Induction Of P53 Target Genes Research Articles

Ectodermal Factor Restricts Mesoderm Differentiation by Inhibiting p53

During gastrulation of the amphibian embryo, specification of the three germ layers, endo-, ecto-, and mesoderm, is regulated by maternal and zygotic mechanisms. Although it is known that mesoderm specification requires the cooperation between TGF-beta signaling and p53 activity and requires maternal factors, essential zygotic factors have been elusive. Here, we report that the Zn-finger protein XFDL156 is an ectodermal, zygotic factor that suppresses mesodermal differentiation. XFDL156 overexpression suppresses mesodermal markers, and its depletion induces aberrant mesodermal differentiation in the presumptive ectoderm. Furthermore, we find that XFDL156 and its mammalian homologs interact with the C-terminal regulatory region of p53, thereby inhibiting p53 target gene induction and mesodermal differentiation. Thus, XFDL156 actively restricts mesodermal differentiation in the presumptive ectoderm by controlling the spatiotemporal responsiveness to p53.

Inhibition of Methyltransferases Results in Induction of G 2 /M Checkpoint and Programmed Cell Death in Human T-Lymphotropic Virus Type 1-Transformed Cells

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia. The HTLV-1-encoded protein Tax transactivates the viral long terminal repeat and plays a critical role in virus replication and transformation. Previous work from our laboratory demonstrated that coactivator-associated arginine methytransferase 1, a protein arginine methytransferase, was important for Tax-mediated transactivation. To further investigate the role of methyltransferases in viral transcription, we utilized adenosine-2,3-dialdehyde (AdOx), an adenosine analog and S-adenosylmethionine-dependent methyltransferase inhibitor. The addition of AdOx decreased Tax transactivation in C81, Hut102, and MT-2 cells. Unexpectedly, we found that AdOx potently inhibited the growth of HTLV-1-transformed cells. Further investigation revealed that AdOx inhibited the Tax-activated NF-kappaB pathway, resulting in reactivation of p53 and induction of p53 target genes. Analysis of the NF-kappaB pathway demonstrated that AdOx treatment resulted in degradation of the IkappaB kinase complex and inhibition of NF-kappaB through stabilization of the NF-kappaB inhibitor IkappaBalpha. Our data further demonstrated that AdOx induced G(2)/M cell cycle arrest and cell death in HTLV-1-transformed but not control lymphocytes. These studies demonstrate that protein methylation plays an important role in NF-kappaB activation and survival of HTLV-1-transformed cells.

MDM2 Antagonist Nutlin-3 Suppresses the Proliferation and Differentiation of Human Pre-Osteoclasts Through a p53-Dependent Pathway

Exposure of human pre-osteoclasts to the MDM2 antagonist Nutlin-3 activated the p53 pathway and significantly decreased the entry of pre-osteoclasts in the S phase in response to RANKL. Moreover, repeated exposure to Nutlin-3 suppressed osteoclastic differentiation, without affecting cell survival at any culture time. The p53 oncosuppressor coordinates an intracellular network involved in protection from malignant transformation and cell cycle control; its activation is tightly regulated by the murine double minute 2 (MDM2) gene and p53-MDM2 interaction can be disrupted by selective small molecule inhibitors, the Nutlins. Although the ability of Nutlins to suppress the growth of wildtype p53 tumors has been clearly established, their biological activity in normal cells and tissues has not been extensively studied. Peripheral blood mononuclear cell pre-osteoclasts were cultured with macrophage-colony stimulating factor (M-CSF) + RANKL or co-cultured with SaOS-2 osteosarcoma cells in the presence of IL-1beta to induce osteoclastic differentiation. Cell cycle was analyzed by BrdU incorporation. The degree of osteoclastic differentiation was monitored at different culture times by TRACP and DAPI staining, as well as by TRACP-5b ELISA. Finally, the role of p53 in mediating the biological activity of Nutlin-3 was studied using specific siRNA. Exposure of human pre-osteoclasts to RANKL induced an early (24 h) increase in the percentage of cells in the S phase, followed by the exit from the cell cycle at later time-points. The simultaneous addition of Nutlin-3 and RANKL dose-dependently decreased the percentage of pre-osteoclasts in the S phase and induced a rapid accumulation of p53 protein coupled with the induction of p53 target genes. Unexpectedly, the administration of Nutlin-3 to pre-osteoclasts at early culture times significantly suppressed the final output of osteoclasts at day 14 of culture. The role of p53 in mediating this biological activity of Nutlin-3 was underscored by gene knockdown experiments, in which the anti-osteoclastic activity of Nutlin-3 was significantly counteracted by siRNA specific for p53. Nutlin-3 also significantly decreased the formation of osteoclasts in a co-culture system of SaOS-2 osteosarcoma and pre-osteoclastic cells. These findings indicate that Nutlin-3 abrogates both pre-osteoclastic proliferation and differentiation through a p53-dependent pathway and may have therapeutic implications for those neoplastic diseases characterized by an abnormal osteoclastic activity.

In vivo p53 response and immune reaction underlie highly effective low-dose radiotherapy in follicular lymphoma

AbstractVery low-dose irradiation (2 × 2 Gy) is a new, effective, and safe local treatment for follicular lymphoma. To understand the biologic mechanisms of this extremely effective response, we compared by microarray the gene-expression profile of patients' biopsies taken before and after radiation. In all patients, a major and consistent induction of p53 target genes was seen. p53 targets involved in cell-cycle arrest and apoptosis showed the same mode of regulation, indicating that, in vivo, both are activated simultaneously. p53 up-regulation and p53-mediated proliferation arrest and apoptosis were substantiated using immunohistochemistry, with activation of both the intrinsic and the extrinsic apoptotic pathways. The other induced genes revealed a whole set of biologically meaningful genes related to macrophage activation and TH1 immune response. Immunohistochemical analysis suggested a specific activation or differentiation of resident macrophages by apoptotic cells. These biologic insights are important arguments to advocate the use of low-dose radiotherapy as an effective palliative treatment for follicular lymphoma. Moreover, this study is the first in vivo report of the radiation-induced p53 apoptotic response in patients and suggests that this apoptotic response is not immunologically silent.

An Essential Role of Human Ada3 in p53 Acetylation

The p53 tumor suppressor protein functions as a critical component of genotoxic stress response by regulating the expression of effector gene products that control the fate of a cell following DNA damage. Unstressed cells maintain p53 at low levels through regulated degradation, and p53 levels and activity are rapidly elevated upon genotoxic stress. Biochemical mechanisms that control the levels and activity of p53 are therefore of great interest. We and others have recently identified hAda3 (human homologue of yeast alteration/deficiency in activation 3) as a p53-interacting protein and enhancer of p53 activity. Here, we show that endogenous levels of p53 and Ada3 interact with each other, and by using inducible overexpression and short hairpin RNA-mediated knockdown strategies we demonstrate that hAda3 stabilizes p53 protein by promoting its acetylation. Use of a p53 mutant with mutations of known p300/CREB-binding protein acetylation sites demonstrated that hAda3-dependent acetylation is required for increase in p53 stability and target gene induction. Importantly, we demonstrate that endogenous hAda3 is essential for DNA damage-induced acetylation and stabilization of p53 as well as p53 target gene induction. Overall, our results establish hAda3, a component of coactivator complexes that include histone acetyltransferase p300/CREB-binding protein, as a critical mediator of acetylation-dependent stabilization and activation of p53 upon genotoxic stress in mammalian cells.

BLIMP1 regulates cell growth through repression of p53 transcription

Tight regulation of p53 is essential for maintaining normal cell growth. Here we report that BLIMP1 acts in an autoregulatory feedback loop that controls p53 activity through repression of p53 transcription. p53 binds to and positively regulates BLIMP1, which encodes for a known B cell transcriptional repressor. Knockdown of BLIMP1 by siRNA results in both apoptosis and growth arrest in human colon cancer cells and cell-cycle arrest in primary human fibroblasts. Interestingly, the levels of both p53 mRNA and protein are substantially increased after BLIMP1 depletion, which is accompanied by the induction of p53 target genes. Importantly, the apoptosis induced by BLIMP1 depletion in HCT116 cells is largely abrogated in cells lacking p53 or in cells depleted in p53 by siRNA. We further demonstrate that BLIMP1 binds to the p53 promoter and represses p53 transcription, and this provides a mechanistic explanation for the induction of p53 response in cells depleted of BLIMP1. Hence, suppression of p53 transcription is a crucial function of endogenous BLIMP1 and is essential for normal cell growth.

E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop

ABSTRACT E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1 , E2f2 , and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21 CIP1 . Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1- , E2f2- , and E2f3- deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21 CIP1 and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

E2f1, E2f2, and E2f3 Control E2F Target Expression and Cellular Proliferation via a p53-Dependent Negative Feedback Loop

E2F-mediated control of gene expression is believed to have an essential role in the control of cellular proliferation. Using a conditional gene-targeting approach, we show that the targeted disruption of the entire E2F activator subclass composed of E2f1, E2f2, and E2f3 in mouse embryonic fibroblasts leads to the activation of p53 and the induction of p53 target genes, including p21(CIP1). Consequently, cyclin-dependent kinase activity and retinoblastoma (Rb) phosphorylation are dramatically inhibited, leading to Rb/E2F-mediated repression of E2F target gene expression and a severe block in cellular proliferation. Inactivation of p53 in E2f1-, E2f2-, and E2f3-deficient cells, either by spontaneous mutation or by conditional gene ablation, prevented the induction of p21(CIP1) and many other p53 target genes. As a result, cyclin-dependent kinase activity, Rb phosphorylation, and E2F target gene expression were restored to nearly normal levels, rendering cells responsive to normal growth signals. These findings suggest that a critical function of the E2F1, E2F2, and E2F3 activators is in the control of a p53-dependent axis that indirectly regulates E2F-mediated transcriptional repression and cellular proliferation.

Functional p53 Signaling in Kaposi's Sarcoma-Associated Herpesvirus Lymphomas: Implications for Therapy

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is associated with Kaposi's sarcoma (KS) as well as primary effusion lymphomas (PEL). The expression of viral proteins capable of inactivating the p53 tumor suppressor protein has been implicated in KSHV oncogenesis. However, DNA-damaging drugs such as doxorubicin are clinically efficacious against PEL and KS, suggesting that p53 signaling remains intact despite the presence of KSHV. To investigate the functionality of p53 in PEL, we examined the response of a large number of PEL cell lines to doxorubicin. Two out of seven (29%) PEL cell lines harbored a mutant p53 allele (BCBL-1 and BCP-1) which led to doxorubicin resistance. In contrast, all other PEL containing wild-type p53 showed DNA damage-induced cell cycle arrest, p53 phosphorylation, and p53 target gene activation. These data imply that p53-mediated DNA damage signaling was intact. Supporting this finding, chemical inhibition of p53 signaling in PEL led to doxorubicin resistance, and chemical activation of p53 by the Hdm2 antagonist Nutlin-3 led to unimpaired induction of p53 target genes as well as growth inhibition and apoptosis.

Excess HDM2 Impacts Cell Cycle and Apoptosis and Has a Selective Effect on p53-dependent Transcription

Mutational inactivation of p53 is only one of the ways that tumors lose p53 function. An alternate route is through overexpression of HDM2, the negative regulator of p53. To further understand how excess HDM2 regulates p53-mediated functions, we generated H1299 cell clones that constitutively express both ectopic HDM2 and tetracycline-regulated inducible p53. We found that over a range of p53 concentrations constitutively expressed HDM2 did not affect the levels of p53 protein. Nevertheless, cells with excess HDM2 displayed numerous changes in their response to p53. After DNA damage, such cells had both increased p53-mediated G2 arrest and reduced cell death. They also showed selective impairment of p53 target gene induction in that some p53 targets were unaffected whereas others were markedly less well induced in the presence of extra HDM2 protein. We also found that excess HDM2 was correlated with reduced p53 acetylation but did not affect p53 association with target promoters in vivo. Indeed, there was no significant difference in the amount of HDM2 associated with p53 at target promoters that differed in their expression depending on the presence of extra HDM2. Thus, HDM2 can selectively down-regulate the transcription function of p53 without either degrading p53 or affecting the interaction of p53 with target promoters.

Denatured and Reversibly Cationized p53 Readily Enters Cells and Simultaneously Folds to the Functional Protein in the Cells

Cationization is a powerful strategy for internalizing a protein into living cells. On the other hand, a reversibly cationized denatured protein through disulfide bonds is not only soluble in water but also able to fold to the native conformation in vitro. When these advantages in cationization were combined, we developed a novel method to deliver a denatured protein into cells and simultaneously let it fold to express its function within cells. This "in-cell folding" method enhances the utility of recombinant proteins expressed in Escherichia coli as inclusion bodies; that is, the recombinant proteins in inclusion bodies are solubilized by reversible cationization through cysteine residues by disulfide bonds with aminopropyl methanethiosulfonate or pyridyldithiopropionylpolyethylenimine and then incubated with cells without an in vitro folding procedure. As a model protein, we investigated human tumor-suppressor p53. Treatment of p53-null Saos-2 cells with reversibly cationized p53 revealed that all events examined as indications of the activation of p53 in cells, such as reduction of disulfide bonds followed by tetramer formation, localization into the nucleus, induction of p53 target genes, and induction of apoptosis of cells, occurred. These results suggest that reversible cationization of a denatured protein through cysteine residues is an alternative method for delivery of a functional protein into cells. This method would be very useful when a native folded protein is not readily available.

RETRACTED: hnRNP K: An HDM2 Target and Transcriptional Coactivator of p53 in Response to DNA Damage

In response to DNA damage, mammalian cells trigger the p53-dependent transcriptional induction of factors that regulate DNA repair, cell-cycle progression, or cell survival. Through differential proteomics, we identify heterogeneous nuclear ribonucleoprotein K (hnRNP K) as being rapidly induced by DNA damage in a manner that requires the DNA-damage signaling kinases ATM or ATR. Induction of hnRNP K ensues through the inhibition of its ubiquitin-dependent proteasomal degradation mediated by the ubiquitin E3 ligase HDM2/MDM2. Strikingly, hnRNP K depletion abrogates transcriptional induction of p53 target genes and causes defects in DNA-damage-induced cell-cycle-checkpoint arrests. Furthermore, in response to DNA damage, p53 and hnRNP K are recruited to the promoters of p53-responsive genes in a mutually dependent manner. These findings establish hnRNP K as a new HDM2 target and show that, by serving as a cofactor for p53, hnRNP K plays key roles in coordinating transcriptional responses to DNA damage.

Cyclin-dependent Kinase Inhibitors Uncouple Cell Cycle Progression from Mitochondrial Apoptotic Functions in DNA-damaged Cancer Cells

DNA damage results in transcriptional induction of p53 target genes, including the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) (CDKN1A) and the proapoptotic Bcl-2 family member p53 up-regulated modulator of apoptosis (PUMA). Depending on the cellular context, p21(Cip1) and PUMA mediate cell cycle arrest and apoptosis, respectively. By imposing cell cycle arrest at the expense of apoptosis, p21(Cip1) can sharply reduce the effectiveness of DNA-damaging anticancer agents in colorectal cancer cells. We investigated the link between cell cycle progression and the onset of apoptosis in DNA-damaged cells by analyzing the activation of the apoptotic cascade in p21(Cip1)-deficient HCT116 colorectal cancer cells. DNA damage induced a similar level of p53 activation and PUMA induction in p21(Cip1)-deficient cells compared with wild-type isogenic counterparts. p21(Cip1) did not act as a direct blocker of PUMA. However, only p21(Cip1)-deficient cells showed extensive cytochrome c release, mitochondrial membrane depolarization, and caspase activation. An increase in caspase activation occurred as these cells reached M-phase and incurred polyploidy. When ectopically expressed in p21(Cip1)-deficient HCT116 cells, p21(Cip1), its family member p27(Kip1), and the structurally unrelated CDK inhibitor p16(Ink4a) were similarly effective at causing cell cycle arrest and inhibiting DNA damage-induced apoptotic events such as cytochrome c release, mitochondrial membrane depolarization, and activation of the caspase cascade. These observations suggest that by blocking dysregulated cell cycle progression, CDK inhibitors can influence the sensitivity of the mitochondria to proapoptotic signals in DNA damage-induced cancer cells.

The histone deacetylase inhibitor FK228 increases the efficacy of adenovirus-mediated p53 family gene therapy in in Vitro and in Vivo human cancer models

3160 Background: p53 gene therapy is being tested clinically for the treatment of human cancer, however some cancer models (in vivo and in vitro) are resistant to p53. We have shown that p73/p63, the newly discovered p53 family members, is effective in some of colorectal cancer cells resistant to p53-mediated apoptosis. FK228 is a natural histone deacetylase inhibitors that is known to enhance adenovirus transgene expression. Before testing whether a combination of the adenovirus (Ad)-mediated transfer of the p53 family genes with FK228 could be developed in clinical trials, preclinical experiments were performed. Methods: The effect of FK228 on adenoviral transduction, coxsakie-adenovirus receptor (CAR) expression, and acetylation of exogenous p53 family proteins were investigated. Apoptosis was examined by flow cytometry, PARP cleavage, and TUNEL analyses. The Induction of p53-target genes in FK228-treated cells was measured using Western blot. The in vivo effects of FK228 on the anti-tumor activity of Ad-mediated transfer of the p53 family genes were determined by using human cancer xenograft models. Results: Cells treated with FK228 prior to infection had a 3–7-fold increase in transgene expression from a beta-galactosidase- or GFP-expressing Ad. The enhanced viral transduction was due in part to the up-regulation of the CAR protein. Pretreatment with FK228 enhanced apoptosis induced by p53 family through increased expression of exogenous proteins. Additionally, FK228 induced hyperacetylation of exogenous p53 or p63. The levels of certain p53-targets, including NOXA and PIG3, were significantly increased compared with treated with Ad alone. Together, the combination of FK228 and Ad-p53 or Ad-p63 induced Bax translocation to the mitochondria. In human cancer xenograft models, FK228 significantly increased the therapeutic effectiveness of p53 gene therapy. Conclusions: FK228 increased expression of exogenous p53 family gene transfer, apoptosis, and antitumor mechanisms. These results support a clinical combination of FK228 with p53 gene therapy in cancer patients. No significant financial relationships to disclose.

Inhibitory effect of c-Myc on p53-induced apoptosis in leukemia cells. Microarray analysis reveals defective induction of p53 target genes and upregulation of chaperone genes

We have previously demonstrated that c-Myc impairs p53-mediated apoptosis in K562 human leukemia cells, which lack ARF. To investigate the mechanisms by which c-Myc protects from p53-mediated apoptosis, we used K562 cells that conditionally express c-Myc and harbor a temperature-sensitive allele of p53. Gene expression profiles of cells expressing wild-type conformation p53 in the presence of either uninduced or induced c-Myc were analysed by cDNA microarrays. The results show that multiple p53 target genes are downregulated when c-Myc is present, including p21WAF1, MDM2, PERP, NOXA, GADD45, DDB2, PIR121 and p53R2. Also, a number of genes that are upregulated by c-Myc in cells expressing wild-type conformation p53 encode chaperones related to cell death protection as HSP105, HSP90 and HSP27. Both downregulation of p53 target genes and upregulation of chaperones could explain the inhibition of apoptosis observed in K562 cells with ectopic c-Myc. Myc-mediated impairment of p53 transactivation was not restricted to K562 cells, but it was reproduced in a panel of human cancer cell lines derived from different tissues. Our data suggest that elevated levels of Myc counteract p53 activity in human tumor cells that lack ARF. This mechanism could contribute to explain the c-Myc deregulation frequently found in cancer.

COX-2 regulates p53 activity and inhibits DNA damage-induced apoptosis

We have previously shown that p53 induces cyclooxygenase-2 (COX-2) expression and COX-2 inhibits p53- or genotoxic stress-induced apoptosis. However, the COX-2 effects have been demonstrated indirectly by the use of a selective inhibitor, NS-398, and the molecular mechanisms by which COX-2 inhibits apoptosis have not been identified. In the present study, we demonstrated that COX-2 inhibits genotoxic stress-induced apoptosis by using an adenoviral COX-2 overexpression system. In addition, we found that COX-2 regulates the transcription function of p53 as evidenced by suppression of p53 target gene induction by COX-2 cotransfection. Furthermore, COX-2 interacted with p53 in vitro and in vivo, which was inhibited by the treatment with NS-398. Taken together, these results suggest a novel function of COX-2 that inhibits DNA damage-induced apoptosis through direct regulation of p53 function.

YY1 inhibits the activation of the p53 tumor suppressor in response to genotoxic stress.

The tumor suppressor p53 regulates cell-cycle progression and apoptosis in response to genotoxic stress, and inactivation of p53 is a common feature of cancer cells. The levels and activity of p53 are tightly regulated by posttranslational modifications, including phosphorylation, ubiquitination, and acetylation. Here, we demonstrate that the transcription factor Yin Yang 1 (YY1) interacts with p53 and inhibits its transcriptional activity. We show that YY1 disrupts the interaction between p53 and the coactivator p300 and that expression of YY1 blocks p300-dependent acetylation and stabilization of p53. Furthermore, expression of YY1 inhibits the accumulation of p53 and the induction of p53 target genes in response to genotoxic stress. YY1 also interacts with Mdm2 and the expression of YY1 promotes the assembly of the p53-Mdm2 complex. Consequently, YY1 enhances Mdm2-mediated ubiquitination of p53. Inactivation of endogenous YY1 enhances the accumulation of p53 as well as the expression of p53 target genes in response to DNA damage, and it sensitizes cells to DNA damage-induced apoptosis. Hence, our results demonstrate that YY1 regulates the transcriptional activity, acetylation, ubiquitination, and stability of p53 by inhibiting its interaction with the coactivator p300 and by enhancing its interaction with the negative regulator Mdm2. YY1 may, therefore, be an important negative regulator of the p53 tumor suppressor in response to genotoxic stress.

Transfection of a dominant-negative mutant NF-kB inhibitor (IkBm) represses p53-dependent apoptosis in acute lymphoblastic leukemia cells: interaction of IkBm and p53.

To investigate the possible role of inhibiting NF-kB activation in sensitizing tumor cells to chemotherapy-induced apoptosis, we transfected the dominant-negative mutant inhibitor of NF-kB (IkBm) into the EU-1 cell line, an acute lymphoblastic leukemia (ALL) line with constitutive NF-kB activation. Overexpression of IkBm significantly reduced constitutive NF-kB activity in EU-1 cells, resulting in decreased cell growth. In response to apoptosis induced by chemotherapeutic drugs, IkBm-transfected cells (EU-1/IkBm) exhibited increased sensitivity to vincristine (VCR), whereas sensitivity to doxorubicin (Dox) was not changed as compared to neo-transfected control (EU-1/neo) cells. To further evaluate the link between IkBm and sensitivity to Dox and VCR, we demonstrated that both endogenous IkBalpha and ectopic IkBm bind to p53. In response to Dox, the cytosolic p53.IkBalpha complex rapidly dissociated due to downregulation of IkBalpha. However, the p53.IkBm complex did not dissociate under these conditions. Although treatment of EU-1/IkBm cells with Dox increased the expression of p53, the nondissociating p53.IkBm complex resulted in decreased p53 function, as demonstrated by absence of cell-cycle arrest and induction of p53 target genes. Contrastingly, VCR-induced cell death neither downregulated IkBalpha nor induced p53, as shown by the lack of NF-kB activation and p53-mediated gene expression in VCR-treated cells. Our data suggest that IkBm simultaneously downregulates NF-kB activation and sequesters p53 in the cytoplasm, thus enhancing NF-kB-regulated apoptosis but blocking p53-dependent apoptosis.

Impaired function of p53R2 in Rrm2b-null mice causes severe renal failure through attenuation of dNTP pools.

p53R2, which is regulated by tumor suppressor p53, is a small subunit of ribonucleotide reductase. To determine whether it is involved in DNA repair by supplying deoxyribonucleotides (dNTPs) for resting cells in vivo, we generated a strain of mice lacking Rrm2b (encoding p53R2). These mice developed normally until they were weaned but from then on had growth retardation and early mortality. Pathological examination indicated that multiple organs had failed, and all Rrm2b-null mice died from severe renal failure by the age of 14 weeks. TUNEL staining showed a greater number of apoptotic cells in kidneys of 8-week-old Rrm2b-/- mice relative to wild-type mice. p53 was activated in kidney tissues of Rrm2b-/- mice, leading to transcriptional induction of p53 target genes. Rrm2b-/- mouse embryonic fibroblasts (MEFs) became immortal much earlier than Rrm2b+/+ MEFs. dNTP pools were severely attenuated in Rrm2b-/- MEFs under oxidative stress. Rrm2b deficiency caused higher rates of spontaneous mutation in the kidneys of Rrm2b-/- mice. Our results suggest that p53R2 has a pivotal role in maintaining dNTP levels for repair of DNA in resting cells. Impairment of this pathway may enhance spontaneous mutation frequency and activate p53-dependent apoptotic pathway(s) in vivo, causing severe renal failure, growth retardation and early mortality.

Induction and bypass of p53 during productive infection by polyomavirus.

Lytic infection by polyomavirus leads to elevated levels of p53 and induction of p53 target genes p21Cip1/WAF1 (p21) and BAX. This is seen both in polyomavirus-infected primary mouse cell cultures and in kidney tissue of infected mice. Stabilization of p53 and induction of a p53 response are accompanied by phosphorylation of p53 on serine 18, mimicking a DNA damage response. Stabilization of p53 does not depend on p19Arf interaction with mdm2. Cells infected by a mutant virus defective in binding pRb and in inducing G(1)-to-S progression show a greatly diminished p53 response. However, cells infected by wild-type virus and blocked from entering S phase by addition of mimosine still show a p53 response. These results suggest a role of E2F target genes in inducing a p53 response. Polyomavirus large T antigen coprecipitates with p53 phosphorylated on serine 18 and also with p21Cip1/WAF1. Implications of these and other findings on possible mechanisms of induction and override of p53 functions during productive infection by polyomavirus are discussed.