James J. Manfredi

The Mdm2–p53 relationship evolves: Mdm2 swings both ways as an oncogene and a tumor suppressor

Mdm2 has been well characterized as a negative regulator of the tumor suppressor p53. Recent studies have shown that Mdm2 is activated in response to a variety of oncogenic pathways independent of p53. Although its role as an oncogene via suppression of p53 function remains clear, growing evidence argues for p53-independent effects, as well as the remarkable possibility that Mdm2 has tumor suppressor functions in the appropriate context. Hence, Mdm2 is proving to be a key player in human cancer in its own right, and thus an important target for therapeutic intervention.

The p53 Tumor Suppressor Participates in Multiple Cell Cycle Checkpoints

The process of cell division is highly ordered and regulated. Checkpoints exist to delay progression into the next cell cycle phase only when the previous step is fully completed. The ultimate goal is to guarantee that the two daughter cells inherit a complete and faithful copy of the genome. Checkpoints can become activated due to DNA damage, exogenous stress signals, defects during the replication of DNA, or failure of chromosomes to attach to the mitotic spindle. Abrogation of cell cycle checkpoints can result in death for a unicellular organism or uncontrolled proliferation and tumorigenesis in metazoans (Nyberg et al., 2002 ). The tumor suppressor p53 plays a critical role in each of these cell cycle checkpoints and is reviewed here. J. Cell. Physiol. 209: 13–20, 2006.

A conserved intronic response element mediates direct p53-dependent transcriptional activation of both the human and murine bax genes.

Both the human and the mouse bax promoters contain p53 binding sites which are sufficient to confer p53-dependent transcriptional activation in a heterologous setting. Nevertheless in the context of the bax promoter, these sites do not mediate a p53-dependent response, suggesting that bax may not be a direct transcriptional target of p53. Here, data are presented identifying a conserved p53 response element in the first intron of both the human and the murine bax genes. This element both in isolation and in the context of the first intron conferred p53-dependent transcriptional activation upon a minimal promoter. Electrophoretic mobility shift assays demonstrated that this sequence also is capable of mediating sequence specific binding to p53. p53 effectively activated transcription through both human and murine bax gene reporter constructs, whereas deletion of the intronic response element abrogated the p53-responsiveness of both reporters. Interestingly, tumor-derived mutants of p53 which are defective in inducing an apoptotic response retain the ability to activate transcription via the bax intronic p53 site. Since these mutants are transcriptionally inactive on the p53 site in the bax promoter, the ability of these mutants to up-regulating endogenous bax mRNA levels supports a role for the intronic element in p53-dependent up-regulation of bax expression. Taken together, these results show the requirement for a novel intronic element in the p53-dependent transcriptional activation of bax, and demonstrate that bax is indeed a direct and evolutionarily conserved transcriptional target of p53

Another fork in the road--life or death decisions by the tumour suppressor p53.

In response to cellular stress signals, the tumour suppressor p53 accumulates and triggers a host of antineoplastic responses. For instance, DNA damage activates two main p53‐dependent responses: cell cycle arrest and attendant DNA repair or apoptosis (cell death). It is broadly accepted that, in response to DNA damage, the function of p53 as a sequence‐specific transcription factor is crucial for tumour suppression. The molecular determinants, however, that favour the initiation of either a p53‐dependent cell cycle arrest (life) or apoptotic (death) transcriptional programme remain elusive. Gaining a clear understanding of the mechanisms controlling cell fate determination by p53 could lead to the identification of molecular targets for therapy, which could selectively sensitize cancer cells to apoptosis. This review summarizes the literature addressing this important question in the field. Special emphasis is given to the role of the p53 response element, post‐translational modifications and protein–protein interactions on cell fate decisions made by p53 in response to DNA damage.

Evidence against a Role for SV40 in Human Mesothelioma

SV40 has been implicated in the etiology of 40% to 60% of human mesotheliomas. These studies could have important medical implications concerning possible sources of human infection and potential therapies if human tumors are induced by this agent. We did PCR-based analysis to detect SV40 large T antigen DNA in human mesotheliomas. None of 69 tumors in which a single copy gene was readily amplified contained detectable SV40 large T antigen sequences. Under these conditions, it was possible to detect one copy of integrated SV40 DNA per cell in a mixture containing a 5,000-fold excess of normal cells using formalin-fixed preparations. Kidney, a known reservoir of SV40 in monkeys, from some of these individuals were also negative for SV40 large T antigen sequences. A subset of mesotheliomas was analyzed for SV40 large T antigen expression by immunostaining with a highly specific SV40 antibody. These tumors as well as several human mesothelioma cell lines previously reported to contain SV40 large T antigen were negative for detection of the virally encoded oncoprotein. Moreover, mesothelioma cell lines with wild-type p53 showed normal p53 function in response to genotoxic stress, findings inconsistent with p53 inactivation by the putative presence of SV40 large T antigen. Taken together, these findings strongly argue against a role of SV40 by any known transformation mechanism in the etiology of the majority of human malignant mesotheliomas.

Constitutive Expression of the Cyclin-dependent Kinase Inhibitor p21 Is Transcriptionally Regulated by the Tumor Suppressor Protein p53

The tumor suppressor protein p53 has been implicated in the response of cells to DNA damage. Studies to date have demonstrated a role for p53 in the transcriptional activation of target genes in the cellular response to DNA damage that results in either growth arrest or apoptosis. In contrast, here is demonstrated a role for p53 in regulating the basal level of expression of the cyclin-dependent kinase inhibitor p21 in the absence of treatment with DNA-damaging agents. Wild-type p53-expressing MCF10F cells had detectable levels of p21 mRNA and protein, whereas the p53-negative Saos-2 cells did not. Saos-2 cells were infected with recombinant retrovirus to establish a proliferating pool of cells with a comparable constitutive level of expression of wild-type p53 protein to that seen in untreated MCF10F cells. Restoration of wild-type but not mutant p53 expression recovered a basal level of expression of p21 in these cells. Constitutive expression of luciferase reporter constructs containing the p21 promoter was inhibited by co-transfection with the human MDM2 protein or a dominant-negative p53 protein and was dependent on the presence of p53 response elements in the reporter constructs. Furthermore, p53 in nuclear extracts of untreated cells was capable of binding to DNA in a sequence-specific manner. These results implicate a role for p53 in regulating constitutive levels of expression of p21 and demonstrate that the p53 protein is capable of sequence-specific DNA binding and transcriptional activation in untreated, proliferating cells.

One Mechanism for Cell Type-specific Regulation of thebax Promoter by the Tumor Suppressor p53 Is Dictated by the p53 Response Element

Key to the function of the tumor suppressor p53 is its ability to activate the transcription of its target genes, including those that encode the cyclin-dependent kinase inhibitor p21 and the proapoptotic Bax protein. In contrast to Saos-2 cells in which p53 activated both the p21 andbax promoters, in MDA-MB-453 cells p53 activated thep21 promoter, but failed to activate the baxpromoter. Neither phosphorylation of p53 on serines 315 or 392 nor an intact C terminus was required for p53-dependent activation of the bax promoter, demonstrating that this differential regulation of bax could not be explained solely by modifications of these residues. Further, this effect was not due to either p73 or other identified cellular factors competing with p53 for binding to its response element in the bax promoter. p53 expressed in MDA-MB-453 cells also failed to activate transcription through the p53 response element of the bax promoter in isolation, demonstrating that the defect is at the level of the interaction between p53 and its response element. In contrast to other p53 target genes, like p21, in which p53-dependent transcriptional activation is mediated by a response element containing two consensus p53 half-sites, activation by p53 of the bax element was mediated by a cooperative interaction of three adjacent half-sites. In addition, the interaction of p53 with its response element from the bax promoter, as compared with its interaction with its element from the p21promoter, involves a conformationally distinct form of the protein. Together, these data suggest a potential mechanism for the differential regulation of p53-dependent transactivation of thebax and p21 genes.

p53 and Apoptosis: It's Not Just in the Nucleus Anymore

The tumor suppressor p53 triggers apoptosis in response to a variety of stress stimuli. Its role as a transcription factor modulating gene expression has been clearly implicated in this process. Two recent publications now argue for an additional direct role of p53 at the mitochondria in inducing apoptosis.

E2F7, a novel target, is up-regulated by p53 and mediates DNA damage-dependent transcriptional repression

The p53 tumor suppressor protein is a transcription factor that exerts its effects on the cell cycle via regulation of gene expression. Although the mechanism of p53-dependent transcriptional activation has been well-studied, the molecular basis for p53-mediated repression has been elusive. The E2F family of transcription factors has been implicated in regulation of cell cycle-related genes, with E2F6, E2F7, and E2F8 playing key roles in repression. In response to cellular DNA damage, E2F7, but not E2F6 or E2F8, is up-regulated in a p53-dependent manner, with p53 being sufficient to increase expression of E2F7. Indeed, p53 occupies the promoter of the E2F7 gene after genotoxic stress, consistent with E2F7 being a novel p53 target. Ablation of E2F7 expression abrogates p53-dependent repression of a subset of its targets, including E2F1 and DHFR, in response to DNA damage. Furthermore, E2F7 occupancy of the E2F1 and DHFR promoters is detected, and expression of E2F7 is sufficient to inhibit cell proliferation. Taken together, these results show that p53-dependent transcriptional up-regulation of its target, E2F7, leads to repression of relevant gene expression. In turn, this E2F7-dependent mechanism contributes to p53-dependent cell cycle arrest in response to DNA damage.

Disruption of G1-phase phospholipid turnover by inhibition of Ca2+-independent phospholipase A2 induces a p53-dependent cell-cycle arrest in G1 phase.

The G1 phase of the cell cycle is characterized by a high rate of membrane phospholipid turnover. Cells regulate this turnover by coordinating the opposing actions of CTP:phosphocholine cytidylyltransferase and the group VI Ca2+-independent phospholipase A2 (iPLA2). However, little is known about how such turnover affects cell-cycle progression. Here, we show that G1-phase phospholipid turnover is essential for cell proliferation. Specific inhibition of iPLA2 arrested cells in the G1 phase of the cell cycle. This G1-phase arrest was associated with marked upregulation of the tumour suppressor p53 and the expression of cyclin-dependent kinase inhibitor p21cip1. Inactivation of iPLA2 failed to arrest p53-deficient HCT cells in the G1 phase and caused massive apoptosis of p21-deficient HCT cells, suggesting that this G1-phase arrest requires activation of p53 and expression of p21cip1. Furthermore, downregulation of p53 by siRNA in p21-deficient HCT cells reduced the cell death, indicating that inhibition of iPLA2 induced p53-dependent apoptosis in the absence of p21cip1. Thus, our study reveals hitherto unrecognized cooperation between p53 and iPLA2 to monitor membrane-phospholipid turnover in G1 phase. Disrupting the G1-phase phospholipid turnover by inhibition of iPLA2 activates the p53-p21cip1 checkpoint mechanism, thereby blocking the entry of G1-phase cells into S phase.