A point mutation of human p53, which was not detected as a mutation by a yeast functional assay, led to apoptosis but not p21Waf1/Cip1/Sdi1 expression in response to ionizing radiation in a human osteosarcoma cell line, Saos-2.
OBJECTIVE
The 123A point mutation of p53 showed increased radiosensitivity, whereas other mutations (143A, 175H, and 273H) were not affected. To determine the reason for increased radiosensitivity of the 123A mutation, the response of the transformant of 123A mutation to ionizing radiation (IR) was examined and compared to those of transformants with the wild type p53 or other point mutations (143A, 175H, and 273H).


METHODS
Stable transformants with a mutant or wild type p53 made by introducing cDNA into the human osteosarcoma cell line, Saos-2, which lacks an endogenous p53 were used. The transcriptional activity of mutant p53 was examined using a yeast functional assay. The transformants were examined for the accumulation of p53, the induction of p21Waf1/Cip1/Sdi1 (hereafter referred to as p21), and the other response of p53-responsive genes (MDM2, Bax, and Bcl-2) by Western blotting. Apoptosis was analyzed by detection of DNA fragmentation.


RESULTS
The 123A point mutation of p53 was detected as a wild type in the yeast functional assay. The 123A mutant accumulated p53 in response to IR. The 123A mutant did not induce p21, but normally responded to MDM2, Bax, and Bcl-2. The 123A mutant entered apoptosis earlier than the wild type p53 transformant, and induced Fas at earlier in response to IR.


CONCLUSIONS
The 123A mutant led to apoptosis, but not p21 expression in response to IR. The occurrence of apoptosis, but not induction of p21, corresponded to the radiosensitivity in the transformant. The early occurrence of apoptosis in 123A transformants may depend on the early induction of Fas.
Okaichi
K
K
Department of Radiation Biophysics, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Japan. okaichi@net.nagasaki-u.ac.jp
Wang
L H
LH
Sasaki
J
J
Saya
H
H
Tada
M
M
Okumura
Y
Y
D017209:Apoptosis / Q000235:genetics*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D005786:Gene Expression Regulation; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D012516:Osteosarcoma / Q000235:genetics* / Q000503:physiopathology; D017354:Point Mutation / Q000235:genetics / Q000502:physiology*; D011836:Radiation Tolerance / Q000235:genetics*; D011853:Radiobiology; D014407:Tumor Cells, Cultured / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D009363:Neoplasm Proteins; D016159:Tumor Suppressor Protein p53