Publications (6)32.73 Total impact

  • DA Fennell · A Pallaska · M Corbo · F E Cotter
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    ABSTRACT: Robust quantitative estimation of average whole cell mitochondrial dysfunction is a useful tool for assessing sensitivity to apoptotic stimuli induced either by novel agents, or following manipulation of apoptotic threshold by pharmacological or functional genomics approaches. We have mathematically modelled the kinetics of whole cell mitochondrial membrane potential depolarisation within a population of cells as a Bernouli transition. An exponential distribution enables the median latency preceding mitochondrial membrane potential dissipation to be derived. The kinetic model can be fitted to in vitro single cell resolution data derived from kinetic flow cytometric studies by non-linear regression. We propose that kinetic determination of cumulative frequency distributions provides a useful approach for estimating apoptosis sensitivity across cell populations over short time-frames.
    No preview · Article · Apr 2005 · APOPTOSIS
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    ABSTRACT: Cholangiocarcinoma cells express high levels of the antiapoptotic proteins Bcl-X(L) and Mcl-1 and are markedly chemo- and radioresistant. Mitochondria have emerged as central players in apoptosis. Antiapoptotic members of the Bcl-2 protein family localise to the outer mitochondrial membrane and regulate mitochondrial release of apoptogenic proteins. Mitochondrial benzodiazepine receptor (mBzR) ligands have been shown to reverse Bcl-2 action and facilitate apoptosis. We evaluated the ability of the mBzR antagonist Pk11195 to overcome preapoptotic mitochondrial dysfunction in Egi-1 and Tfk-1, two human cholangiocarcinoma cell lines expressing high levels of Bcl-X(L) and Mcl-1. Cells growing in culture were used to perform in vitro experiments over 48-96 hours following treatment. The cytotoxic agents used were 5 fluorouracil 10 microM and etoposide (Vp16) 10 microM, together with ultraviolet and 0.5-1 Gy x ray irradiation with or without 75 microM Pk11195. Apoptosis and mitochondrial dysfunction were measured at single cell resolution by flow cytometry using the mitochondrial fluorochrome DiOC6(3). Severe combined immunodeficient non-obese diabetic (SCID-NOD) mice with subcutaneous xenografts using the Egi-1 and Tfk-1 cell lines were treated with etoposide with or without addition of Pk11195 over a 72 hour period during which time the xenograft growth patterns were monitored. In vitro, the effect of Pk11195 on induction of apoptosis in cholangiocarcinoma cells following stimulation by chemotherapy or radiotherapy was found to be both time and dose dependent, with Pk11195 increasing rates of apoptosis by 50-95%. Intraperitoneal administration of Pk11195 in combination with Vp16 was found to increase the growth inhibiting effects of Vp16 on xenografts during the treatment phase. PK11195 75 microM on its own had no intrinsic cytotoxic efficacy. This is the first study to demonstrate that functional antagonism of coexpressed Bcl-X(L) and Mcl-1 proteins using the mBzR antagonist Pk11195 can facilitate apoptosis in cholangiocarcinoma following chemotherapy and radiotherapy.
    Preview · Article · Nov 2002 · Gut
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    ABSTRACT: Graft-versus-host disease (GvHD) is a multistep immune process involving lymphocyte activation, proliferation and target cell killing. We have devised a novel method for the selective depletion of alloreactive cells from haematopoietic stem cell grafts which retains a pool of immunocompetent lymphocytes possessing antiviral activity with the potential to hasten immune reconstitution. The method is based upon the expression of the activation antigen CD69 on responding donor lymphocytes in a cytokine-modified mixed lymphocyte culture (mMLC) and depletion of these cells by paramagnetic bead sorting. We have previously demonstrated the in vitro efficacy of this system for the removal of alloreactive cells in both human leucocyte antigen-mismatched and -matched settings. Here, we describe a non-obese diabetic/severe combined immunodeficient murine model of aggressive GvHD in which we have tested its in vivo efficacy. Murine recipients of infusions of non-manipulated major histocompatibility complex class I and class II mismatched donor T cells suffered rapid onset of acute, and generally fatal, GvHD. This model is akin to aggressive clinical transfusion-related GvHD. Recipients of lymphocyte infusions depleted of CD69+ alloreactive donor cells ex vivo and monitored for 10 weeks post infusion demonstrated significantly improved survival (71.4% compared with 12.5% in the non-manipulated group) and the absence of clinical GvHD despite the presence of circulating donor lymphocytes.
    Full-text · Article · Aug 2002 · British Journal of Haematology
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    DA Fennell · M Corbo · A Pallaska · F E Cotter
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    ABSTRACT: Resistance to apoptosis is a major obstacle preventing effective therapy for malignancy. Mitochondria localized anti-death proteins of the Bcl-2 family play a central role in inhibiting apoptosis and therefore present valid targets for novel therapy. The peripheral benzodiazepine receptor (PBR) shares a close physical association with the permeability transition pore complex (PTPC), a pivotal regulator of cell death located at mitochondrial contact sites. In this study we investigated the cytotoxicity of the PBR ligand, PK11195, in the micromolar concentration range. PK11195 induced antioxidant inhibitable collapse of the inner mitochondrial membrane potential (DeltaPsi(m)) and mitochondrial swelling in HL60 human leukaemia cells, but not in SUDHL4 lymphoma cells (which exhibited a higher level of reduced glutathione and relative tolerance to chemotherapy or pro-oxidant induced DeltaPsi(m)dissipation). PK11195 induced the production of hydrogen peroxide that was not inhibited by Bcl-2 transfection, nor depletion of mitochondrial DNA. ROS production was however blocked by protonophore, implicating a requirement for DeltaPsi(m). Our findings suggest that PK11195-induced cytotoxicity relies upon Bcl-2 resistant generation of oxidative stress; a process only observed at concentrations several orders of magnitude higher that required to saturate its receptor.
    Preview · Article · Jun 2001 · British Journal of Cancer

  • No preview · Article · Jul 2000 · British Journal of Cancer
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    ABSTRACT: Graft versus host disease (GvHD) is a multi-step immune process involving lymphocyte activation and proliferation. We have devised a novel method of selectively depleting alloreactive cells from haematopoietic stem cell grafts while retaining a pool of immunocompetent non alloreactive lymphocytes possessing anti-viral and possibly anti-leukaemic activity and capable of hastening immune reconstitution. This method involved identifying the alloreactive cells thought to initiate GvHD by means of an activation marker, CD69 in an ex-vivo system and depleting these cells by paramagnetic bead sorting. The temporal dynamics of CD69 expression in an allogeneic setting was first initially assessed to determine the optimal marker and time for depletion. Using flow cytometric analysis and cell proliferation data, the engineered graft was shown to retain only 12% of its original alloreactivity while preserving 78% of its 3(r)(d) party reactivity. This system has been tested on mismatched and histocompatibility matched donor/patient pairs. This depletion strategy was then tested in a NOD/SCID murine GvHD model in which 5-10 x 106 unmanipulated or allodepleted T cells from a CBA (H-2(k)) mouse were injected intraperitoneally into non-lethally irradiated (250 cGy) NOD/SCID (H-2(g)7) recipients. This allodepletion strategy protected against death from lethal GvHD in a complete MHC mismatched setting (survival 71.4% in versus 12.5% in unmanipulated group). Furthermore, by using tetrameric HLA-CMV peptide complexes we have demonstrated that the non-alloreactive fraction using this strategy retained 90% of the specific anti-CMV activity, suggesting that these grafts would protect from CMV reactivation. In addition, the alloreactive cells are easily recoverable in this allodepletion strategy and ready for access as donor lymphocyte infusions in cases of frank relapse post-transplant.
    No preview · Article · Jan 2000