SINGLE-CELL MONITORING OF GROWTH ARREST AND MORPHOLOGICAL-CHANGES INDUCED BY TRANSFER OF WILD-TYPE P53 ALLELES TO GLIOBLASTOMA CELLS

Mutation of the p53 tumor suppressor gene is one of the earliest ident ified genetic lesions during malignant progression of human astrocytom as. To assess the functional significance of these mutations, wild-typ e (WT) p53 genes were introduced into glioblastoma cell lines having m utant, WT, or null endogenous p53 alleles. Populations of cells with m utant or null endogenous p53 alleles and exogenous WT p53 were spontan eously selected in culture for cells expressing only mutant p53 or no p53, which then displayed a growth and tumorigenic phenotype identical to the parental cells. To determine the phenotypic consequences of WT p53 expression before the occurrence of mutations, we developed a sin gle cell assay to monitor WT p53-dependent transcription activity. Tra nsfer and expression of exogenous WT p53 genes to cells with endogenou s mutant or deleted, but not WT, p53 alleles caused growth arrest and morphological changes, including increased cell size and acquisition o f multiple nuclei. This supports the hypothesis that genetic lesions o f the p53 gene play an important role in the genesis of astrocytomas. Furthermore, the high sensitivity of the episomal single cell reporter strategy developed here has potential clinical applications in the ra pid screening of patients for germ-line mutations of the p53 gene or a ny other gene with known targets for transcriptional transactivation.