p53-mediated transcription induces resistance of DNA to UV inactivation.

A possible role of p53-dependent transcription in the induction of DNA repair was explored by transfecting a UV-irradiated chloramphenicol acetyl transferase (CAT) reporter plasmid (pRGC.FOS.CAT), containing a minimal FOS promoter driven by a consensus p53 binding site, into a p53 negative-mouse cell line [(10)1]. When a p53-expressing plasmid (pSV.p53) was cotransfected into these cells, CAT expression levels persisted even after prolonged UV irradiation. In comparison, CAT expression from pSV2.CAT, which lacks a p53-responsive element in its SV40 promoter, dropped off much more precipitously after UV irradiation in the absence or presence of WT p53 expression. A similar sharp drop was observed with three other constructs when the reporter gene was under the control of the ras, beta-actin or fos promoter. Mouse cells (A1-5) that constitutively express a temperature-sensitive mutant (135 AV) of mouse p53 also generated, at 32 degrees C, higher levels of enzyme expressed from UV-irradiated pRGC.FOS.CAT than from UV-irradiated pSV2.CAT. The frequency of cyclobutane pyrimidine dimers in UV-irradiated pRGC.FOS.CAT was determined with T4 endo V, and the probability of having an undamaged CAT coding strand was calculated by the Poisson distribution for various times of UV-irradiation. The observed relative CAT expression levels from irradiated pSV2.CAT and pRGC.FOS.CAT in the absence of p53 were consistent with those numbers. These results show that WT p53-mediated transcription directs a resistance of the transcribed DNA to UV inactivation and reactivates the reporter gene. Furthermore, some single point substitution mutants of p53 that maintain a near normal ability to activate transcription had lost their ability to extend CAT gene expression after UV irradiation. Conversely, other mutants with reduced transcriptional activity retained this ability. This indicates that although resistance to UV inactivation is transcriptionally-dependent, these two activities are genetically distinct. These data, taken together, suggest that the transcription of UV-damaged DNA by a p53-dependent process promotes its repair.

p53 binds to a novel recognition sequence in the proximal promoter of the rat muscle creatine kinase gene and activates its transcription.

The rat muscle creatine kinase (CKM) gene promoter is unusual since it is one of the few cellular promoters containing a p53 response element which is located proximally (bp -168 to -57) to the transcription start site. We have previously shown that p53wt transactivates transcription in vivo of rat CKM, in CV-1 monkey kidney cells, through this 112 bp promoter-proximal fragment which contains at least five degenerate p53-binding elements. In this report, we employed the gel-shift assay and demonstrated that recombinant, immunoaffinity-purified mouse p53wt binds to this 112 bp CKM sequence and activates the in vitro transcription of the proximal CKM promoter by nuclear extracts from CV-1 cells. Also, a competitor plasmid containing this 112 bp CKM fragment interferred with the in vivo transactivation of CKM by p53. This CKM fragment, when cloned upstream of the rat brain creatine kinase (CKB) promoter, mediated the p53 transactivation of CKB. Analyses of p53wt and a series of missense mutants (altered in conserved region II of p53) showed that binding of p53 to the CKM promoter was required but was not sufficient for transactivation. The results are discussed in relation to the possible role of p53wt in the expression of CKM in cell types which may not express the myogenic transcription factors.

Mouse p53 represses the rat brain creatine kinase gene but activates the rat muscle creatine kinase gene.

The creatine kinases (CK) regenerate ATP for cellular reactions with a high energy expenditure. While muscle CK (CKM) is expressed almost exclusively in adult skeletal and cardiac muscle, brain CK (CKB) expression is more widespread and is highest in brain glial cells. CKB expression is also high in human lung tumor cells, many of which contain mutations in p53 alleles. We have recently detected high levels of CKB mRNA in HeLa cells and, in this study, have tested whether this may be due to the extremely low amounts of p53 protein present in HeLa cells. Transient transfection experiments showed that wild-type mouse p53 severely repressed the rat CKB promoter in HeLa but not CV-1 monkey kidney cells, suggesting that, in HeLa but not CV-1 cells, p53 either associates with a required corepressor or undergoes a posttranslational modification necessary for CKB repression. Conversely, mouse wild-type p53 strongly activated the rat CKM promoter in CV-1 cells but not in HeLa cells, suggesting that, in CV-1 cells, p53 may associate with a required coactivator or is modified in a manner necessary for CKM activation. The DNA sequences required for p53-mediated modulations were found to be within bp -195 to +5 of the CKB promoter and within bp -168 to -97 of the CKM promoter. Moreover, a 112-bp fragment from the proximal rat CKM promoter (bp -168 to -57), which contained five degenerate p53-binding elements, was capable of conferring p53-mediated activation on a heterologous promoter in CV-1 cells. Also, this novel p53 sequence, when situated in the native 168-bp rat CKM promoter, conferred p53-mediated activation equal to or greater than that of the originally characterized far-upstream (bp -3160) mouse CKM p53 element. Therefore, CKB and CKM may be among the few cellular genes which could be targets of p53 in vivo. In addition, we analyzed a series of missense mutants with alterations in conserved region II of p53. Mutations affected p53 transrepression and transactivation activities differently, indicating that these activities in p53 are separable. The ability of p53 mutants to transactivate correlated well with their ability to inhibit transformation of rat embryonic fibroblasts by adenovirus E1a and activated Ras.

p53 mutants with changes in conserved region II: three classes with differing antibody reactivity, SV40 T antigen binding and ability to inhibit transformation of rat cells.

The wild-type p53 gene is able to inhibit the ability of a number of oncogenes to induce transformation, but this inhibition is frequently lost when the p53 gene undergoes mutation. In this study, we generated mutated mouse p53 proteins containing conservative amino acid substitutions at residues 127 to 138 and at 140. The mutant proteins fell into three distinct classes. Class I proteins reacted well with PAb246 (246+), poorly with PAb240 (240-), bound to SV40 T antigen (T+), and inhibited the transformation of rat embryo fibroblasts by adenovirus E1a and activated ras. Class II proteins were 246-, 240+, T-, and failed to inhibit transformation. Class III proteins were 246+, 240-, but T-, and varied in their ability to inhibit transformation. The existence and properties of the Class III proteins suggest that the residues mutated in this class are important for binding to T antigen. They also lead us to conclude that the maintenance of an apparent wild-type structure is not sufficient for p53 to inhibit transformation and that the loss of T antigen binding does not necessarily result in loss of transformation inhibition. We propose the existence of a third activity important for the ability of p53 to inhibit transformation.

Characterization of the in vitro interaction between SV40 T antigen and p53: mapping the p53 binding site.

An efficient in vitro system for generating soluble complexes between simian virus 40 T antigen and the cellular protein p53 was developed. A p53 cDNA was inserted 3' to the SP6 promoter in pGEM-1 (Promega-Biotec) and transcribed by SP6 polymerase. In vitro translation of the cRNA generated p53 which was immunoprecipitable with all five monoclonal antibodies tested (PAb122, PAb421, PAb242, PAb246, and PAb248). The p53 sedimented at about 8-10 S in sucrose gradients, possibly corresponding to a tetramer. T-antigen-p53 complexes were produced by the addition of immunoaffinity-purified T antigen to p53-containing translation lysates. Equivalent amounts of p53 were immunoprecipitated with the anti-T-antigen antibodies PAb416, PAb419, and PAb101, suggesting that in vitro made p53 complexed mostly to a population of T-antigen molecules that had matured at least 15 min in the cell. The complexes sedimented at 18-20 S in sucrose gradients. In order to map the p53 binding site on T antigen, p53 was complexed in vitro to labeled proteolytic fragments of T antigen. A 46K fragment, spanning residues 131-517, was immunoprecipitated with the anti-p53 monoclonal PAb122 and therefore is likely to contain the p53 binding site. This region contains T-antigen sequences necessary for the efficient transformation of nonpermissive cells and for the induction of cellular rRNA synthesis. It also contains the binding sites for DNA polymerase alpha and ATP. We suggest a possible role for T-p53 complexes in T-antigen-associated functions.

Intracellular location and kinetics of complex formation between simian virus 40 T antigen and cellular protein p53.

The intracellular location and kinetics at which the simian virus 40 T antigen and the cellular protein p53 associate with one another were determined for simian virus 40-transformed mouse (215) and rat (14B) cells. Cells were labeled under pulse-chase conditions and fractionated into nuclear and cytoplasmic components, and the proteins were immunoprecipitated with monoclonal antibodies (pAb 416, 101, and 122). We found that newly made T antigen and p53 migrated to the nucleus of these cells independently; that is, in uncomplexed form. Newly made p53 was transported to the nucleus more rapidly than T antigen in both cell lines and formed a complex with a mature form of T antigen recognizable by pAb 101. This association was very rapid in both cell lines (t 1/2, 5 to 15 min). In contrast, the time course of complex formation between newly made T antigen and the p53 in the nucleus varied with the ratio of T antigen to p53 of the cell line studied. In 215 cells, where the ratio was 3.6, the kinetics were quite slow (t 1/2, 30 min), whereas in 14B cells, where the ratio was 1.7, they were quite rapid (t 1/2, 5 min). We suggest that a competition between newly made and uncomplexed T antigen for the p53 in the nucleus is the major determinant of the rate of complex formation for newly made T antigen. Our studies indicate that this macromolecular interaction is extremely dynamic.