Alteration of p53 protein in C3H/10T1/2 cells morphologically transformed by γ-rays in stationary phase

The panel of 70 transformed clones was isolated after exposure of C3H/10T1/2 cells in stationary phase to low-moderate doses (1-4 Gy) of (137) Cs gamma-rays. Two widely different dose rates were used: high (HDR, 0.66 Gy/min) and low (LDR, 4.8 x 10(-4) Gy/min). Immunohistochemical analyses were performed by cellular staining with three types of monoclonal anti-p53 antibodies, Ab-l (PAb421), Ab-3 (PAb240) and Ab-4 (PAb246) in order to identify wild-type and altered conformation of the p53 protein in cell nuclei. The gamma-ray exposure led to induction of altered p.53 protein in the majority of morphologically transformed clones. For LDR exposure the percentage of clones with changed p53 protein was 79 (11/14), 71 (12/17) and 100 (6/6) for the exposure doses of 2, 3 and 3.6 Gy, respectively. For HDR exposure the percentage of such clones was 60 (3/5). 40 (4/10) and 87 (13/15) for 1, 2 and 3 Gy, respectively. Moreover, in some transformed clones. especially in those induced by higher gamma-ray doses, p53 protein in cell nuclei was not found. It was demonstrated by lack of the staining with Ab-l antibody which can detect both mutant and wild-type of p53 protein. An altered conformation of p53 protein was detected, using Ab-3 antibody which does not react with its native conformation, in 27% (18/67) of all radiation-induced clones. A native conformation of p53 protein was recognized by Ab-4 antibody in 33% (10/30) of clones induced by HDR, and in 22% (8/37) of clones induced by LDR exposure. Our study shows that alterations of p53 protein in cell nuclei is a frequent feature of morphological transformants induced by ionizing radiation in C3H/10T1/2 cells, and that these alterations occur independently of dose rate. (C) 1998 Elsevier Science B.V.