title: Extracellular lipid-mediated signaling in tumor-cell activation and pseudopod protrusion.
abstract: We have pioneered an in vitro pseudopod-generation model wherein suspended tumor cells are stimulated to form pseudopods into glass micropipettes in response to soluble collagen type IV (CIV). Pertussis toxin and removing intracellular calcium were found previously to be inhibitory to that process. We now extend those observations to dissect the roles of transmembrane calcium influx and circulating fatty acids on pseudopod extension. Removal of fatty acids from BSA in basal media resulted in abrogation of pseudopod formation, while reconstitution of free fatty acids restored cell pseudopod protrusion. We thus hypothesized that fatty acids may provide necessary pseudopod stimulatory signals. Addition of lysophosphatidic acid (LPA) to the fatty acid-free CIV solution or in an opposite pipette without CIV permitted approximately 50% pseudopod recovery in all pipette directions in a dose-dependent fashion. Thapsigargin (TG), an agent that releases internal calcium stores and causes opening of store-operated calcium channels, restored pseudopod protrusion up to 80% in CIV with fatty acid-free albumin. [Ca(2+)](i) release was non-additive when cells were stimulated by TG and LPA, suggesting overlapping [Ca(2+)](i) stores. The combination of TG and LPA in fatty acid-free albumin fully restored the pseudopod response to CIV. Addition of EGTA to chelate stimulatory media calcium blocked the pseudopod response to CIV in the presence of fatty acids. This indicates that pseudopod protrusion requires transmembrane calcium entry. Thus, extracellular lipids and calcium mobilization are required to complement CIV in pseudopod protrusion from suspended cells.
authors.affiliation: Department of Bioengineering, Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: L
authors.initials: L
authors.lastname: Hodgson
authors.forename: E C
authors.initials: EC
authors.lastname: Kohn
authors.forename: C
authors.initials: C
authors.lastname: Dong
mesh_terms: D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure*; D003470:Culture Media; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000494:pharmacology*; D008246:Lysophospholipids / Q000494:pharmacology; D008545:Melanoma / Q000503:physiopathology / Q000648:ultrastructure*; D012710:Serum Albumin, Bovine; D015398:Signal Transduction / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D015220:Calcium Channels; D003470:Culture Media; D005230:Fatty Acids, Nonesterified; D008055:Lipids; D008246:Lysophospholipids; D012710:Serum Albumin, Bovine; D019284:Thapsigargin; D002118:Calcium