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Marion MacFarlane

Publications and source records attributed to Marion MacFarlane.

11 recordsLinked to original sources
PubMed2005

TRAIL receptor-selective mutants signal to apoptosis via TRAIL-R1 in primary lymphoid malignancies.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its agonistic antibodies, which are currently in early clinical trials for treating various malignancies, induce apoptosis through triggering of either TRAIL-R1 or TRAIL-R2. Based on studies using agonistic monoclonal antibodies, we recently proposed that primary chronic lymphocytic leukemic cells seem to signal apoptosis primarily through TRAIL-R1. We have now synthesized mutant forms of TRAIL specific for TRAIL-R1 or TRAIL-R2. The selectivity of these mutants to induce apoptosis in cell lines is due to selective binding to their cognate receptors resulting in apoptosis via formation of a death-inducing signaling complex. Using these mutants, we now unequivocally show that primary cells from patients with chronic lymphocytic leukemia and mantle cell lymphoma signal to apoptosis almost exclusively through TRAIL-R1. Thus, no significant therapeutic benefit can be anticipated from treating such patients with agents currently in clinical trials that signal predominantly through TRAIL-R2, such as HGS-ETR2 or Apo2L/TRAIL. Our study highlights the necessity to determine whether primary cells from a particular tumor signal via TRAIL-R1 or TRAIL-R2. Such information will provide a rational approach to optimize TRAIL therapy.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Caspase activation inhibits proteasome function during apoptosis.

The ubiquitin/proteasome system regulates protein turnover by degrading polyubiquitinated proteins. To date, all studies on the relationship of apoptosis and the proteasome have emphasized the key role of the proteasome in the regulation of apoptosis, by virtue of its ability to degrade regulatory molecules involved in apoptosis. We now demonstrate how induction of apoptosis may regulate the activity of the proteasome. During apoptosis, caspase activation results in the cleavage of three specific subunits of the 19S regulatory complex of the proteasome: S6' (Rpt5) and S5a (Rpn10), whose role is to recognize polyubiquitinated substrates of the proteasome, and S1 (Rpn2), which with S5a and S2 (Rpn1) holds together the lid and base of the 19S regulatory complex. This caspase-mediated cleavage inhibits the proteasomal degradation of ubiquitin-dependent and -independent cellular substrates, including proapoptotic molecules such as Smac, so facilitating the execution of the apoptotic program by providing a feed-forward amplification loop.

Apoptosis↗
PubMed2004

Pro-apoptotic proteins released from the mitochondria regulate the protein composition and caspase-processing activity of the native Apaf-1/caspase-9 apoptosome complex.

The apoptosome is a large caspase-activating ( approximately 700-1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is approximately 135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9(1-130)) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9(1-130), demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6-8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.

Amino Acid Sequence↗
PubMed2004

Caspase-3 expression is reduced, in the absence of cleavage, in terminally differentiated normal oral epithelium but is increased in oral squamous cell carcinomas and correlates with tumour stage.

Oral carcinomas are known to have a greater apoptotic index than normal oral epithelium, evident as shrinking cells with condensed chromatin. In this study, these morphologically apoptotic cells stained positively for cleaved (active) caspase-3. In normal oral epithelium, cleaved caspase-3 positive-cells were only rarely detected. The terminally differentiated surface epithelial layers did not express cleaved caspase-3. The caspase-3 pro-enzyme showed a gradient of expression in normal oral epithelium, decreasing with differentiation. No expression was detectable in surface epithelial layers. Lack of expression of the major 'executioner' caspase-3 may, at least in part, explain differences in morphology between terminally differentiated and apoptotic cells. In cancers of different tissue origins, caspase-3 pro-enzyme expression can be either increased or decreased compared with normal tissue counterparts. To determine how caspase-3 expression alters during oral carcinogenesis, caspase-3 expression was compared in 39 samples of normal oral epithelium and 54 oral squamous cell carcinomas. Squamous cell carcinomas had more intense caspase-3 staining than normal epithelium (p < 0.001). Moreover, within the oral squamous cell carcinoma series, there was significantly more intense nuclear and cytoplasmic staining with increasing STNMP stage (p = 0.017 and 0.03, respectively). This was a reflection of significant associations with site (S), palpable lymph nodes (N), and differentiation (P). Both caspase-3 staining intensity and the percentage of cells positive for caspase-3 were inversely associated with differentiation. Studies of the mechanisms by which high levels of caspase-3 expression are tolerated in oral carcinoma cells may identify targets that can be used to harness caspase-3 overexpression for therapeutic benefit.

Aged↗
PubMed2003

Protein kinase C modulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by targeting the apical events of death receptor signaling.

We have further examined the mechanism by which phorbol ester-mediated protein kinase C (PKC) activation protects against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. We now report that activation of PKC targets death receptor signaling complex formation. Pre-treatment with 12-O-tetradecanoylphorbol-13-acetate (PMA) led to inhibition of TRAIL-induced apoptosis in HeLa cells, which was characterized by a reduction in phosphatidylserine (PS) externalization, decreased caspase-8 processing, and incomplete maturation and activation of caspase-3. These effects of PMA were completely abrogated by the PKC inhibitor, bisindolylmaleimide I (Bis I), clearly implicating PKC in the protective effect of PMA. TRAIL-induced mitochondrial release of the apoptosis mediators cytochrome c and Smac was blocked by PMA. This, together with the observed decrease in Bid cleavage, suggested that PKC activation modulates apical events in TRAIL signaling upstream of mitochondria. This was confirmed by analysis of TRAIL death-inducing signaling complex formation, which was disrupted in PMA-treated cells as evidenced by a marked reduction in Fas-associated death domain protein (FADD) recruitment, an effect that could not be explained by any change in FADD phosphorylation state. In an in vitro binding assay, the intracellular domains of both TRAIL-R1 and TRAIL-R2 bound FADD: activation of PKC significantly inhibited this interaction suggesting that PKC may be targeting key apical components of death receptor signaling. Significantly, this effect was not confined to TRAIL, because isolation of the native TNF receptor signaling complex revealed that PKC activation also inhibited TNF receptor-associated death domain protein recruitment to TNF-R1 and TNF-induced phosphorylation of IkappaB-alpha. Taken together, these results show that PKC activation specifically inhibits the recruitment of key obligatory death domain-containing adaptor proteins to their respective membrane-associated signaling complexes, thereby modulating TRAIL-induced apoptosis and TNF-induced NF-kappaB activation, respectively.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Fas-associated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis.

Death receptors are a subfamily of the tumor necrosis factor (TNF) receptor subfamily. They are characterized by a death domain (DD) motif within their intracellular domain, which is required for the induction of apoptosis. Fas-associated death domain protein (FADD) is reported to be the universal adaptor used by death receptors to recruit and activate the initiator caspase-8. CD95, TNF-related apoptosis-inducing ligand (TRAIL-R1), and TRAIL-R2 bind FADD directly, whereas recruitment to TNF-R1 is indirect through another adaptor TNF receptor-associated death domain protein (TRADD). TRADD also binds two other adaptors receptor-interacting protein (RIP) and TNF-receptor-associated factor 2 (TRAF2), which are required for TNF-induced NF-kappaB and c-Jun N-terminal kinase activation, respectively. Analysis of the native TNF signaling complex revealed the recruitment of RIP, TRADD, and TRAF2 but not FADD or caspase-8. TNF failed to induce apoptosis in FADD- and caspase-8-deficient Jurkat cells, indicating that these apoptotic mediators were required for TNF-induced apoptosis. In an in vitro binding assay, the intracellular domain of TNF-R1 bound TRADD, RIP, and TRAF2 but did not bind FADD or caspase-8. Under the same conditions, the intracellular domain of both CD95 and TRAIL-R2 bound both FADD and caspase-8. Taken together these results suggest that apoptosis signaling by TNF is distinct from that induced by CD95 and TRAIL. Although caspase-8 and FADD are obligatory for TNF-mediated apoptosis, they are not recruited to a TNF-induced membrane-bound receptor signaling complex as occurs during CD95 or TRAIL signaling, but instead must be activated elsewhere within the cell.

Adaptor Proteins, Signal Transducing↗
PubMed2003

TRAIL-induced signalling and apoptosis.

Prominent among cell surface molecules capable of initiating apoptosis are members of the tumour necrosis factor (TNF) family of ligands and receptors. Death-inducing members of the TNF receptor family each contain a cytoplasmic 'death domain' (DD): a protein-protein interaction motif critical for engaging downstream components of the signal transduction machinery. Following activation of these receptors by their cognate ligands, a death-inducing signalling complex (DISC) is formed by recruitment of cytoplasmic DD-containing proteins to the receptor DD. The best-studied death-inducing ligand-receptor pairs are TNF/TNF receptor-1 (TNFR1) and CD95L/CD95 (Fas, Apo-1). A more recently identified member of the TNF ligand family is TNF-related apoptosis-inducing ligand (TRAIL), Apo-2L, which induces apoptosis in a number of tumour cell lines, yet is relatively non-toxic to normal cells. Consistent with its lack of toxicity, TRAIL is constitutively expressed in many human tissues. This raises the conundrum of why tumour cells are sensitive to TRAIL-mediated apoptosis and normal tissues resistant. One possibility lies in the existence of a family of four membrane-bound TRAIL receptors (TRAIL-R1-R4), which although able to bind TRAIL, differ in their ability to transduce the death signal. Recently, this hypothesis has been challenged based on poor correlations between TRAIL receptor expression and TRAIL sensitivity. This suggests that other factors, such as TRAIL-induced NF-kappaB activation or death inhibitors including c-FLIP, are involved in determining differential sensitivity to TRAIL. We have investigated TRAIL sensitivity in primary tumour cells together with TRAIL-induced signalling pathways in both sensitive and resistant cell lines. Our results and their implications for the potential use of TRAIL in cancer therapy are discussed.

Animals↗
PubMed2002

Mechanisms of resistance to TRAIL-induced apoptosis in primary B cell chronic lymphocytic leukaemia.

Primary B cells from B cell chronic lymphocytic leukaemia (B-CLL) were resistant to the novel selective cytotoxic agent, TNF-related apoptosis-inducing ligand (TRAIL). Low levels of the death-inducing TRAIL receptors, TRAIL-R1 and TRAIL-R2 but not the putative 'decoy' receptors, TRAIL-R3 and TRAIL-R4, were expressed on the surface of B-CLL cells. Resistance to TRAIL was upstream of caspase-8 activation, as little or no caspase-8 was processed in TRAIL-treated B-CLL cells. Low levels of a TRAIL death-inducing signalling complex (DISC) were formed in these cells, accompanied by the recruitment of endogenous FADD, caspase-8 and c-FLIP(L) but not c-FLIP(S). Both caspase-8 and c-FLIP(L) were cleaved to form two stable intermediates of approximately 43 kDa, which remained associated with the DISC. Caspase-8 was not further processed to its active heterotetramer. Thus the resistance of B-CLL cells to TRAIL may be due partly to low surface expression of the death receptors resulting in low levels of DISC formation and also to the high ratio of c-FLIP(L) to caspase-8 within the DISC, which would prevent further activation of caspase-8. Our results highlight the possibility of sensitising B-CLL cells to TRAIL by modulation of c-FLIP levels or by upregulation of surface expression of death receptors.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Proteasome-mediated degradation of Smac during apoptosis: XIAP promotes Smac ubiquitination in vitro.

During apoptosis, Smac (second mitochondria-derived activator of caspases)/DIABLO, an IAP (inhibitor of apoptosis protein)-binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to the death-inducing ligand, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), results in rapid Smac release from mitochondria, which occurs before or in parallel with loss of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor demonstrating that this event is caspase-dependent and modulated by Bcl-2 family members. Following release, Smac is rapidly degraded by the proteasome, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as a ubiquitin-protein ligase (E3) in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP are essential for ubiquitination, suggesting that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of mitochondrial-released Smac. Thus, in addition to its well characterized role in inhibiting caspase activity, XIAP may also protect cells from inadvertent mitochondrial damage by targeting pro-apoptotic molecules for proteasomal degradation.

Apoptosis↗
PubMed2002

Bcl-2 and Bcl-xL inhibit CD95-mediated apoptosis by preventing mitochondrial release of Smac/DIABLO and subsequent inactivation of X-linked inhibitor-of-apoptosis protein.

Bcl-2 and Bcl-x(L) are reported to inhibit CD95-mediated apoptosis in "type II" but not in "type I" cells. In the present studies, we found that stimulation of CD95 receptors, with either agonistic antibody or CD95 ligand, resulted in the activation of caspase-8, which in turn processed caspase-3 between its large and small subunits. However, in contrast to control cells, those overexpressing either Bcl-2 or Bcl-x(L) displayed a distinctive pattern of caspase-3 processing. Indeed, the resulting p20/p12 caspase-3 was not active and did not undergo normal autocatalytic processing to form p17/p12 caspase-3, because it was bound to and inhibited by endogenous X-linked inhibitor-of-apoptosis protein (XIAP). Importantly, Bcl-2 and Bcl-x(L) inhibited the release of both cytochrome c and Smac from mitochondria. However, since Smac alone was sufficient to promote caspase-3 activity in vitro by inactivating XIAP, we proposed the existence of a death receptor-induced, Smac-dependent and apoptosome-independent pathway. This type II pathway was subsequently reconstituted in vitro using purified recombinant proteins at endogenous concentrations. Thus, mitochondria and associated Bcl-2 and Bcl-x(L) proteins may play a functional role in death receptor-induced apoptosis by modulating the release of Smac. Our data strongly suggest that the relative ratios of XIAP (and other inhibitor-of-apoptosis proteins) to active caspase-3 and Smac may dictate, in part, whether a cell exhibits a type I or type II phenotype.

Apoptosis↗