ASGSB 2003 Annual Meeting Abstracts


MODELED MICROGRAVITY INDUCES REACTIVE NITROGEN AND OXYGEN SPECIES (RNS AND ROS) TO DAMAGE CYTOSKELETON AND INDUCE APOPTOSIS.  J. M. Jessup, M. Schmidt, S. Lin, R. Samara, L. Laguinge. Dept of Oncology, Georgetown University Medical Center, Washington, D.C.

   The human colon carcinoma MIP-101 cell line develops more apoptosis in both actual and modeled microgravity in rotating wall vessels (RWVs) than in stationary 3-D spheroids or monolayer cultures.  We postulated that since mitotic spindle checkpoint proteins were decreased in rotated 3-D cultures that iNOS upregulation with production of RNS and ROS depolymerized microtubules caused apoptosis.  MIP-101 cells were cultured in standard 2-D monolayers, as Static 3-D spheroids on polyHEMA-coated dishes or as Rotated 3-D cultures in the RWV.  At 24 hr microtubules were clumped in RWV cultures but intact in stationary cultures by confocal microscopy.  Annexin V-FITC stained 45% of Rotated 3-D cells at 24 hr of culture compared to ≤ 10% for either stationary control.  Examination of intracellular RNS by DAF-FM fluorometry revealed that Rotated 3-D cultures developed ~200-fold more RNS than either monolayer or Static 3-D after 24 hr of culture.  Intracellular ROS measured by CM-H2DCDFA fluorometry revealed that ROS was increased in both Static and Rotated 3-D cultures after 24 hr by ~150-fold compared to monolayer cultures.  Turbulent but not laminar flow shear stress for 24 hr increased RNS in MIP-101 cell monolayers 96-fold and neither shear stress increased ROS by more than 15%.  Finally, treatment of MIP-101 cells in RWV cultures with the nonspecific NMMA and specific 1400w iNOS inhibitor decreased apoptosis by Annexin V-staining by 50% at 72 hr.  Protein nitration was higher in 24 hr extracts of RWV cultures than in stationary culture extracts and iNOS inhibition decreased this protein nitration to baseline.  Thus, we conclude that MIP-101 cells in the RWV increase iNOS activity to produce intracellular RNS and ROS that causes protein nitration and microtubule depolymerization with apoptosis.  MIP-101 cells in the RWV behave as though they experience turbulent shear stress while intracellular ROS is increased during shape change from 2-D to 3-D growth.  Inhibition of iNOS may be an important component of future countermeasures.

(Supported by NASA NAG 9-1366 and DHHSR01 CA42857-15)


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