ABSTRACT
Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein µ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed µ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of µ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for µ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of µ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing µ1, indicating that caspases were not required. Additionally, µ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.
Subject(s)
Apoptosis/physiology , Capsid Proteins/metabolism , Mammalian orthoreovirus 3/pathogenicity , Orthoreovirus, Mammalian/pathogenicity , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism , Animals , CHO Cells , Capsid Proteins/genetics , Capsid Proteins/pharmacology , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspases/genetics , Caspases/metabolism , Cell Line , Cricetinae , Cricetulus , Cytochromes c/genetics , Cytochromes c/metabolism , Cytosol/metabolism , Fibroblasts/virology , HeLa Cells , Humans , Intracellular Membranes/metabolism , Mice , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/geneticsABSTRACT
Industrial chemicals in a variety of applications are often found in highly populated areas and their presence carries risks. The threat of serious consequences from inadvertent or intentional events involving hazardous chemicals is a possibility. Extremism and/or other illicit activities pose environmental threats from chemical exposures. We present here a review of the threat of ocular injury in small-and large-scale chemical releases and discuss mechanisms of damage and repair to the eyes. The emerging field of proteomics has been described in relation to its potential role in the assessment of ocular changes following chemical exposures and management of ocular trauma.
Subject(s)
Accidents, Occupational , Burns, Chemical/etiology , Chemical Warfare Agents/toxicity , Disasters , Eye Burns/chemically induced , Eye/drug effects , Hazardous Substances/toxicity , Proteomics/methods , Acids/toxicity , Alkalies/toxicity , Ammonia/toxicity , Burns, Chemical/metabolism , Burns, Chemical/physiopathology , Burns, Chemical/therapy , Chlorine Compounds/toxicity , Eye/metabolism , Eye/physiopathology , Eye Burns/metabolism , Eye Burns/physiopathology , Eye Burns/therapy , Eye Proteins/metabolism , Humans , Isocyanates/toxicity , Mechlorethamine/toxicity , Mustard Gas/toxicity , Proteins/metabolism , Risk Assessment , Wound HealingABSTRACT
Overexpression of the cell-surface glycosphingolipid G(M3) is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G(M3) epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G(M3)-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3A8, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K(d)) ranging from 4.2 x 10(-7) to 2.1 x 10(-5) M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G(M3) panning reagent can be used to isolate a fully human scFv that is highly specific for native G(M3) on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer.
