Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells.
Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.
Hiwasa
T
T
Department of Biochemistry, School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8670, Japan. hiwasa@med.m.chiba-u.ac.jp
Arase
Y
Y
Kikuno
K
K
Hasegawa
R
R
Sugaya
S
S
Kita
K
K
Saido
T
T
Yamamori
H
H
Maki
M
M
Suzuki
N
N
D017209:Apoptosis / Q000528:radiation effects*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D002461:Cell Line, Transformed; D015853:Cysteine Proteinase Inhibitors / Q000096:biosynthesis* / Q000235:genetics; D053938:DNA Fragmentation / Q000528:radiation effects; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000096:biosynthesis; D011493:Protein Kinase C / Q000096:biosynthesis; D011836:Radiation Tolerance; D014466:Ultraviolet Rays
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D007527:Isoenzymes; C033668:calpastatin; D011493:Protein Kinase C