although TRAIL induced some apoptosis in adenoma cultures, the carcinoma cell lines were significantly more sensitive (P<0.001). This finding was recapitulated in an in vitro model of tumour progression in which conversion of the adenoma cell line AA/C1 to a tumorigenic phenotype was associated with increased TRAIL sensitivity (P<0.001). Increased TRAIL sensitivity during colorectal carcinogenesis has been previously attributed to changes in the balance between TRAIL receptors TRAIL-R1 and -R2 and "decoy" receptors TRAIL-R3 and -R4 during malignant progression. To address this, cell surface receptor expression was measured by flow cytometry. In summary, during colorectal carcinogenesis, there is a marked increase in sensitivity to TRAIL-induced apoptosis associated with progression from benign to malignant tumour that could be exploited for colon cancer therapy, but alterations in cell surface TRAIL receptor expression may not be the primary reason for this change.

Adenoma↗
PubMed2005

Chronic lymphocytic leukemic cells exhibit apoptotic signaling via TRAIL-R1.

Clinical trials have been initiated with Apo2L/TRAIL (Genentech) and agonistic mAbs to TRAIL receptors, -R1 and -R2 (Human Genome Sciences). The apoptosis-inducing ability of these mAbs and different TRAIL preparations, in the presence or absence of histone deacetylase inhibitors (HDACi), varied markedly against primary chronic lymphocytic leukaemia (CLL) cells and various tumor cell lines, demonstrating an unanticipated preferential apoptotic signaling via either TRAIL-R1 or -R2. Contrary to literature reports that TRAIL-induced apoptosis occurs primarily via signaling through TRAIL-R2, CLL cells, in the presence of HDACi, undergo predominantly TRAIL-R1-mediated apoptosis. Consequently, Apo2L/TRAIL, which signals primarily through TRAIL-R2, is virtually devoid of activity against CLL cells. To maximize therapeutic benefit, it is essential to ascertain whether a primary tumor signals via TRAIL-R1/-R2, prior to initiating therapy. Thus combination of an agonistic TRAIL-R1 Ab and an HDACi, such as the anticonvulsant sodium valproate, could be of value in treating CLL.

Adult↗
PubMed2004

Histone deacetylase inhibitors potentiate TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in lymphoid malignancies.

New therapies are required for chronic lymphocytic leukemia (CLL), an incurable disease characterized by failure of mature lymphocytes to undergo apoptosis. Activation of cell surface death receptors, such as via TRAIL receptor ligation, may provide a novel therapeutic target for various malignancies. However, CLL and other lymphoid malignancies are resistant to TRAIL. We report that low concentrations of histone deacetylase (HDAC) inhibitors, such as depsipeptide, which alone failed to induce apoptosis, markedly sensitize CLL cells and other primary lymphoid malignancies to TRAIL-induced apoptosis. These combinations caused little or no toxicity to normal lymphocytes. HDAC inhibitors sensitized resistant cells to TRAIL-induced apoptosis by facilitating formation of an active death-inducing signalling complex (DISC), leading to the rapid activation of caspase-8. The facilitated DISC formation also occurred in the absence of TRAIL-R2 upregulation. Thus, the combination of HDAC inhibitors and TRAIL may be valuable in the treatment of various hemopoietic malignancies.

Apoptosis↗
PubMed2003

Macrophage-mediated clearance of cells undergoing caspase-3-independent death.

Little is known of the functions of caspases in mediating the surface changes required for phagocytosis of dying cells. Here we investigate the role played by the effector caspase, caspase-3 in this process using the caspase-3-defective MCF-7 breast carcinoma line and derived caspase-3-expressing transfectants. Our results indicate that, while certain typical features of apoptosis induced by etoposide--namely classical morphological changes and the ability to degrade DNA into oligonucleosomal fragments - are caspase-3-dependent, loss of cell adhesion to plastic and the capacity to interact with, and to be phagocytosed by, human monocyte-derived macrophages - both by CD14-dependent and CD14-independent mechanisms--do not require caspase-3. Furthermore, both etoposide-induced caspase-3-positive and -negative MCF-7 cells suppressed proinflammatory cytokine release by macrophages. These results demonstrate directly that cell surface changes that are sufficient for anti-inflammatory clearance by human macrophages can be regulated independently of stereotypical features of the apoptosis programme that require caspase-3.

Annexin A5↗
PubMed2002

The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet.

Mitochondria are 'life-essential' organelles for the production of metabolic energy in the form of ATP. Paradoxically mitochondria also play a key role in controlling the pathways that lead to cell death. This latter role of mitochondria is more than just a 'loss of function' resulting in an energy deficit but is an active process involving different mitochondrial proteins. Cytochrome c was the first characterised mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be actively implicated in apoptotic cell death. Since then, other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process. Members of the Bcl-2 protein family control the integrity and response of mitochondria to apoptotic signals. The molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins is still elusive. This review summarises and evaluates the current knowledge concerning the complex role of released mitochondrial proteins in the apoptotic process.

Animals↗
PubMed2001

CARDINAL, a novel caspase recruitment domain protein, is an inhibitor of multiple NF-kappa B activation pathways.

Proteins possessing the caspase recruitment domain (CARD) motif have been implicated in pathways leading to activation of caspases or NF-kappaB in the context of apoptosis or inflammation, respectively. Here we report the identification of a novel protein, CARDINAL, that contains a CARD motif and also exhibits a high degree of homology to the C terminus of DEFCAP/NAC, a recently described member of the Apaf-1/Nod-1 family. In contrast with the majority of CARD proteins described to date, CARDINAL failed to promote apoptosis or NF-kappaB activation. Rather, CARDINAL potently suppressed NF-kappaB activation associated with overexpression of TRAIL-R1, TRAIL-R2, RIP, RICK, Bcl10, and TRADD, or through ligand-induced stimulation of the interleukin-1 or tumor necrosis factor receptors. Co-immunoprecipitation experiments revealed that CARDINAL interacts with the regulatory subunit of the IkappaB kinase (IKK) complex, IKKgamma (NEMO), providing a molecular basis for CARDINAL function. Thus, CARDINAL is a novel regulator of NF-kappaB activation in the context of pro-inflammatory signals.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Modulation of tumor necrosis factor apoptosis-inducing ligand- induced NF-kappa B activation by inhibition of apical caspases.

Tumor necrosis factor (TNF) apoptosis-inducing ligand (TRAIL), a member of the TNF family, induces apoptosis in many transformed cells. We report TRAIL-induced NF-kappaB activation, concomitant with production of the pro-inflammatory cytokine Interleukin-8 in the relatively TRAIL-insensitive cell line, HEK293. In contrast, TRAIL-induced NF-kappaB activation occurred in HeLa cells only upon pretreatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.fmk), indicating that this was due to a caspase-sensitive component of TRAIL-induced NF-kappaB activation. NF-kappaB activation was mediated by the death receptors, TRAIL-R1 and -R2, but not by TRAIL-R3 or -R4 and was only observed in HeLa cells in the presence of z-VAD.fmk. Receptor-interacting protein, an obligatory component of TNF-alpha-induced NF-kappaB activation, was cleaved during TRAIL-induced apoptosis. We show that receptor-interacting protein is recruited to the native TRAIL death-inducing signaling complex (DISC) and that recruitment is enhanced in the presence of z-VAD.fmk, thus providing an explanation for the potentiation of TRAIL-induced NF-kappaB activation by z-VAD.fmk in TRAIL-sensitive cell lines. Examination of the TRAIL DISC in sensitive and resistant cells suggests that a high ratio of c-FLIP to caspase-8 may partially explain cellular resistance to TRAIL-induced apoptosis. Sensitivity to TRAIL-induced apoptosis was also modulated by inhibition or activation of NF-kappaB. Thus, in some contexts, modulation of NF-kappaB activation possibly at the level of apical caspase activation at the DISC may be a key determinant of sensitivity to TRAIL-induced apoptosis.

Amino Acid Chloromethyl Ketones↗
PubMed2001

The inhibitor of apoptosis protein-binding domain of Smac is not essential for its proapoptotic activity.

Smac/DIABLO, a recently identified inhibitor of apoptosis protein (IAP)-binding protein, is released from the mitochondria during apoptosis and reportedly potentiates apoptosis by relieving the inhibition of IAPs on caspases. We now describe the molecular characterization of Smac beta, an alternatively spliced form of Smac, which lacks the mitochondrial-targeting sequence found in Smac and has a cortical distribution in both human embryonic kidney 293 and breast epithelial tumor MCF-7 cells. Smac beta, which binds IAPs in vitro, does not bind IAPs in intact cells due to cellular processing and removal of its NH(2)-terminal IAP-binding domain. Despite its inability to interact with IAPs in cells, processed Smac beta is proapoptotic, as demonstrated by its ability to potentiate apoptosis induced by both death receptor and chemical stimuli. Furthermore, expression of a NH(2)-terminally truncated Smac mutant (Delta75), which lacks the entire IAP-interacting domain, potentiates apoptosis to the same extent as Smac and Smac beta. Our data support the hypothesis that the main proapoptotic function of Smac and Smac beta is due to a mechanism other than IAP binding.

Amino Acid Sequence↗
PubMed2000

JNK (c-Jun N-terminal kinase) and p38 activation in receptor-mediated and chemically-induced apoptosis of T-cells: differential requirements for caspase activation.

Activation of the stress-activated mitogen-activated protein kinases (MAP kinases), c-Jun N-terminal kinase (JNK) and p38, is necessary for the induction of apoptosis in neuronal cells; however, in other cell types their involvement may be stimulus-dependent. In the present study we investigate the activation of JNK and p38 in a single non-neuronal cell type, undergoing receptor-mediated (tumour necrosis factor-related apoptosis-inducing ligand and CD95) or chemically-induced (lactacystin) apoptosis. In Jurkat T-cells, receptor-mediated and chemically-induced apoptosis resulted in a time-dependent activation of the initiator caspases-8 and -9, respectively. Both types of stimuli resulted in a significant activation of JNK and p38, which closely paralleled the time-dependent induction of apoptosis. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.FMK) inhibited receptor-mediated apoptosis and suppressed JNK and p38 activation. In contrast, inhibition of lactacystin-induced apoptosis with z-VAD.FMK, as assessed by phosphatidylserine exposure and poly(ADP-ribose) polymerase cleavage, did not inhibit activation of JNK or p38, demonstrating that during chemically-induced apoptosis, activation of JNK and p38 is independent of effector caspases. The role of p38 in apoptosis was assessed using the specific p38 inhibitor, SB203580. No effect on the induction of apoptosis or caspase activation was observed, although activation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), an immediate downstream target of p38, was inhibited. Therefore neither p38 activation nor activation of MAPKAPK-2 is critical for induction of either receptor- or chemically-induced apoptosis. Thus, within a single cell type, (1) the mechanism of p38 and JNK activation during apoptosis is stimulus-dependent and (2) activation of the p38 pathway is not required for caspase activation or apoptosis, assessed by phosphatidylserine exposure, but may still be required to elicit other features of the apoptotic phenotype.

Acetylcysteine↗
PubMed2000

Protein complexes activate distinct caspase cascades in death receptor and stress-induced apoptosis.

Caspases play a central role in the execution phase of apoptosis and are responsible for many of the morphological features normally associated with this form of cell death. Caspases can activate one another and consequently can initiate specific caspase cascades. Caspases-8 and -9 appear to be the apical caspases activated in death receptor- and mitochondrial stress-induced apoptosis, respectively. The role of large protein complexes in mediating these pathways is discussed.

Animals↗
PubMed2000

Active caspases and cleaved cytokeratins are sequestered into cytoplasmic inclusions in TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) -induced apoptosis, in transformed human breast epithelial MCF-7 cells, resulted in a time-dependent activation of the initiator caspases-8 and -9 and the effector caspase-7. Cleavage of caspase-8 and its preferred substrate, Bid, preceded processing of caspases-7 and -9, indicating that caspase-8 is the apical initiator caspase in TRAIL-induced apoptosis. Using transient transfection of COOH-terminal-tagged green fluorescent protein fusion constructs, caspases-3, -7, and -8 were localized throughout the cytoplasm of MCF-7 cells. TRAIL-induced apoptosis resulted in activation of caspases-3 and -7, and the redistribution of most of their detectable catalytically active small subunits into large spheroidal cytoplasmic inclusions, which lacked a limiting membrane. These inclusions, which were also induced in untransfected cells, contained cytokeratins 8, 18, and 19, together with both a phosphorylated form and a caspase-cleavage fragment of cytokeratin 18. Similarly, in untransfected breast HBL100 and lung A549 epithelial cells, TRAIL induced the formation of cytoplasmic inclusions that contained cleaved cytokeratin 18 and colocalized with active endogenous caspase-3. We propose that effector caspase-mediated cleavage of cytokeratins, resulting in disassembly of the cytoskeleton and formation of cytoplasmic inclusions, may be a characteristic feature of epithelial cell apoptosis.

Apoptosis↗
PubMed2000

Initiation of Apaf-1 translation by internal ribosome entry.

The apoptotic protease activating factor (Apaf-1) plays a central role in apoptosis: interaction of this protein with procaspase-9 leads to cleavage and activation of this initiator caspase. In common with other mRNAs whose protein products have a major regulatory function, the 5' untranslated region (UTR) of Apaf-1 is long, G-C rich and has the potential to form secondary structure. We have shown that the 5' UTR of Apaf-1 contains an internal ribosome entry segment, located in a 233 nucleotide region towards the 3' end of the leader, and that the translation initiation of this mRNA occurs only by internal ribosome entry. The Apaf-1 IRES is active in almost all human cell types tested, including Human cervical carcinoma (HeLa), Human liver carcinoma (HepG2), Human breast carcinoma (MCF7), Human embryonic kidney (HK293), African Green Monkey kidney (COS7) and Human lung (MRC5). The Apaf-1 IRES initiates translation as efficiently as the HRV IRES, but is less active than the c-myc IRES. We propose that the Apaf-1 IRES ensures that a constant cellular level of Apaf-1 protein is maintained even under conditions where cap-dependent translation is compromised. Oncogene (2000) 19, 899 - 905.

5' Untranslated Regions↗
PubMed2000

Multiple meningiomas: case report and review of the literature.

A case of a 69 year old woman with multiple meningiomas is presented. Since her first presentation with Jacksonian seizures in 1978, she has had 16 meningiomas excised in seven separate surgical procedures. This is extremely unusual due to both the large number of tumours, and the long period over which they have occurred. We review the epidemiology, possible pathogenesis and genetic associations of this condition.

Aged↗