p53 protein accumulation in addition to the transactivation activity is required for p53-dependent cell cycle arrest after treatment of cells with camptothecin.

In this study we characterize the connections between p53-dependent G1 cell cycle arrest, transcriptional activation of the protein and the increase of its intracellular steady-state concentration. Several cell lines expressing wild-type p53 protein were treated with increasing concentrations of DNA-damaging drug camptothecin. Lower doses of the drug caused transcriptional activation of p53, but no accumulation of the protein was detected. Only after a certain threshold dose of camptothecin does the amount of the protein rapidly increase and reach its plateau levels. The threshold dose was different for different cell lines, but the general non-linear profile was similar. Increase of p53 level was accompanied by additional transcriptional activation of some p53 target genes (i.e. waf1), but not the others (mdm2). We demonstrate here that transcriptional activation of p53 after the treatment of camptothecin is not sufficient to cause p53-dependent G1 cell cycle arrest. The latter is observable only after the increase of steady-state level of p53. Low drug concentrations, although accompanied by transcriptional activation of p53, do not cause either p53 protein accumulation nor cell cycle arrest at G1. We propose a model for p53 acting as a part of cellular sensor system detecting the severity of DNA damage.

Monitoring and purification of proteins using bovine papillomavirus E2 epitope tags.

We describe here the use of two newly mapped bovine papillomavirus type 1 (BPV-1) E2 protein epitopes as tags. We constructed several vector plasmids for overexpression as well as for moderate expression of single- or double-tagged proteins in either Escherichia coli or eukaryotic cells. The new tags were fused to several proteins, and the activity of the tagged proteins was tested in different assays. The tags were shown not to interfere with the function of these proteins in vivo and in vitro. Interaction of the monoclonal antibodies 3F12 and 1E2 with their respective epitopes was specific and had high affinity in a variety of conditions. We have demonstrated that the 3F12 antibody-epitope interaction tolerates high salt concentrations up to 2 M. This permits immunoprecipitation and immunopurification of the tagged proteins in high-salt buffers and reduction of the nonspecific binding of the contaminating proteins. We also provide a protocol for DNA binding and DNase I footprinting assays using the tagged, resin-bound DNA-binding proteins. The BPV-1 E2-derived tags can be recommended as useful tools for detection and purification of proteins.

Tumour associated mutants of p53 can inhibit transcriptional activity of p53 without heterooligomerization.

Tumour suppressor protein p53 is the most frequent target of mutations occurring in different types of human cancers. Most of these are point mutations clustered in certain 'hot spots'. Because p53 is a tetramer in solution, it can form heterooligomers when both wild-type and mutant forms of p53 are expressed in the same cell. Inactivation of wt p53 by heterooligomerization has been proposed as a mechanism for dominant negative effect of mutant protein. In this paper we show that other mechanisms can also be involved in the inhibition of transcriptional activity of wt p53 by mutant proteins. In addition to suppressing the wt p53 activity, mutant proteins are also able to suppress the activity of p53 protein unable to oligomerize. Either N- or C-terminus of mutant p53 are needed for this activity. The suppression of transcriptional activity described is restricted to p53-dependent promoters and no effect is seen with the promoter not containing p53 binding site. Point mutants also inhibit the growth suppressing activity of monomeric p53. Our data allow to propose the existence of a cofactor specifically needed for p53-dependent transcription. Depletion of this cofactor could be an alternative mechanism of inactivation of wt p53 by its point mutants.

p53 protein is a suppressor of papillomavirus DNA amplificational replication.

p53 protein was able to block human and bovine papillomavirus DNA amplificational replication while not interfering with Epstein-Barr virus oriP once-per-cell cycle replication. Oligomerization, intact DNA-binding, replication protein A-binding, and proline-rich domains of the p53 protein were essential for efficient inhibition, while the N-terminal transcriptional activation and C-terminal regulatory domains were dispensable for the suppressor activity of the p53 protein. The inhibition of replication was caused neither by the downregulation of expression of the E1 and E2 proteins nor by cell cycle block or apoptosis. Our data suggest that the intrinsic activity of p53 to suppress amplificational replication of the papillomavirus origin may have an important role in the virus life cycle and in virus-cell interactions.

Oligomerization of p53 is necessary to inhibit its transcriptional transactivation property at high protein concentration.

We have previously shown that transactivation by tumor suppressor protein p53 can be inhibited in vivo at elevated protein concentrations. In this study we characterize the structural requirements of this function. We show that oligomerization domain of p53 is involved in loss of transactivation at high protein concentrations: mutants not able to oligomerize are neither able to suppress transactivation, although these transactivating properties can be untouched.

Protein p53 modulates transcription from a promoter containing its binding site in a concentration-dependent manner.

Tumor suppressor protein p53 binds to DNA in a sequence-specific manner and activates transcription from promoters near its binding site. It is also known to repress promoters lacking the p53-binding site. In this study, we demonstrate that p53 can act as a transcriptional activator or repressor in vivo using the same reporter with the DNA-binding site CON and these effects depend on the amount of p53 expressed. Both in Saos2 and Cos7 cells, lower concentrations of p53 lead to activation and higher concentrations lead to repression of the model promoter containing the consensus p53-binding site CON. The N-terminal part of p53 is necessary for the transcriptional activation. It is not needed, however, for the repression of the same promoter, indicating that different domains of p53 are involved in activation and repression.