γ-Rays-generated ROS induce apoptosis via mitochondrial and cell cycle alteration in smooth muscle cells.
OBJECTIVE
γ-rays (IR) cause an increase in intracellular calcium [Ca(2+)], alters contractility and triggers apoptosis via the activation of protein kinase C in intestinal guinea pig smooth muscle cells. The present study investigated the role of the mitochondria in these processes and characterized proteins involved in IR-induced apoptosis.


METHODS
Intestinal smooth muscle cells were exposed to 10-50 Gy from a (60)Co γ-source. Reactive oxygen species (ROS) levels were measured by colourimetry with a fluorescente probe. Protein expression was analyzed by immunoblotting and immunofluorescence.


RESULTS
Apoptosis was inhibited by glutathione, possible by inhibiting the generation or scavenging ROS. Apoptosis was mediated by the mitochondria releasing cytochrome c leading to caspase 3 activation. IR increased the expression of the cyclins A, B2 and E and led to unbalanced cellular growth in an absorption dose-dependent manner. However, radiation did not induce alterations in the mitochondrial ultrastructure or in transmembrane electric potential. In contrast, IR increased the nuclear expression of cytoplasmic proteins and cyclins A and E.


CONCLUSIONS
Smooth muscle cells subjected to IR undergo mitochondrial-mediated apoptosis that involves oncoproteins activation and preserves mitochondrial structure. IR also cause alterations in the expression and localization of both pro- and anti-apoptotic proteins.
Department of Biophysics, Federal University of São Paulo , São Paulo , Brazil.
Sandra
S
Claro
Maria Etsuko Miyamoto
ME
Oshiro
Renato Arruda
RA
Mortara
Edgar Julian
EJ
Paredes-Gamero
Gustavo José Silva
GJ
Pereira
Soraya Soubhi
SS
Smaili
Alice Teixeira
AT
Ferreira
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology* / Q000528:radiation effects; D002453:Cell Cycle / Q000502:physiology / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D006168:Guinea Pigs; D008931:Mitochondria, Muscle / Q000502:physiology* / Q000528:radiation effects; D009119:Muscle Contraction / Q000502:physiology* / Q000528:radiation effects; D009124:Muscle Proteins / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000502:physiology* / Q000528:radiation effects; D011829:Radiation Dosage; D017382:Reactive Oxygen Species / Q000378:metabolism*
D018797:Cell Cycle Proteins; D009124:Muscle Proteins; D017382:Reactive Oxygen Species; D002118:Calcium
ROS; apoptosis; cyclins; mitochondria; smooth muscle cells; γ-ray