Temperature-sensitive ovarian carcinoma cell line (OvBH-1)

OvBH-1 cells from a patient with ovarian clear cell carcinoma were established and their biochemical status was analyzed. Cells grown at 37 degrees C exhibited normal cell cycle distribution, whereas the cells shifted to 31 degrees C were arrested in the G(2) / M phase of the cell cycle. Immunochemical analysis using anti-p53 antibodies (DO-1, PAb240, PAb421, and PAb1620) revealed that only the DO-1 antibody reacted with p53 with a high and similar percentage at both temperatures. PAb240 reacted with a low percentage of cells at 37 degrees C and no reaction was observed at 31 degrees C. PAb421 antibody stained a significantly lower percentage of cells at 37 degrees C than at 31 degrees C. Cells were not stained with PAb1620 antibody and were negative for antibodies against p21(WAF1) and MDM2 proteins independently of the temperature. Sequencing of all coding exons of the p53 gene demonstrated only a neutral genetic polymorphism, i.e. a G-to-A substitution (GAG to GAA) at nucleotide position 13 432. Thus, the observed temperature sensitivity of OvBH-1 cells cannot be ascribed to a p53 primary structure mutation. Based upon immunochemical analyses, we consider, however, that p53 in nuclei of OvBH-1 cells is in a highly unstable conformation. Furthermore, the N-terminal portion of the p53 protein at Ser20 has not been modified, and Lys373 and / or Ser378 of the C-terminus is acetylated and / or phosphorylated. The nuclear location signal of p53 is preserved. Induction of MDM2 protein is uncoupled from the cell regulatory machinery and the induction of p21(WAF1) by p53 is impaired in OvBH-1 cells.