It has been shown previously that mutant p53 can act as an immortalizing gene when cotransfected into primary rat embryo fibroblasts along with a selectable marker. To determine whether a mutation at the p53 locus is a common event in the pathways leading to spontaneous cellular immortalization, 11 clonally derived BALB/c murine embryo fibroblast lines were established by passage on a 3T3 schedule and examined for p53 alterations. By the following criteria, all 11 independently established lines contain at least one mutant allele of p53. Seven of these lines have a PAb240-reactive p53 species and exhibit an extended p53 half-life as determined by pulse-chase analysis. The p53 protein species in a subset of these lines is also capable of complex formation with the constitutive heat shock protein hsc70, p53 cytoplasmic DNAs (cDNAs) from several of these lines have been cloned by reverse transcription of cytoplasmic RNA followed by PCR amplification, and the mutations have been mapped by DNA sequence analysis. Point mutation in conserved domains of p53 appears to be a common alteration in these lines, although one established line carries a 24-bp in-frame deletion of p53. The remaining four cell lines do not express detectable p53 protein. For each line there is a different molecular event underlying the lack of p53 expression: (1) deletion of at least the first 6 exons of both p53 alleles; (2) expression of a single p53 mRNA encoding a stop codon at amino acid position 173; (3) no detectable p53 mRNA; and (4) greatly diminished expression of p53 mRNA. These findings indicate that p53 alteration commonly occurs in spontaneously immortalized BALB/c mouse embryo fibroblasts passaged on a 3T3 schedule and, therefore, may be an important event for the immortalization process.

Irradiation of mammalian cells with UV light results in a dose-dependent accumulation of the p53 tumorsuppressor gene product that is evident within 2 hr. UV treatment causes a dramatic increase in p53-specific transcriptional transactivation activity and an increase in expression of the p53-responsive gene mdm-2. UV-stimulated mdm-2 expression is not directy correlated with the level of p53 protein in a cell because mdm-2 induction is delayed at high UV doses even though p53 levels rise almost immediately. Cells lacking p53 protein do not respond to UV by increasing their expression of mdm-2. The delayed induction of mdm-2 at high UV doses suggests that, in addition to p53 protein levels, other factors contribute to the regulation of mdm-2 expression following UV treatment. The time of induction of mdm-2 in cells treated with UV light correlates with recovery of normal rates of DNA synthesis, presumably after DNA repair. These data indicate a possible role for mdm-2 in cell cycle progression.Mammalian cells respond to irradiation with UV light by transiently decreasing both RNA and DNA synthesis and by inducing expression of several genes whose products are thought to have protective effects against DNA damage (1). The regulation of these UV response genes appears to be mediated by several transcription factors which function after UV exposure (2). The p53 tumor-suppressor gene product is a transcription factor (3,4) that also appears to be involved in the response to UV light. The p53 protein levels increase due to the stabilization ofthis protein in both murine (5, 6) and human (7,8) cells treated with UV light. Although the role of p53 in the response to UV light has not been fully characterized, this tumor-suppressor protein has been shown to act as a cell cycle checkpoint in the response to y irradiation (9,10). fy irradiation induces both a G1 and a G2 phase-specific cell cycle block, and expression of wild-type p53 is necessary for the G1 but not the G2 block. The specific DNA-binding activity of p53 is increased after y irradiation and a DNA damage-inducible, growth arrest-specific gene, GADD45, has been shown to contain a p53 response element (10).Characterization of the role of p53 in the response to 'y irradiation led to the hypothesis that p53 acts as a cell cycle checkpoint, causing a delay in the G1 phase of the cycle during which damage is thought to be repaired (9, 10). It seemed likely that p53 might also act as a checkpoint in the response of cells to UV exposure. In addition, Zhan et al. (8) have recently shown that p53 transcriptional transactivation activity is increased in human cells exposed to UV light. Possible targets for p53 transcriptional transactivation activity in the UV response are the GADD45 gene, which was isolated as a UV response gene (11), and the mdm-2 gene. p53 and mdm-2 appear to form a feedback control loop: while p53The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" ...

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