Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model

Using an improved system for the functional identification of active antise nse fragments, we have isolated antisense fragments which inactivate the p5 3 tumour suppressor gene. These antisense fragments map in two small region s between nt 350 and 700 and nt 800 and 950 of the coding sequence, These a ntisense fragments appear to act by inhibition of p53 mRNA translation both in vivo and in vitro. Expression of these antisense fragments overcame the p53-induced growth arrest in a cell line which expresses a thermolabile mu tant of p53 and extended the in vitro lifespan of primary mouse embryonic f ibroblasts, Continued expression of the p53 antisense fragment contributed to immortalisation of primary mouse fibroblasts, Subsequent elimination of the antisense fragment in these immortalised cells fed to restoration of p5 3 expression and growth arrest, indicating that immortal cells continuously require inactivation of p53, Expression of MDM2 or SV40 large T antigen, b ut not E7 nor oncogenic ras, overcomes the arrest induced by restoration of p53 expression. Functional inactivation of both p21 and bar (by overexpres sion of Bcl2), but not either alone, allowed some bypass of p53-induced gro wth arrest, indicating that multiple transcriptional targets of p53 may med iate its antiproliferative action. The ability to conditionally inactivate and subsequently restore normal gene function may be extremely valuable for genetic analysis of genes for which loss-of-function is involved in specif ic phenotypes.