Ectopic P53 Research Articles

TA-p63-γ regulates expression of ΔN-p63 in a manner that is sensitive to p53

Genetic analysis indicates that TP63 is required for establishment and preservation of self-renewing progenitors within the basal layer of several epithelial structures, however, the specific contributions of transactivating (TA-p63) and dominant-negative (DeltaN-p63) isoforms remain largely undefined. Recent studies have suggested a model in which TA-p63 plays an important role in the establishment of progenitor populations in which expression of DeltaN-p63 contributes to the preservation of self-renewing capacity. Our previous studies indicate that DeltaN-p63 is a transcriptional target of p53, however, the absence of overt epithelial deficiencies in p53-/- mice and reports of increased expression of DeltaN-p63 in p53-/- mice suggest p53-independent mechanisms also contribute to expression of DeltaN-p63. Here, we present data indicating that, prolonged loss of p53 leads to the activation of a p53-independent mechanism for transcriptional regulation of DeltaN-p63. This p53-independent mechanism is sensitive to ectopic p53 but not to a p53 mutant that lacks the transactivation domain. We further show that in cells in which p53 is expressed TA-p63-gamma protein is destabilized in a manner that is p53 dependent and sensitive to pharmacologic inhibition of the 26S proteosome. Consistent with this observation, we demonstrate that loss of p53 leads to the stabilization of TA-p63-gamma that is reversible by ectopic p53. Finally, we present evidence that disruption of TA-p63-gamma expression leads to decreased expression of DeltaN-p63 and that overexpression of TA-p63-gamma was sufficient to enhance the activity of the DeltaN-p63 promoter. Taken together, our studies indicate that TA-p63-gamma is capable of activating expression of DeltaN-p63 and that this mechanism may account for p53-independent expression of DeltaN-p63.

WOX1 Is Essential for Tumor Necrosis Factor-, UV Light-, Staurosporine-, and p53-mediated Cell Death, and Its Tyrosine 33-phosphorylated Form Binds and Stabilizes Serine 46-phosphorylated p53

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, undergoes Tyr33 phosphorylation at its first N-terminal WW domain and subsequent nuclear translocation in response to sex steroid hormones and stress stimuli. The activated WOX1 binds tumor suppressor p53, and both proteins may induce apoptosis synergistically. Functional suppression of WOX1 by antisense mRNA or a dominant negative abolishes p53-mediated apoptosis. Here, we determined that UV light, anisomycin, etoposide, and hypoxic stress rapidly induced phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (phospho-WOX1) and their binding interactions in several tested cancer cells. Mapping by yeast two-hybrid analysis and co-immunoprecipitation showed that phospho-WOX1 physically interacted with Ser46-phosphorylated p53. Knockdown of WOX1 protein expression by small interfering RNA resulted in L929 fibroblast resistance to apoptosis by tumor necrosis factor, staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, UV light could not induce p53 protein expression in these WOX1 knockdown cells, although p53 mRNA levels were not reduced. Suppression of WOX1 by dominant negative WOX1 (to block Tyr33 phosphorylation) also abolished UV light-induced p53 protein expression. Time course analysis showed that the stability of ectopic wild type p53, tagged with DsRed, was decreased in WOX1 knockdown cells. Inhibition of MDM2 by nutlin-3 increased the binding of p53 and WOX1 and stability of p53. Together, our data show that WOX1 plays a critical role in conferring cellular sensitivity to apoptotic stress and that Tyr33 phosphorylation in WOX1 is essential for binding and stabilizing Ser46-phosphorylated p53.

P21 WAF1 modulates NF-κB signaling and induces anti-apoptotic protein Bcl-2 in Tax-expressing rat fibroblast

Of the cell cycle-associated genes regulated by human T-cell leukemia virus type-1 (HTLV-1) Tax, cyclin-dependent kinase (CDK) inhibitor p21 WAF1 is upregulated in HTLV-1-infected cells. Previously, we reported that p21 WAF1 stimulated Tax-dependent NF-κB activation which influences a variety of cellular processes, including proliferation, differentiation, and apoptosis. In HTLV-1-infected cells, Tax is primarily involved in the constitutive activation of NF-κB signaling. Here, we demonstrate that p21 WAF1 affects Tax-dependent NF-κB signaling by inducing p100/52, an NF-κB-related protein. W4, a Tax-transformed rat fibroblast cell line, exhibits the constitutive activation of NF-κB signaling, potentially mediated by overexpression of RelB. Ectopic expression of p21 WAF1 in W4 cells, which lack endogenous expression due to methylation of the p21 WAF1 promoter, induces the expression of p100/52. Bcl-2 expression was also upregulated by ectopic p21 WAF1 in this cell line, suggesting that p21 WAF1 plays an important role in the regulation of apoptosis by modulating NF-κB signaling in Tax-expressing rat fibroblasts. We also address the expression of NF-κB-related proteins in HTLV-1-infected cells.

A role for p21 (WAF1) in the cAMP-dependent differentiation of F9 teratocarcinoma cells into parietal endoderm

Combined treatment of teratocarcinoma F9 cells with retinoic acid and dibutyryl-cAMP induces the differentiation into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation, concomitantly with the appearance of p21 in the immunoprecipitated CDK2–cyclin E complex. The induction of differentiation markers could not be achieved by expression of ectopic p21 alone and still required treatment with differentiation agents. Clones of F9 cells transfected with sense or antisense p21 cDNA constructs revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker, or increased fraction of cells in sub-G1 phase of the cell cycle, respectively. Consistent with this observation, whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1–1303) fusion protein on the GAL sites-containing TM promoter. This implies that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. As differentiation of F9 cells into parietal endoderm-like cells requires the cAMP signaling, the results together suggest that the cyclin-dependent kinase inhibitor p21 may promote specifically this pathway in F9 cells.

The Tumor Suppressor p53 Abrogates Smad-dependent Collagen Gene Induction in Mesenchymal Cells

The pleiotropic cytokine transforming growth factor-beta (TGF-beta) is a potent inducer of collagen synthesis and is implicated in the pathogenesis of fibrosis. Acting in concert with transcriptional coactivators p300/CBP, the Smads mediate TGF-beta stimulation of collagen synthesis in human dermal fibroblasts. Little information exists regarding positive and negative modulation of physiological TGF-beta responses. Because the tumor suppressor p53 is implicated in connective tissue homeostasis, here we examined the regulation of collagen gene expression by p53. Forced expression of ectopic p53 in dermal fibroblasts repressed basal and TGF-beta-stimulated collagen gene expression, whereas the absence of cellular p53 was associated with significantly enhanced transcriptional activity of the Type I collagen gene (COL1A2) and collagen synthesis. Ectopic expression of p53 also repressed TGF-beta stimulation of promoter activity driven by minimal Smad-binding elements, suggesting that p53 modulated Smad-dependent intracellular signaling. Inhibition was not due to altered levels, phosphorylation, or nuclear translocation of cellular Smads. Treatment of fibroblasts with etoposide, a potent inducer of cellular p53, abrogated TGF-beta stimulation of COL1A2 promoter activity and collagen synthesis in a p53-dependent manner. Overexpression of the transcriptional coactivator p300 rescued TGF-beta stimulation of COL1A2 promoter activity in fibroblasts overexpressing p53. Furthermore, the ligand-induced interaction of cellular Smad3 with p300 or with its cognate Smad-binding DNA element and recruitment of p300 to the DNA-protein complex assembled on the Smad-binding element were markedly reduced in p53-overexpressing fibroblasts. Collectively, these results indicate, for the first time, that p53 is a potent and selective endogenous repressor of TGF-beta-regulated collagen gene expression in dermal fibroblasts. The ligand-dependent interaction of Smad3 with p300 may be one of the targets of p53-mediated inhibition of TGF-beta responses. These findings suggest that a novel and important physiologic function for the tumor suppressor p53 is the regulation of fibrotic cellular responses.

Inhibition of MDM2-mediated p53 Ubiquitination and Degradation by Ribosomal Protein L5

The oncoprotein MDM2 associates with ribosomal proteins L5, L11, and L23. Both L11 and L23 have been shown to activate p53 by inhibiting MDM2-mediated p53 suppression. Here we have shown that L5 also activates p53. Overexpression of L5 stabilized ectopic p53 in H1299 cells and endogenous p53 in U2OS cells. Consequently, L5 enhanced p53 transcriptional activity and induced p53-dependent G1 cell cycle arrest. Furthermore, like L11 and L23, L5 also remarkably inhibited MDM2-mediated p53 ubiquitination. The interaction of L5 with MDM2 was also enhanced by treatment with a low dose of actinomycin D. Actinomycin D-induced p53 was inhibited by small interference RNA against L5. By reciprocal co-immunoprecipitation, we further showed that there were at least two MDM2-ribosomal protein complexes in cells: MDM2-L5-L11-L23 and p53-MDM2-L5-L11-L23. We propose that the MDM2-L5-L11-L23 complex functions to inhibit MDM2-mediated p53 ubiquitination and thus activates p53.

Ordered Cooperative Functions of PRMT1, p300, and CARM1 in Transcriptional Activation by p53

Transcriptional coactivators that modify histones represent an increasingly important group of regulatory factors, although their ability to modify other factors as well precludes common assumptions that they necessarily act by histone modification. In an extension of previous studies showing a role for acetyltransferase p300/CBP in p53 function, we have used systems reconstituted with recombinant chromatin templates and (co)activators to demonstrate (1) the additional involvement of protein arginine methyltransferases PRMT1 and CARM1 in p53 function; (2) both independent and ordered cooperative functions of p300, PRMT1, and CARM1; and (3) mechanisms that involve direct interactions with p53 and, most importantly, obligatory modifications of corresponding histone substrates. ChIP analyses have confirmed the ordered accumulation of these (and other) coactivators and cognate histone modifications on the GADD45 gene following ectopic p53 expression and/or UV irradiation. These studies thus define diverse cofactor functions, as well as underlying mechanisms involving distinct histone modifications, in p53-dependent gene activation.

Cdk6-cyclin D3 activity in murine ES cells is resistant to inhibition by p16(INK4a).

Through a screen aimed at identifying genes that are specifically upregulated in embryomic stem (ES) cells but not primitive ectoderm, we identified cyclin D3. This was surprising since cyclin D activity is generally believed to be inactive in ES cells even though retinoblastoma tumor suppressor protein (pRb) accumulates in a predominantly hyperphosphorylated state. Cdk6 is the major catalytic partner for cyclin D3 in ES cells and exhibits robust pRb kinase activity that is downregulated during the early stages of ES embryoid body differentiation. To investigate the basis underlying the insensitivity of ES cells to ectopic p16 expression, we show that Cdk6-cyclin D3 complexes are not subject to inhibition by p16, similar to Cdk-viral cyclin complexes. These observations show that specificity exists between Cdk4/6-cyclin D complexes and their ability to be targeted by p16. Our data suggest that Cdk6-cyclin D3 activity in other cell types, including tumors, may also be refractory to p16-mediated growth inhibition and raises the possibility of additional specificity within the INK4 family.

TIAF1 and p53 functionally interact in mediating apoptosis and silencing of TIAF1 abolishes nuclear translocation of serine 15-phosphorylated p53.

TIAF1 is a TGF-beta 1-induced factor that protects L929 fibroblasts from TNF-mediated apoptosis. In contrast, overexpressed TIAF1 induces growth inhibition and apoptosis of monocytic U937 and various nonfibroblast cells. TIAF1-mediated apoptosis of U937 cells involves upregulation of p53, p21, and Smad2/4, but downregulation of ERK phosphorylation. To determine whether p53 and TIAF1 functionally interact in regulating cell death, ectopic TIAF1 and p53 were shown to induce apoptosis of U937 cells in both synergistic and antagonistic manners. At optimal levels both TIAF1 and p53 mediated apoptosis cooperatively. Also, both proteins suppressed adherence-independent growth of L929 cells. In contrast, initiation of apoptosis by overexpressed TIAF1 was blocked by low doses of p53, and vice versa. Furthermore, ectopic p53 blocked an ongoing apoptosis in U937 cells stably expressing TIAF1. Yeast two-hybrid analyses failed to demonstrate the binding of p53 with TIAF1, suggesting an unidentified protein that links the p53/TIFA1 interaction. Suppression of TIAF1 expression by siRNA could not inhibit Ser15 phosphorylation in p53 in response to UV and etoposide. However, nuclear translocation of these Ser15-phosphorylated p53 was significantly reduced in TIAF1-silenced cells. Taken together, TIAF1 and p53 functionally interact in regulating apoptosis, and TIAF1 is likely to participate in the nuclear translocation of activated p53.

Induction of p53-regulated genes in lung cancer cells: implications of the mechanism for adenoviral p53-mediated apoptosis.

The p53 gene is the most frequently mutated gene in human cancers. Loss of functional p53 leads to impaired responses of cancer cells to apoptosis induction and to poor prognosis in patients with certain types of cancer. Cancer gene therapies using ectopic expression of wild-type p53 to force cancer cells through the apoptotic pathway have been tested extensively preclinically and clinically, and genes in various cell lines have been reported to be regulated upon ectopic p53 overexpression. However, the effect of p53 on many other p53-dependent and apoptosis-related genes remains unclear, as does the mechanism of p53-induced apoptosis in human cancers. In this study, we used real-time reverse transcription polymerase chain reaction to evaluate the changes in expression of various p53-dependent and apoptosis-related genes in five human non-small-cell lung cancer cell lines with varying p53 statuses after adenoviral p53 treatment. We found that Ad/p53 induced the expression of the proapoptotic genes PUMA, Bak, Bax, and Fas in a cell type- and time-dependent manner. Among these genes, PUMA was upregulated the most dramatically and broadly. However, when a specific siRNA construct against PUMA was employed, we observed no attenuation of apoptosis in H1299 cells. Our data suggest that Ad-p53 induces the expression of a variety of proapoptotic genes and that lack of induction in one of these genes does not block Ad/p53-mediated cell killing in human lung cancer cells.

P21/CDKN1A Mediates Negative Regulation of Transcription by p53

The tumor suppressor p53 regulates transcription positively and negatively, depending on the target gene. Whereas p53 induces transcription through direct interaction with promoter DNA, the mechanism of p53-mediated transcriptional repression is less well understood. Early reports described the alleviation of p53-mediated repression by inhibitors of apoptosis, suggesting that negative regulation of transcription might occur only in conjunction with programmed cell death. More recently, it has been proposed that certain genes, such as survivin, are repressed by direct association of p53 with their promoters, followed by recruitment of a repressor complex. We show here that p53-mediated negative regulation of transcription could occur independently of apoptosis. In contrast, the amino-terminal transactivation domain of p53 was required for negative regulation of transcription. Similarly, the p53 homologue p73 diminished the expression of survivin and stathmin, depending on its transactivation domain. Mutation of the putative p53 binding site within the survivin promoter did not impair its repression. These observations raised the hypothesis that activation of an effector gene might be required for repression by p53. Strikingly, when the p53-inducible p21/CDKN1A gene was deleted, p53 no longer repressed any one among 11 genes that it down-regulates otherwise. Most of these genes were also repressed by ectopic p21 in the absence of p53. Overexpressed c-Myc reduced the transcription of p21/CDKN1A and impaired p53-mediated repression but did not abolish repression by ectopic p21. Taken together, these results strongly suggest that increased expression of p21/CDKN1A is necessary and sufficient for the negative regulation of gene expression by p53.

Response of Ewing tumor cells to forced and activated p53 expression.

The EWS-FLI1 transcription factor is consistently expressed in 85% of Ewing tumors (EFT). In heterologous cells, EWS-FLI1 induces p53-dependent cell cycle arrest or apoptosis. It has been speculated that the p53 tumor suppressor pathway may be generally compromised in EFT despite only rare p53 mutations. In order to test for functional integrity of this pathway, we have investigated a series of EFT cell lines that differ from each other with respect to their endogenous p53 and INK4A gene status for their response to ectopic p53 expression and to stimulation of endogenous p53 activity by X-ray treatment. Significant interindividual and intratumoral variations in the apoptotic propensity of EFT cell lines to transient expression of ectopic p53 were observed, which was independent of the level of p53 expression. In cell lines with a low apoptotic incidence, apoptosis was delayed and the surviving fraction showed a prolonged growth arrest. Complete resistance to p53-induced apoptosis in two cell lines established from the same patient was associated with a high BCL2/BAX ratio and low levels of APAF1. Sensitivity to X-rays showed a trend towards a higher apoptotic rate in wild-type (wt) p53 expressing than in p53 mutant cells. However, one wt p53-expressing EFT cell line was completely refractory to irradiation-stimulated cell death despite high apoptotic responsiveness to ectopic p53. No difference in Ser15 phosphorylation and the transcriptional activation of p53 targets was observed in wt p53 EFT cell lines irrespective of the induction of cell death or growth arrest. All together, our results demonstrate that despite significant variability in the outcome, cell death or cell cycle arrest, the p53 downstream pathway and the DNA damage signaling pathway are functionally intact in EFT.

Parc: A Cytoplasmic Anchor for p53

Nuclear localization of p53 is essential for its tumor suppressor function. Here, we have identified Parc, a Parkin-like ubiquitin ligase, as a cytoplasmic anchor protein in p53-associated protein complexes. Parc directly interacts and forms a ∼1 MDa complex with p53 in the cytoplasm of unstressed cells. In the absence of stress, inactivation of Parc induces nuclear localization of endogenous p53 and activates p53-dependent apoptosis. Overexpression of Parc promotes cytoplasmic sequestration of ectopic p53. Furthermore, abnormal cytoplasmic localization of p53 was observed in a number of neuroblastoma cell lines; RNAi-mediated reduction of endogenous Parc significantly sensitizes these neuroblastoma cells in the DNA damage response. These results reveal that Parc is a critical regulator in controlling p53 subcellular localization and subsequent function.

Conditional expression of the tumor suppressor p16 in a heterotopic glioblastoma model results in loss of pRB expression.

We have expressed the tumor suppressor p16 under the control of a tetracycline-sensitive promoter in two human glioblastoma cell lines which do not contain endogenous p16. Ectopic p16 expression led to a stable but reversible G1 phase cell cycle arrest, reduced the growth of both cell lines in cell culture, and almost abolished their in vitro tumorigenicity. U-87MG-tTA-p16 glioblastoma cells consistently formed tumors after subcutaneous injection into the flanks of nude mice. p16 expression in these tumors was strictly dependent on the presence or absence of tetracycline in the drinking water. Ectopic p16 reduced the tumor take rate (in vivo tumorigenicity) of U-87MG-tTA-p16 cells from 18/20 (90%) to 5 tumors/12 (42%) tumor cell injections. p16 positive and negative tumors differed with respect to their Ki67 labeling indices (34 +/- 4% vs. 52 +/- 6% , P < 0.001, student's t-test). These data are consistent with an in vitro and in vivo glioma suppressor role for p16. Interestingly, we observed a secondary reduction of pRB expression in tumors (and cell cultures) exposed to p16 for > or = 10 (6) days. pRB is p16's major downstream target. Hence, this finding might explain, why p16 expression neither significantly affected the morphology nor led to a reduction of size or growth rate of the tumors. Loss of pRB following p16 expression might severely limit the potential benefit of p16 gene therapy for glioblastoma.

P53 Inhibits Adriamycin-Induced Down-Regulation of Cyclin D1 Expression in Human Cancer Cells

The tumor suppressor p53 gene product is an essential component of the cytotoxic pathway triggered by DNA-damaging stimuli such as chemotherapeutic agents and ionizing radiation. We previously demonstrated that adenovirus-mediated wild-type p53 gene transfer could enhance the cytotoxic actions of chemotherapeutic drugs both in vitro and in vivo; however, the molecular mechanism of this chemosensitization is still unclear. Cyclin D1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle. Here we show that infection with an adenovirus vector expressing the wild-type p53 gene (Ad-p53) caused an increase in cyclin D1 protein levels in human colorectal cancer cell lines DLD-1 and SW620; treatment with the anti-cancer drug adriamycin, however, down-regulated their cyclin D1 protein expression in a dose-dependent manner. The suppression of cyclin D1 expression following adriamycin treatment could be blocked by simultaneous Ad-p53 infection. Furthermore, DLD-1 and SW620 cells transfected with the cyclin D1 expression construct displayed increased sensitivity to adriamycin compared to that of the vector-transfected control. Our results suggest that ectopic wild-type p53 gene transfer results in increased cyclin D1 expression and, consequently, sensitizes human colorectal cancer cells to chemotherapeutic agents.

Regulation of cell cycle and productivity in NS0 cells by the over‐expression of p21CIP1

We have constructed NS0 myeloma cell lines that inducibly express the p21CIP1 cyclin dependent kinase inhibitor, using the Lacswitch system. Ectopic p21(CIP1) protein expression was rapidly induced within 12 h of addition of IPTG, causing G1-phase arrest and almost complete inhibition of cell proliferation. The production of a chimeric IgG4 antibody, expressed constitutively from an independent promoter, was found to be significantly increased by more than 4-fold in p21CIP1-arrested cells. This study demonstrates for the first time the successful construction of anchorage-independent and proliferation-controlled NS0 cell lines with enhanced secreted chimeric antibody production independent of the inducible promoter activity used to achieve cytostasis.

Accelerated degradation of cellular FLIP protein through the ubiquitin-proteasome pathway in p53-mediated apoptosis of human cancer cells.

Apoptosis is a morphologically distinct form of programmed cell death that plays a major role in cancer treatments. This cellular suicide program is known to be regulated by many different signals from both intracellular and extracellular stimuli. Here we report that p53 suppressed expression of the cellular FLICE-inhibitory protein (FLIP) that potentially blocks apoptotic signaling in human colon cancer cell lines expressing mutated and wild-type p53. In contrast, the expression of the death receptor KILLER/DR5 (TRAIL-R2) had no effect on FLIP expression, although exogenous p53 is known to induce KILLER/DR5 expression. In line with these observations, FLIP-negative cancer cells were sensitive to both p53- and KILLER/DR5-mediated apoptosis, whereas cells containing high levels of FLIP underwent apoptotic cell death when triggered by ectopic p53 expression but not by KILLER/DR5 expression. Treating the cells with a specific inhibitor of the proteasome inhibited the decrease of FLIP by p53, suggesting that p53 enhances the degradation of FLIP via a ubiquitin-proteasome pathway. Thus, the data indicate that p53-mediated downregulation of FLIP may explain the potent sensitization of human cancer cells to the apoptotic suicide program induced by wild-type p53 gene transfer.

Transcriptional Regulation of the Human DNA Polymerase δ Catalytic Subunit Gene POLD1 by p53 Tumor Suppressor and Sp1

The DNA polymerase delta catalytic subunit gene (POLD1) was studied as a transcriptional target of p53. Northern blotting showed that a significantly decreased steady-state level of POLD1 mRNA was associated with increased wild-type p53 expression in cells treated with methyl methanesulfonate. When ectopic wild-type p53 expression was induced to a physiologically relevant level in "tet-off" cultured cells in which p53 expression was tightly regulated by tetracycline, it was found that POLD1 steady-state mRNA was repressed by about 65%. Transient cotransfection experiments using a POLD1 promoter luciferase reporter construct showed that: (i) POLD1 promoter activity was inhibited by transfected wild-type p53 plasmid to a maximum of about 86%; (ii) p53 mediated a large part of the transcriptional repression through a sequence-specific interaction with a site identified as the P4 site of the POLD1 promoter; (iii) tumor-derived p53 mutations in the p53 DNA-binding domain completely abolished the p53 transrepression activity. Moreover, transfection assays demonstrated that p53 was able to repress Sp1-stimulated POLD1 promoter activity and that this repression was largely due to the loss of the sequence-specific interaction between Sp1 protein and the P4 Sp1-binding site, which overlaps the P4 p53-binding site. Finally, gel shift assays suggested that p53 competes with Sp1 protein for binding to the P4 sequence of the POLD1 promoter.

Inhibition of HIF-1- and wild-type p53-stimulated transcription by codon Arg175 p53 mutants with selective loss of functions

Overexpression of ectopic mutant p53 represses wild-type p53-stimulated transcription, known as a dominant negative effect. On the other hand, overexpression of wild-type p53 can repress transcription stimulated by several transcription factors, including hypoxia-inducible factor-1 (HIF-1). Using a panel of well-characterized Arg175 p53 mutants we found that only mutants (Tyr175, Trp175, Asp175 and Phe175) which have completely lost their ability to transactivate repress wild-type p53-stimulated Bax, p21 and PG13 promoter constructs. In contrast, Asn175, Gln175, Leu175 and Pro175 mutants which partially retained transactivating functions did not exert dominant negative effects against PG13 and p21 promoter constructs. However, these latter mutants failed to activate Bax and, instead, exerted a dominant negative effect on a Bax-Luc promoter construct. We conclude that a dominant negative effect is promoter selective as a consequence of selective loss of transactivating function. Albeit less potent than wild-type p53, all Arg175 p53 mutants retained partial ability to repress HIF-1-stimulated transcription. We propose that transrepression and the dominant negative effect have similar mechanisms and may involve competition with transcription factors (wild-type p53, HIF-1, etc.) for cofactors such as p300. Thus, a p53(22/23) mutant, which is deficient in p300 binding, did not exert dominant negative effects. Like transrepression, the dominant negative effect required overexpression of mutant p53 and, therefore, is not dominant. In the presence of a wild-type p53 allele, levels of endogenous mutant p53 protein were low in heterozygous cells. Endogenous mutant p53 became overexpressed only after loss of the second p53 allele. Therefore, endogenous mutant p53s are unable to display a dominant negative effect. This explains why loss of the second p53 allele is required to eliminate p53 functions in cancer cells.

Reprimo, a New Candidate Mediator of the p53-mediated Cell Cycle Arrest at the G2 Phase

A novel gene, Reprimo, in which induction in cells exposed to X-irradiation is dependent on p53 expression, has been isolated. Ectopic p53 expression results in the induction of its mRNA. Reprimo is a highly glycosylated protein and, when ectopically expressed, it is localized in the cytoplasm and induces G(2) arrest of the cell cycle. In the arrested cells, both Cdc2 activity and nuclear translocation of cyclin B1 are inhibited, suggesting the involvement of Reprimo in the Cdc2.cyclin B1 regulation pathway. Thus, Reprimo may be a new member involved in the regulation of p53-dependent G(2) arrest of the cell cycle.