Munna L. Agarwal

The p53 Network

Loss of control of genomic stability is central in the development of cancer, and p53, by regulating normal responses to DNA damage and other forms of genotoxic stress, is a key element in maintaining genomic stability. Thus, it is no surprise that functional p53 is lost in about half of all human cancers. What about the other half? One possibility is that p53-independent regulatory mechanisms have been lost. Another is that inactivation of p53-dependent pathways can occur at any of several different points and that p53 itself is merely the most common target. For example, the p53 inhibitor Mdm2 is overexpressed in tumors independently of the p53 mutation. Here, we review pathways that signal in to p53, in response to different forms of stress, and pathways that signal out, triggered by activated p53. It is clear that p53 is the central component of a complex network of signaling pathways and that the other components of these pathways pose alternative targets for inactivation. For additional recent reviews, see Refs. 1 and 2.

Role of p53 and NF-κB in epigallocatechin-3-gallate-induced apoptosis of LNCaP cells

We have recently shown that oral consumption of green tea polyphenols inhibits prostate carcinogenesis in transgenic mouse model of prostate cancer and suggested that induction of apoptosis in prostate cancer cells is responsible for these effects. Much of the chemopreventive effects of green tea are attributed to its major polyphenolic constituent (−) epigallocatechin-3-gallate (EGCG). In the present study, we report that EGCG-induced apoptosis in human prostate carcinoma LNCaP cells is mediated via modulation of two related pathways: (a) stabilization of p53 by phosphorylation on critical serine residues and p14ARF-mediated downregulation of murine double minute 2(MDM2) protein, and (b) negative regulation of NF-κB activity, thereby decreasing the expression of the proapoptotic protein Bcl-2. EGCG-induced stabilization of p53 caused an upregulation in its transcriptional activity, thereby resulting in activation of its downstream targets p21/WAF1 and Bax. Thus, EGCG had a concurrent effect on two important transcription factors p53 and NF-κB, causing a change in the ratio of Bax/Bcl-2 in a manner that favors apoptosis. This altered expression of Bcl-2 family members triggered the activation of initiator capsases 9 and 8 followed by activation of effector caspase 3. Activation of the caspases was followed by poly (ADP-ribose) polymerase cleavage and induction of apoptosis. Taken together, the data indicate that EGCG induces apoptosis in human prostate carcinoma cells by shifting the balance between pro- and antiapoptotic proteins in favor of apoptosis.

Tocotrienol-rich fraction of palm oil activates p53, modulates Bax/Bcl2 ratio and induces apoptosis independent of cell cycle association

Anti-cancer properties of palm oil have been attributed to the presence of tocotrienols and carotenoids. Studies from various laboratories have shown that tocotrienol-rich fraction (TRF) of palm oil inhibits cell growth and induces apoptosis in both pre-neoplastic and neoplastic cells. However, the mechanism by which TRF induces apoptosis remains largely unknown. Since several chemopreventive agents have been shown to utilize p53 pathway in negative regulation of cell growth, using human colon carcinoma RKO cells which express wild type p53, we investigated the effect of TRF on components of p53 signaling network. Treatment of cells with TRF resulted in a dose- and time- dependent inhibition of growth and colony formation. Further, TRF treatment of RKO cells resulted in the induction of WAF1/p21 which appears to be independent of cell cycle regulation and is transcriptionally upregulated in p53 dependent fashion. These results were further confirmed by using cells that express luciferase from a p53 responsive promoter where TRF treatment leads to activation of p53 reporter activity. TRF treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1. This altered expression of Bcl2 family members triggered the activation of initiator caspase-9 followed by activation of effector caspase-3. These signaling cascades lead to condensed chromatin, DNA fragmentation and shrinkage of cell membrane resulting into apoptosis. Our data suggest that TRF-induced apoptosis in colon carcinoma cells is mediated by p53 signaling network which appears to be independent of cell cycle association.

Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

ABSTRACT 7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21waf1, or Stat1, we found that optimal 7kchol-induced apoptosis requires p21waf1 and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser727 but not Tyr701 was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-β) blunted Ser727 phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-β receptor subunit exhibited blunted apoptosis. IFN-β alone did not induce apoptosis; thus, 7kchol-induced release of IFN-β was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21waf1 was much less than in wild-type cells; introducing transient expression of p21waf1 restored apoptosis. Stat1 and p21waf1 were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.

Defective induction but normal activation and function of p53 in mouse cells lacking poly-ADP-ribose polymerase.

Poly-ADP-ribose polymerase (PARP) and p53 are both induced by DNA damage and each has been proposed to mediate the normal cellular response to damage. We find that embryo fibroblasts from PARP-null mice have a ∼twofold lower basal level of p53 and that the induction of p53 in response to DNA damage or nucleotide depletion is more than twofold less than in normal mouse cells. These factors combine to decrease the induced level of the p53 protein in PARP-deficient cells by 4u2009–u20095-fold, compared to normal cells. However, there is virtually no decrease in the induction of p53 activity in PARP-deficient cells, as assayed with a p53-responsive promoter. Furthermore, cells lacking PARP arrest normally in G1 after DNA damage, in contrast to cells lacking p53, where this checkpoint is absent. Other p53-dependent properties, such as the mitotic spindle checkpoint and permissivity for gene amplification, are also normal in PARP-deficient cells. We conclude that the induced level of the p53 protein is governed by a combination of PARP-dependent and PARP-independent pathways and that the activation of p53 is largely PARP-independent. The results are consistent with a model in which the regulation of gene expression by p53 involves both increases in the amount of the protein and activation of p53 as a transcription factor.

p130/p107/p105Rb-dependent transcriptional repression during DNA-damage-induced cell-cycle exit at G2

The progression of normal cells from G2 into mitosis is stably blocked when their DNA is damaged. Tumor cells lacking p53 arrest only transiently in G2, but eventually enter mitosis. We show that an important component of the stable G2 arrest in normal cells is the transcriptional repression of more than 20 genes encoding proteins needed to enter into and progress through mitosis. Studies from a number of labs including our own have shown that, by inducing p53 and p21/WAF1, DNA damage can trigger RB-family-dependent transcriptional repression. Our studies reported here show that p130 and p107 play a key role in transcriptional repression of genes required for G2 and M in response to DNA damage. For plk1, repression is partially abrogated by loss of p130 and p107, and is completely abrogated by loss of all three RB-family proteins. Mouse cells lacking RB-family proteins do not accumulate with a 4N content of DNA when exposed to adriamycin, suggesting that all three RB-family proteins contribute to G2 arrest in response to DNA damage. Stable arrest in the presence of functional p53-to-RB signaling is probably due to the ability of cells to exit the cell cycle from G2, a conclusion supported by our observation that KI67, a marker of cell-cycle entry, is downregulated in both G1 and G2 in a p53-dependent manner.

p53 inhibits entry into mitosis when DNA synthesis is blocked

Human and mouse fibroblasts with normal p53 fail to enter mitosis when DNA synthesis is blocked by aphidicolin or hydroxyurea. Isogenic p53-null fibroblasts do enter mitosis with incompletely replicated DNA, revealing that p53 contributes to a checkpoint that ensures that mitosis does not occur until DNA synthesis is complete. When treated with N-(phosphonacetyl)-L-aspartate (PALA), which inhibits pyrimidine nucleotide synthesis, leading to synthesis of damaged DNA from highly unbalanced dNTP pools, p53-null cells enter mitosis after they have completed DNA replication, but cells with wild-type p53 do not, revealing that p53 also mediates a checkpoint that monitors the quality of newly replicated DNA.

The role of p53 in regulating genomic stability when DNA and RNA synthesis are inhibited

In addition to its induction by DNA damage, p53 is induced by drugs that starve cells for DNA and RNA precursors, or by inhibitors of DNA or RNA polymerase. In normal cells, the induction of p53 by dNTP starvation serves a protective role, mediating rapid, reversible cell-cycle arrest without DNA damage. In most cell lines, this first line of defense is missing, so that starvation for dNTPs causes DNA to break, thus increasing the probability of genomic instability, chromosome deletions and gene amplification. The mechanism of how p53 is induced remains unclear.

MYC Abrogates p53-Mediated Cell Cycle Arrest in N-(Phosphonacetyl)-L-Aspartate-Treated Cells, Permitting CAD Gene Amplification

ABSTRACT Genomic instability, including the ability to undergo gene amplification, is a hallmark of neoplastic cells. Similar to normal cells, “nonpermissive” REF52 cells do not develop resistance toN-(phosphonacetyl)-l-aspartate (PALA), an inhibitor of the synthesis of pyrimidine nucleotides, through amplification of cad, the target gene, but instead undergo protective, long-term, p53-dependent cell cycle arrest. Expression of exogenous MYC prevents this arrest and allows REF52 cells to proceed to mitosis when pyrimidine nucleotides are limiting. This results in DNA breaks, leading to cell death and, rarely, to cad gene amplification and PALA resistance. Pretreatment of REF52 cells with a low concentration of PALA, which slows DNA replication but does not trigger cell cycle arrest, followed by exposure to a high, selective concentration of PALA, promotes the formation of PALA-resistant cells in which the physically linked cad and endogenous N-myc genes are coamplified. The activated expression of endogenous N-myc in these pretreated PALA-resistant cells allows them to bypass the p53-mediated arrest that is characteristic of untreated REF52 cells. Our data demonstrate that two distinct events are required to form PALA-resistant REF52 cells: amplification ofcad, whose product overcomes the action of the drug, and increased expression of N-myc, whose product overcomes the PALA-induced cell cycle block. These paired events occur at a detectable frequency only when the genes are physically linked, ascad and N-myc are. In untreated REF52 cells overexpressing N-MYC, the level of p53 is significantly elevated but there is no induction of p21 waf1 expression or growth arrest. However, after DNA is damaged, the activated p53 executes rapid apoptosis in these REF52/N-myc cells instead of the long-term protective arrest seen in REF52 cells. The predominantly cytoplasmic localization of stabilized p53 in REF52/N-myc cells suggests that cytoplasmic retention may help to inactivate the growth-suppressing function of p53.

Regulation of p53 expression by the RAS-MAP kinase pathway

Activation of MAP kinase leads to the activation of p53-dependent pathways, and vice-versa. Although the amount of p53 protein increases in response to MAP kinase-dependent signaling, the basis of this increase is not yet fully understood. We have isolated the mutant cell line AP14, defective in p53 expression, from human HT1080 fibrosarcoma cells, which have an activated ras allele. The expression of p53 mRNA and protein is ∼10-fold lower in AP14 cells than in the parental cells. The high constitutive phosphorylation and activities of the MAP kinases ERK1 and ERK2 in HT1080 cells are greatly reduced in AP14 cells, although the levels of these proteins are unchanged, suggesting that the defect in the mutant cells affects the steady-state phosphorylation of ERK1 and ERK2. Overexpression of ERK2 in AP14 cells restored both MAP kinase activity and p53 expression, and incubation of the mutant cells with the phosphatase inhibitor orthovanadate resulted in strong coordinate elevation of MAP kinase activity and p53 expression. The levels of expression of the p53-regulated gene p21 parallel those of p53 throughout, showing that basal p21 expression depends on p53. The levels of p53 mRNA increased by 5–8-fold when activated ras was introduced into wild-type cells, and the levels of the p53 and p21 proteins decreased substantially in wild-type cells treated with the MEK inhibitor U0216. We conclude that MAP kinase-dependent pathways help to regulate p53 levels by regulating the expression of p53 mRNA.