Several publications and patent documents are referenced in this application to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
The p53 tumor suppressor protein is a nuclear phosphoprotein that functions in cell-cycle arrest, programmed cell death (apoptosis), inhibition of tumor growth, and preservation of genetic stability. The p53 gene is inactivated by mutation in over 60% of human tumor samples analyzed. This gene continues to hold distinction as the most frequently mutated gene in human cancer. The strong selection for mutation of p53 in cancer cells can be explained by the normal functions of the protein. p53 protein is normally expressed at very low levels in cells and is latent in activity. The protein is post translationally stabilized and activated as a transcription factor by a number of stimuli that are detrimental to the normal cell, notably genotoxic stress, hypoxia, and the mutational activation of oncogenes like c-myc and Ha-ras. The result of p53 induction in a cell is either growth arrest in the G1 phase of the cell cycle, or programmed cell death (apoptosis). The choice of fate depends upon a number of factors, including cell type, level of p53 induced, and environmental factors, such as the presence of cytokines or increased expression of bc12 family members. It is currently accepted that it is the ability of p53 to induce cell death, not G1 growth arrest, that underlies the powerful selection for mutation of the p53 gene in tumorigenesis.
The best-understood activity of p53 remains its ability to function as a sequence-specific transcriptional activator. Following a stimulus such as DNA damage, p53 protein is post-translationally stabilized and activated for DNA binding. This protein then binds in a sequence-specific manner to promoters or enhancers containing the consensus element 5′ Pu Pu Pu C A/T T/A G Py Py Py 3′. (SEQ ID NO: 12) p53 binding to these elements is believed to recruit the core transcriptional apparatus and thereby enhance the transcription of such (p53-response) genes.
Despite the ability of p53 to transactivate pro-apoptotic genes like bax and IGF-BP3, in the past several years compelling indications have accumulated that p53 has a pro-apoptotic activity that is independent of its transactivation function. Recently it has been shown that physiological induction of wt p53 can lead to apoptosis in the absence of trans-activation of p53 response genes.
In addition to its well-characterized function as a transcriptional activator, p53 has a poorly-understood activity as a transcriptional repressor. Previous studies investigating this function of p53 were performed using transient over-expression assays where non-physiological levels of p53 and candidate promoters are introduced into cells. In this type of setting, a large number of promoters are repressed by p53. In no case, however, have these endogenous genes been found to be repressed by p53. Therefore, in these types of assays, transcriptional repression by p53 is non-physiological, and impossible to distinguish from transcriptional “squelching”. During transcriptional “squelching”, a powerful transactivator like p53 represses transcription of other promoters non-specifically by more efficiently recruiting basal transcription factors, such as TBP-associated factors (TAFs).
To determine whether p53 can function as a sequence-specific transcriptional repressor, differences in gene expression in cells containing an inducible p53 protein have been analyzed (Murphy et al., Genes & Devel. 10:2971–2980, 1996). Several genes were identified that exhibit decreased expression following wt p53 induction in cell lines containing a well-characterized temperature-sensitive mutant of p53. One of these genes encodes the microtubule-associated protein Map4. It was found that p53 induction leads to repression of Map4 at the level of transcriptional initiation (Murphy et al., Genes & Devel. 10:2971–2980, 1996). This transcriptional repression is manifested at both the RNA and protein levels, and approaches a 90% reduction at the RNA level following p53 induction for 24 hours. Further, physiological induction of p53, by DNA damaging agents or ionizing radiation, leads to significant repression of Map4 in both normal and tumor cells containing wt p53. In cells with mutant p53, Map4 levels are unaffected by such treatments (Murphy et al., Genes & Devel. 10:2971–2980, 1996). This and other data indicate that independent of it's cell cycle effects, wt p53 represses the expression of Map4. However, the precise mechanism by which p53 represses Map4 expression remains to be elucidated.
The Map4 gene is transcriptionally repressed following p53 induction in cells that undergo either G1 arrest or apoptosis in response to p53. Significantly, it has been found that removing Map4 from transcriptional repression by p53 significantly interferes with p53 dependent apoptosis (Murphy et al., Genes & Devel. 10:2971–2980, 1996), thereby directly placing Map4 in a pathway influential in p53-mediated programmed cell death.
Following the cloning of Map4 as the first endogenous gene repressed by physiological induction of wt p53, several other genes have been cloned that exhibit decreased expression following p53 induction. These genes include those encoding DNA topoisomerase IIα, wee1, DP-1, presenilin 1, and others. It is clear from these studies that there will exist other candidate p53-repressed genes. Significantly, repression of at least one of these genes, presenilin 1, has been demonstrated to contribute to the progression of apoptosis. Further, the combined data from several sources suggest a perfect correlation between the ability of p53 to induce apoptosis, and its ability to repress the expression of genes like Map4.
It is clear from the foregoing that the expression repression function of p53 is a significant part of p53's central role in controlling cell growth and death. In order to utilize this function in a practical way, more information is needed relative to the physical interactions among p53 and its targets for repression in cells.