Search

We recently established transgenic animals (WAP-SV-T/t) carrying the early coding region of Simian Virus 40 (SV40) under the transcriptional control of the whey acidic milk protein promoter (WAP), which restricts the expression of the transgene to mammary gland epithelial cells (ME-cells). SV40 T/t-antigen synthesis causes premature mammary gland involution during late pregnancy by inducing apoptosis and leads to development of mammary tumors after the first lactation period in both p53 positive (WAP-SV-T/t) and p53 negative double transgenic animals (WAP-SV-T/t.p53-/-). The high apoptotic rate persists in all of the T/t-antigen positive breast tumor cells, as well as in established ME-tissue culture cell lines. ME-cells which spontaneously switch off the expression of the WAP-SV-T/t transgene do not undergo apoptosis. However, these cells again exhibit an extensive DNA fragmentation when SV40 T/t-antigen synthesis is reintroduced, which indicates that it is the expression of T/t antigen which is the critical factor for induction of apoptosis. In addition, we isolated several ME-cell lines from different breast tumors which have spontaneously lost the T/t-antigen yet remain maximally transformed. Strikingly, these cells contain a missense mutation of the p53 gene at codon 242 (p53(242)), which substitutes alanine for glycine. This mutation increases p53 stability and it reduces the transactivating function of p53, albeit without affecting the ability of the protein to interact with the DNA. This indicates that p53 missense mutations are selected for in breast tumors initially expressing T/t-antigen. Therefore, the p53(242) mutation is sufficient to maintain the transformed state after the ME-cells have switched off the WAP-SV-T/t transgene. Interestingly, the p53 minus state per se is not sufficient to induce ME-cell transformation since homozygous null mice for the p53 gene (p53-/-) fail to develop breast cancer.

p300 and CBP are highly related nuclear proteins, which have been implicated in transcriptional responses to disparate extracellular and intracellular signals. There are at least two very good reasons for which p300 and CBP have attracted the attention of the scientific world. First, they belong to an unique class of transcription co-activators possessing histone acetyltransferase activity and therefore have the potential to reveal basic aspects pertaining to regulation of chromatin structure. Second, p300 and CBP deliver essential functions in virtually all known cellular programs, including the decision to grow, to differentiate, or to commit suicide by apoptosis. Consistent with the complexity of these processes, a multitude of intracellular factors physically interact with p300 and CBP. Thus, the task of many investigations has been the understanding of how these proteins receive signals in the cells, what induces their recruitment in a given signal transduction pathway, and what determines the final outcome of their individual activity. This review will focus on mechanistic and theoretical questions pertaining to the mode of action of p300 and CBP posed by works performed in animal and in vitro model systems.

p300 is a nuclear phosphoprotein likely to be involved in the control of cell growth. Here we show that SV40 large T antigen (Tag) forms a specific complex with p300. In various Tag-expressing cell lines, the affinity of Tag for p300 was restricted to a newly identified unphosphorylated but ubiquitinated form of the protein. Further, Tag did not associate with p300 in an SV40 Tag-producing cell line (REV2) in which the original transformed phenotype (SV52) is reverted. Biochemical studies demonstrate that both the phosphorylation and the ubiquitination profile of p300 are altered in REV2 with respect to the wild-type fully transformed SV52 parental cells, wherein Tag-p300 complexes are readily detected. In contrast to Tag, the adenovirus early expression product E1a interacts with both phosphorylated and unphosphorylated forms of p300. In addition, when REV2 cells were infected with adenovirus, E1a-p300 complexes were detected, suggesting that the p300 expressed in REV2 has lost the affinity for Tag, but not for E1a. We then compared the ability of Tag and E1a to affect the transcription levels of the cAMP-responsive promoter (CRE), which is modulated in vivo by p300, in REV2 cells. We found that Tag repressed the CRE promoter in all of the cell lines in which Tag-p300 complexes were detected, but not in REV2 cells. In contrast, E1a efficiently inhibited CRE-directed transcription in this cell line. The data thus indicate that the different specificities exhibited by Tag and E1a towards the various forms of p300 are reflected in vivo as a difference in the ability of these viral oncoproteins to modulate the expression of CRE-containing genes.