Clonal variation of p53 expression and proliferative phenotype in A253 squamous carcinoma cells.

Loss of normal p53 tumor-suppressor gene function is characteristic of the majority of squamous carcinomas. During the course of gene transfer studies in the human squamous carcinoma cell line, A253, which does not express p53 mRNA or protein, we incidentally observed increased levels of p53 expression in up to 20% of clonal cell lines derived from parental A253 cells. p 53-expressing A253 cells (A253-p53) were also isolated by dilutional cloning. Nuclear p53 protein was identified by immunohistochemistry in A253-p53 cells in a wild-type pattern, and p53 mRNA (2.5 kb) was demonstrated by northern blot. Mutational analysis of the p53 gene in A253-p53 cells revealed no evidence for mutations in exons 5-9. A253-p53 cells could be distinguished from native A253 cells by prolonged doubling times (2-5 fold) and by a marked reduction of [3H]-thymidine uptake. Whereas A253 cells were unresponsive to the growth-inhibitory effects of TGF-beta, EGF-stimulated A253-p53 cells responded to TGF-beta with markedly reduced DNA synthetic rates. A253-p53 cells cocultured with A253 demonstrated enhanced cell growth and DNA synthesis rates compared to control A253-p53 cells. Finally, A253-p53 cells show reduced expression of c-fos, fibronectin, thrombospondin and parathyroid hormone-related protein (PTHrP) mRNAs. PTHrP measured by RIA in conditioned medium was approximately 300 pM for A253 but undetectable for A253-p53. We conclude that the A253 cell line contains a subpopulation of cells which express high levels of "wild-type-like" p53 protein. This results in dramatic changes in gene expression and a slower-growing phenotype in vitro.