- and -Protein Kinase C Activity during Smooth Muscle Cell Apoptosis in Response to -Radiation

Sandra Claro, Celia Akemi Kanashiro, Maria Etsuko Miyamoto Oshiro, Alice Teixeira Ferreira, and Raouf Ali Khalil

Department of Biophysics, Federal University of São Paulo, São Paulo, Brazil (S.C., M.E.M.O., A.T.F.); Department of Physiology, University of Medical Sciences of Alagoas, Alagoas, Brazil (C.A.K.); and Division of Vascular Surgery, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts (R.A.K.)

Abstract: The use of -radiation in treatment of pelvic cancer is associated with injury of healthy surrounding tissues and disorders of intestinal motility; however, the cellular mechanisms involved are unclear. We tested the hypothesis that exposure of visceral smooth muscle cells (SMCs) to -radiation induces apoptosis via activation of specific protein kinase C (PKC) isoforms. Cultured SMCs and slices from guinea pig ileum smooth muscle longitudinal layer (GPISMLL) were exposed to 10 to 50 Gy. Flow cytometry in -radiated SMCs showed increased percentage of cells in the sub-G₀/G₁ phase, a hallmark of apoptosis. -Radiation-induced reduction in cell survival was partially but significantly alleviated with the PKC inhibitors. Sections of -irradiated GPISMLL showed DNA fragmentation and apoptotic bodies analyzed by the terminal deoxynucleotidyl transferase dUTP nick-end labeling method, whereas the plasma and nuclear membranes were preserved. Confocal microscopy in -radiated SMCs labeled with annexin V-fluorescein showed an increase in apoptotic cells and phosphatidylserine externalization. Contraction of GPISMLL strips in response to KCl and acetylcholine was reduced in tissues exposed to 30 and 50 Gy. -Radiation of GPISMLL caused an increase in PKC activity in the particulate fraction, a decrease in the cytosolic fraction, and increased particulate/cytosolic PKC activity ratio. Western blot analysis revealed significant amounts of - and -PKC in the cytosolic fraction of control GPISMLL. -Radiation caused an increase in the amount of - and -PKC in the particulate fraction and a decrease in the cytosolic fraction. Data suggest that -radiation induces apoptosis, growth arrest, and contractile dysfunction in visceral SMCs of GPISMLL via activation and translocation of - and -PKC isoforms.

Address correspondence to: Sandra Claro, Departamento de Biofísica, Universidade Federal de São Paulo, Escola Paulisa de Medicina, Rua Botucatu 862, Vila Clementino, CEP 04023-062 São Paulo, Brazil. E-mail: sandra{at}biofis.epm.br