PubMed Health⌕ Search

Biomedical subjects

S Tomlinson

Publications and source records attributed to S Tomlinson.

At least 19 recordsLinked to original sources
PubMed2006

Upregulation of two BH3-only proteins, Bmf and Bim, during TGF beta-induced apoptosis.

Transforming growth factor-beta (TGFbeta)-activated signalling pathways can lead to apoptosis, growth arrest or promotion of malignant behaviour, dependent on cellular context. The molecular mechanisms involved in TGFbeta-induced apoptosis remain controversial; although changes in gene expression are thought to be pivotal to the process, several different candidate apoptotic initiators and mediators have been proposed. Smad4, a critical component of the TGFbeta-induced transcriptional machinery, is shown here to be essential for induction of apoptosis. Gene expression analysis identified the proapoptotic Bcl-2 family members, Bmf and Bim, as induced by TGFbeta, dependent on both Smad4 and p38 function and the generation of reactive oxygen species. TGFbeta-induced Bmf and Bim localize to cellular membranes implicated in apoptosis. Inhibition of the TGFbeta-induced expression of both these proteins together provides significant protection of cells from apoptosis. The TGFbeta-triggered cell death programme thus involves induction of multiple BH3-only proteins during the induction of apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report.

As of January 2005, there were 1020 gene therapy clinical trials ongoing worldwide with 675 or 66.2% devoted to cancer gene therapy. The majority are occurring in the US and Europe (http://www.wiley.co.uk/genetherapy/clinical/). At the present time, to our knowledge there are no trials that employ gene delivery of Fas Ligand (FasL). As an important note, and in contrast to somatic cell therapy trials, there are no reported deaths due to therapeutic vector administration in any cancer gene therapy trial. That said, from our studies and from the published literature, the issue of gene delivery remains the major obstacle to successfully employing gene therapy for cancer treatment. Numerous laboratories are studying this with many different approaches. My co-workers and I have focused on the delivery issue by using various approaches that address tumor targeting and transgene expression. In addition, we are focusing on enhancing tumor cell killing via the bystander effect and through use of small molecules to enhance bystander activity.

Adenoviridae↗
PubMed2001

Complement-independent, peroxide-induced antibody lysis of platelets in HIV-1-related immune thrombocytopenia.

Immunologic thrombocytopenia is seen commonly in HIV-1 infection. The pathogenesis of this problem has been unclear, but it is associated with circulating immune complexes that contain platelet membrane components and anti-platelet membrane GPIIIa49-66 IgG antibodies. These antibodies cause acute thrombocytopenia when injected into mice. We now show that purified anti-GPIIIa49-66 causes platelet fragmentation, in vitro in the absence of complement, and in vivo in wild-type and C3-deficient mice. The mechanism of complement-independent platelet lysis is shown to be caused by the antibody-induced generation of H202, as indicated by in vitro experiments with inhibitors of reactive oxygen species, and in vivo studies carried out with p47phox-deficient mice. Thus, a novel mechanism of immunologic platelet clearance is described in which an anti-platelet IgG causes platelet fragmentation via the induction of reactive oxygen species.

Animals↗
PubMed2001

Targeting of functional antibody-decay-accelerating factor fusion proteins to a cell surface.

Recombinant soluble complement inhibitors hold promise for the treatment of inflammatory disease and disease states associated with transplantation. Targeting complement inhibitors to the site of complement activation and disease may enhance their efficacy and safety. Data presented show that targeting of decay-accelerating factor (DAF, an inhibitor of complement activation) to a cell surface by means of antibody fragments is feasible and that cell-targeted DAF provides significantly enhanced protection from complement deposition and lysis compared with soluble untargeted DAF. An extracellular region of DAF was joined to an antibody combining site with specificity for the hapten dansyl, at the end of either C(H)1 or C(H)3 Ig regions. The recombinant IgG-DAF chimeric proteins retained antigen specificity and bound to dansylated Chinese hamster ovary cells. Both soluble C(H)1-DAF and C(H)3-DAF were effective at inhibiting complement-mediated lysis of untargeted Chinese hamster ovary cells at molar concentrations within the range reported by others for soluble DAF. However, when targeted to a dansyl-labeled cell membrane, C(H)1-DAF was significantly more potent at inhibiting complement deposition and complement-mediated lysis. Cell-bound C(H)1-DAF also provided cells with protection from complement lysis after removal of unbound C(H)1-DAF. Of further importance, the insertion of a nonfunctional protein domain of DAF (the N-terminal short consensus repeat) between C(H)1 and the functional DAF domain increased activity of the fusion protein. In contrast to C(H)1-DAF, C(H)3-DAF was not significantly better at protecting targeted versus untargeted cells from complement, indicating that a small targeting vehicle is preferable to a large one. We have previously shown that for effective functioning of soluble complement inhibitor CD59, binding of CD59 to the cell surface close to the site of complement activation is required. Significantly, such a constraint did not apply for effective DAF function.

Animals↗
PubMed2001

A novel epitope of CD59 expressed by primitive human hematopoietic progenitors.

OBJECTIVE: The aim of this study was to determine the identity of the cell surface molecule on primitive hematopoietic cells recognized by monoclonal antibody HCC-1. MATERIALS AND METHODS: Screening of a cDNA expression library prepared from human bone marrow stromal cells with HCC-1 yielded a single cDNA, which when expressed in FDCP-1 cells, resulted in the specific acquisition of HCC-1 binding. The cDNA demonstrated complete identity with CD59, a phosphoinositol glycan-linked membrane protein that protects cells against autologous complement attack. The ubiquitous expression of CD59 is in marked contrast to the restricted reactivity of HCC-1. Studies were performed to examine the basis for the novel specificity of HCC-1 for CD59. The epitope on CD59 identified by HCC-1 was mapped using a series of rat/human CD59 chimeric proteins. Immunoprecipitation analyses were performed to determine whether CD59 associates with other membrane proteins. RESULTS: Mutagenesis of Asn18 did not alter the binding of HCC-1 to CD59, suggesting that N-linked carbohydrates are not responsible for the binding specificity of HCC-1. The epitope for HCC-1 was shown to differ from that identified by previously described CD59 antibodies, encompassing residues A31, L33, R55, and L59. An 80 kDa protein co-immunoprecipitated with CD59 in the HCC-1(-) cell line HL-60 but not in HCC-1(+) K562 cells. CONCLUSION: Collectively, these data support the hypothesis that the unique specificity of HCC-1 for CD59 is due in part to recognition of a novel epitope, which is masked as a result of association with an as yet unidentified 80 kDa protein.

Adult↗
PubMed2001

Trypanosome lytic factors: novel mediators of human innate immunity.

A novel trypanosome lytic factor (TLF) has been characterized that protects humans from infection by Trypanosoma brucei brucei. The mechanism of trypanolysis is unknown; contrary to one hypothesis, TLF does not kill trypanosomes by generating oxygen radicals. However, these trypanosomes become human-infective when they express a serum-resistance-associated gene.

Animals↗
PubMed2000

CD59 expressed on a tumor cell surface modulates decay-accelerating factor expression and enhances tumor growth in a rat model of human neuroblastoma.

It has been hypothesized that complement inhibitors expressed on the surface of tumor cells prevent effective immune-mediated clearance. Whereas there are in vitro data to support this hypothesis, the species-selective activity of complement inhibitors has been a hindrance to investigating the role of membrane-bound complement inhibitors in rodent models of human cancer. The CD59-positive LAN-1 human neuroblastoma cell line was significantly more sensitive to lysis by rat complement than by human complement, illustrating the species selectivity of endogenously expressed complement inhibitors. Transfection of LAN-1 cells with rat CD59, an inhibitor of the terminal cytolytic membrane attack complex, effectively protected the cells from lysis by rat complement in vitro. When LAN-1 cells stably expressing rat CD59 were inoculated into immune-deficient rats, the onset of tumor growth and the rate of tumor growth were significantly enhanced compared with those of control-transfected LAN-1 cells. These data show directly that the expression of a complement inhibitor on a tumor cell promotes tumor growth. Flow cytometric analysis revealed that the endogenous expression of decay-accelerating factor (DAF), an inhibitor of complement activation, was up-regulated on the surface of cells after in vivo growth. Of further interest, higher levels of DAF were present on CD59-transfected cells than on control-transfected cells derived from tumors. Increased DAF expression correlated with decreased complement deposition on the tumor cell surface. These results show that expression of complement inhibitors on a tumor cell has functional consequences with regard to complement deposition in vivo and indicate that CD59 can indirectly effect complement activation and C3 deposition in vivo via a link between CD59 and DAF expression.

Animals↗
PubMed2000

Müller glia cells reorganize reaggregating chicken retinal cells into correctly laminated in vitro retinae.

Müller cells, that belong to the family of radial glia cells, have central functions during retinogenesis. They form a stabilizing scaffold, they are candidate targets for the mediation of extraneous retinogenetic factors, and they are an important source for retina-borne retinogenetic factors. Reaggregate cultures allow the analysis of retinogenesis from dispersed cells to fully laminated tissues. Reaggregating cells from the embryonic chick retina reassemble to reversed laminated cellular spheres including constituents of all retinal layers, yet the outer nuclear layer is represented by internal rosettes. Using spheroids, we tested whether Müller cells have a decisive function in establishing retinal polarity and in determining the lamination pattern. To this end, we established confluent monolayers of highly enriched Müller cells derived from E6 or E13 chicken retinas, and then let dispersed E5.5 retinal cells reaggregate either in the absence of these monolayers or on top of them. In the presence of Müller cells, the reversed lamina polarity of rosetted spheroids progressively transformed within a week into correctly laminated retinal spheres, whereas all initial rosettes vanished. Moreover, photoreceptors formed a regular outer nuclear layer, as visualized by the rod-specific CERN901 antibody. In correctly laminated spheroids, staining for vimentin and glutamine synthetase was much more pronounced than in rosetted spheroids; in particular, a well-established inner limiting membrane stood out wherever the retinal lamination was complete. Because these effects can be similarly achieved by supernatants derived from Müller cells, direct cell-cell contacts or cellular replenishment from the monolayer do not account for these effects. We conclude that Müller cells are involved in the establishment of a correct retinal lamination and in the arrangement of the cells in the reaggregate cultures. In particular, rosette formation is counteracted and the formation of an inner limiting membrane is induced. Because rosettes are objects of concern in several ophthalmological defects, these results are highly relevant, both biomedically and also for normal retinogenesis.

Animals↗
PubMed2000

Surface antigen expression and complement susceptibility of differentiated neuroblastoma clones.

Human neuroblastoma cell lines typically consist of heterogenous subpopulations of cells that are morphologically and biochemically distinct. The cell types are characterized as neuroblastic (N-type), substrate-adherent Schwann-like (S-type), or intermediate (I). These cell types can undergo spontaneous or induced transdifferentiation in vitro. We investigated the complement sensitivity of different neuroblastoma cell lines and of matched sets of cloned N- and S-type neuroblastoma cell lines. Human neuroblastoma cell lines that consisted predominantly of a neuroblastic phenotype were shown to be significantly more susceptible to human complement-mediated lysis than cell lines of other cancer types. Complement sensitivity of neuroblastoma cell lines was correlated with low levels of CD59, decay-accelerating factor, and membrane cofactor protein expression. We found that cloned S-type neuroblastoma cells were much more resistant to complement-mediated lysis than cloned N-type cells. The increased complement resistance of S-type cells was shown to be due to increased expression of membrane-bound complement inhibitors. CD59 was the single most important protein in providing S-type cells with protection from complement lysis. S-type cells were also found to express lower levels of GD2, a target antigen for a complement activating monoclonal antibody currently in clinical trials for neuroblastoma immunotherapy. The ability of S-type cells to evade complement, and the ability of S-type cells to differentiate into the more tumorigenic N-type cells, may represent a mechanism of tumor survival and regrowth, a phenomenon often observed with this cancer.

Antibodies, Blocking↗
PubMed2000

An investigation into the mechanism of trypanosome lysis by human serum factors.

African trypanosomes are the causative agents of sleeping sickness in humans and of Nagana in cattle. The infectivity of African trypanosome species for humans appears to be defined by their susceptibility to two lytic factors in human serum; trypanosome lytic factor (TLF)1, a subclass of human high density lipoprotein (HDL) and TLF2, a high molecular weight protein complex. Available evidence indicates that following receptor mediated uptake, TLF is targeted to the lysosome where the low pH triggers a TLF-dependant peroxidase activity resulting in the formation of reactive oxygen radicals with consequent lipid peroxidation and destruction of the lysosomal membrane. Nearly all previous work on the mechanism of parasite lysis has been performed using TLF1. In this study, we directly test the hypothesis that TLF1 and TLF2 kill Trypanosoma brucei by a mechanism involving oxidative stress. We found no evidence for lipid peroxidation in trypanosomes exposed to high concentrations of trypanolytic HDL (impure TLF1), although lipid peroxidation was detected in parasites exposed to low concentrations of low molecular weight peroxides. Neither HDL, TLF1 nor TLF2 generated detectable levels of intracellular reactive oxygen intermediates. Various antioxidants also had no effect on TLF1 or TLF2-mediated lysis, although the antioxidants catalase and superoxide dismutase were effective at inhibiting peroxide generation and parasite lysis in control systems. Various metal chelating agents and protease inhibitors were also tested without effect. These data provide strong evidence against a peroxidative mechanism being involved in TLF-mediated lysis.

Animals↗
PubMed1999

Identification of the individual residues that determine human CD59 species selective activity.

Formation of the cytolytic membrane attack complex of complement on host cells is inhibited by the membrane-bound glycoprotein, CD59. The inhibitory activity of CD59 is species restricted, and human CD59 is not effective against rat complement. Previous functional analysis of chimeric human/rat CD59 proteins indicated that the residues responsible for the species selective function of human CD59 map to a region contained between positions 40 and 66 in the primary structure. By comparative analysis of rat and human CD59 models and by mutational analysis of candidate residues, we now identify the individual residues within the 40-66 region that confer species selective function on human CD59. All nonconserved residues within the 40-66 sequence were substituted from human to rat residues in a series of chimeric human/rat CD59 mutant proteins. Functional analysis revealed that the individual human to rat residue substitutions F47A, T51L, R55E, and K65Q each produced a mutant human CD59 protein with enhanced rat complement inhibitory activity with the single F47A substitution having the most significant effect. Interestingly, the side chains of the residues at positions 47, 51, and 55 are all located on the short single helix (residues 47-55) of CD59 and form an exposed continuous strip parallel to the helix axis. A single human CD59 mutant protein containing rat residue substitutions at all three helix residues produced a protein with species selective activity comparable to that of rat CD59. We further found that synthetic peptides spanning the human CD59 helix sequence were able to inhibit the binding of human CD59 to human C8, but had little effect on the binding of rat CD59 to rat C8.

Amino Acid Sequence↗
PubMed1999

Reply

Explore the source record for details and available documents.

Journal Article↗
PubMed1999

Protection of human breast cancer cells from complement-mediated lysis by expression of heterologous CD59.

CD59, decay accelerating factor (DAF) and membrane cofactor protein (MCP) are widely expressed cell surface glycoproteins that protect host cells from the effects of homologous complement attack. Complement inhibitory activity of these proteins is species-selective. We show that the human breast cancer cell line MCF7 is relatively resistant to lysis by human complement, but is effectively lysed by rat or mouse complement. CD59, DAF and MCP were all shown to be expressed by MCF7. The species-selective nature of CD59 activity was used to demonstrate directly the effectiveness of CD59 at protecting cancer cells from complement-mediated lysis. cDNAs encoding rat and mouse CD59 were separately transfected into MCF7 cells, and cell populations expressing high levels of the rodent CD59 were isolated by cell sorting. Data show that rat and mouse CD59 were highly effective at protecting transfected MCF7 cells from lysis by rat and mouse complement, respectively. Data further reveal that rat CD59 is not effective against mouse complement, whereas mouse CD59 is effective against both mouse and rat complement. These studies establish a model system for relevant in vivo studies aimed at determining the effect of complement regulation on tumourigenesis, and show that for effective immunotherapy using complement-activating anti-tumour antibodies, the neutralization of CD59 and/or other complement inhibitory molecules will probably be required.

Animals↗