P53-induced Cell Cycle Arrest Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

The Multifunctional Sorting Protein PACS-2 Regulates SIRT1-Mediated Deacetylation of p53 to Modulate p21-Dependent Cell-Cycle Arrest

SIRT1 regulates the DNA damage response by deacetylating p53, thereby repressing p53 transcriptional output. Here, we demonstrate that the sorting protein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate the DNA damage response. PACS-2 knockdown cells failed to efficiently undergo p53-induced cell-cycle arrest in response to DNA damage. Accordingly, p53 acetylation was reduced both in PACS-2 knockdown cells and thymocytes from Pacs-2(-/-) mice, thereby blunting induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A). The SIRT1 inhibitor EX-527 or SIRT1 knockdown restored p53 acetylation and p21 induction as well as p21-dependent cell-cycle arrest in PACS-2 knockdown cells. Trafficking studies revealed that cytoplasmic PACS-2 shuttled to the nucleus, where it interacted with SIRT1 and repressed SIRT1-mediated p53 deacetylation. Correspondingly, in vitro assays demonstrated that PACS-2 directly inhibited SIRT1-catalyzed p53 deacetylation. Together, these findings identify PACS-2 as an in vivo mediator of the SIRT1-p53-p21 axis that modulates the DNA damage response.

SPARC silencing inhibits the growth of acute myeloid leukemia transformed from myelodysplastic syndrome via induction of cell cycle arrest and apoptosis

Secreted protein acidic and rich in cysteine (SPARC) plays key roles in erythropoiesis; haploinsufficiency of SPARC is implicated in the progression of the 5q- syndrome. However, the role of SPARC in other subtypes of myelodysplastic syndrome (MDS) is not fully understood, particularly in the del(5q) type with a complex karyotype, which has a high risk to transform into acute myeloid leukemia (AML). In the present study, we investigated the role of SPARC in the proliferation and apoptosis of SKM-1 cells, an acute myeloid leukemia cell line transformed from an MDS cell line. SKM-1 cells were infected with SPARC-RNAi-LV or NC-GFP-LV lentivirus. Apoptosis and cell cycle profiling were assessed by flow cytometry, and cell proliferation was evaluated by MTS assay. The mRNA and protein expression levels of SPARC, p53, caspase-3, caspase-9 and Fas were detected by RT-PCR, real-time PCR and western blot assay. The SPARC shRNA constructed by us led to a significant reduction in SPARC expression in SKM-1 cells. SPARC knockdown inhibited the proliferation of SKM-1 cells by inducing cell cycle arrest at the G1/G0 phase and apoptosis. SPARC knockdown elevated the expression of p53, caspase-9, caspase-3 and Fas at both the mRNA and protein levels. SPARC silencing inhibited the growth of AML transformed from MDS by activating p53-induced apoptosis and cell cycle arrest. These data indicate that SPARC acts as an oncogene in transformed MDS/AML and is a potential therapeutic target in MDS/AML.

Novel simplified yeast-based assays of regulators of p53-MDMX interaction and p53 transcriptional activity

Yeast has proven to be an efficient model system for functional and pharmacological studies of the p53 tumour suppressor protein. In this work, the human p53-MDMX regulatory pathway was reconstituted in yeast. Additionally, by using the known inhibitor of p53-MDMX interaction, SJ-172550, the efficacy of a simplified yeast-based screening assay to search for inhibitors of p53-MDMX interaction is demonstrated for the first time. Moreover, further insights on p53 transcriptional activity in yeast are provided. In particular, it is shown that the reported wild-type (wt) p53-induced yeast growth inhibition and cell cycle arrest is associated with actin depolarization and with an increase of actin mRNA and protein expression levels. The increase of actin protein levels was not observed with the p53 R273H mutant (a loss of function p53 mutation hotspot) and was further intensified with the toxic p53 V122A mutant (reported to exhibit higher transcriptional activity than wt p53 for selected p53 target sequences). Moreover, it is shown that the wt p53-induced actin protein levels are modulated by natural (MDM2 and MDMX) and chemical (pifithrin-α, nutlin-3a and SJ-172550) regulators of p53 activity. Furthermore, wt p53 could stimulate transcription from a minimal promoter containing a fragment of the ACT1 upstream sequence. Thus, ACT1 is proposed as a putative endogenous p53 target gene. This finding may open the way for the development of simpler yeast p53 transactivation assays, not based on artificial reporter constructs, for the analysis of the impact of mutants, cofactors and small molecules on p53 transcriptional activity.

Abstract 18726: Single Cell Expression Profiling Reveals Up-Regulation of Radiation Sensitive Genes After Cardiac Computed Tomographic Angiography

Introduction: High dose radiation activates radiation sensitive genes involved in the DNA damage response pathway, which is responsible for tumor suppression. Whether low dose radiation from medical imaging tests, such as cardiac computed tomographic angiography (CTA), changes the expression of these genes is unknown. Hypothesis: We hypothesize that low dose radiation alters the gene expression of 3 genes involved in tumor suppression: 1) MDM2 (a negative regulator of p53-induced cell cycle arrest and apoptosis), 2) BAX (a mediator of p53-induced apoptosis), and 3) DDB2 (a mediator of DNA repair). Methods: To determine the optimal time point for collection of blood in vivo, we performed an in vitro study. Blood from healthy volunteers (n=4) received either irradiation with a single dose of 25 mGy or 5,000 mGy, or received sham-irradiation. Whole blood was maintained at 37° for 6, 12, 24, and 38 hours following irradiation. Blood was collected from adult patients immediately before and 24 hours after CTA (n=10) or echocardiography (n=5). After separation of lymphocytes, gene expression was analyzed by single cell polymerase chain reaction. Results: The in vitro study showed significant up-regulation of MDM2, BAX, and DDB2 after low and high dose irradiation compared with sham-irradiated controls (Figure 1A, B, C). Similarly, up-regulation of these genes was noted in patients undergoing CTA (3-15 mSv) for various indications but not in patients undergoing echocardiography (Figure 1D). Of note, only 50% of patients had changes in gene expression. Conclusions: Low dose radiation from CTA alters the expression of genes involved in regulating tumor suppression. These findings, however, are seen in only 50% of patients. Further investigation is needed to determine how to identify these “radiosensitive” patients.

Intact p53-Dependent Responses in miR-34–Deficient Mice

MicroRNAs belonging to the miR-34 family have been proposed as critical modulators of the p53 pathway and potential tumor suppressors in human cancers. To formally test these hypotheses, we have generated mice carrying targeted deletion of all three members of this microRNA family. We show that complete inactivation of miR-34 function is compatible with normal development in mice. Surprisingly, p53 function appears to be intact in miR-34–deficient cells and tissues. Although loss of miR-34 expression leads to a slight increase in cellular proliferation in vitro, it does not impair p53-induced cell cycle arrest or apoptosis. Furthermore, in contrast to p53-deficient mice, miR-34–deficient animals do not display increased susceptibility to spontaneous, irradiation-induced, or c-Myc–initiated tumorigenesis. We also show that expression of members of the miR-34 family is particularly high in the testes, lungs, and brains of mice and that it is largely p53-independent in these tissues. These findings indicate that miR-34 plays a redundant function in the p53 pathway and suggest additional p53-independent functions for this family of miRNAs.

Viral oncoprotein LMP1 disrupts p53-induced cell cycle arrest and apoptosis through modulating K63-linked ubiquitination of p53

Disruption of the gatekeeper p53 tumor suppressor is involved in various virus-associated tumorigeneses, with aberrant ubiquitination as the major cause of p53 abnormalities in virus-associated tumors. Of note, wild-type p53 is accumulated in Epstein-Barr virus (EBV)-associated tumors, especially in nasopharyngeal carcinoma (NPC). We have previously identified that p53 is accumulated and phosphorylated by EBV oncoprotein latent membrane protein 1 (LMP1) in NPC. Here, we further found that LMP1 promoted p53 accumulation via two distinct ubiquitin modifications. LMP1 promoted p53 stability and accumulation by suppressing K48-linked ubiquitination of p53 mediated by E3 ligase MDM2, which is associated with its phosphorylation at Ser20, while increasing the levels of total cellular ubiquitinated p53. LMP1 also induced K63-linked ubiquitination of p53 by interacting with tumor necrosis factor receptor-associated factor 2 (TRAF2), thus contributing to p53 accumulation. Furthermore, LMP1 rescued tumor cell apoptosis and cell cycle arrest mediated by K63-linked ubiquitination of p53. Collectively, these results demonstrate aberrant ubiquitin modifications of p53 and its biological functions by viral protein LMP1, which has broad implications to the pathogenesis of multiple EBV-associated tumors.

Reprimo (RPRM) Is a Novel Tumor Suppressor in Pituitary Tumors and Regulates Survival, Proliferation, and Tumorigenicity

Reprimo (RPRM), initially identified as a downstream effector of p53-induced cell cycle arrest at G(2)/M, is a putative tumor suppressor silenced in some types of cancer. In microarrays, the RPRM transcript was repressed 26-fold in gonadotrope (null cell) human pituitary tumors compared with normal pituitary but in the absence of changes in p53. Inhibition of RPRM mRNA was confirmed by RT-PCR in all gonadotrope tumors, most GH samples, and variably in other tumor types. Human pituitary tumors showed no evidence of abnormal promoter hypermethylation as a mechanism of RPRM repression. RPRM stable expression in gonadotrope (LβT2) and GH (GH3) pituitary cells resulted in decreased rates of cell proliferation by 55 and 30%, respectively; however, RPRM reexpression did not alter G(2)/M transition. In addition, RPRM increased rates of apoptosis in response to growth factor deprivation as assessed by caspase-3 cleavage and nuclear condensation. Clonagenic assays showed a 5.3- and 3.7-fold suppression of colony growth in RPRM-overexpressing LβT2 and GH3 cells, respectively, supporting its role as a tumor suppressor. In cells stably expressing RPRM mRNA, protein levels were actively suppressed due to rapid degradation through ubiquitination and proteasomal targeting. Growth factor withdrawal, as a model of cellular stress, stabilized RPRM protein levels. Together these data suggest that RPRM is transiently up-regulated at a posttranscriptional level in times of cellular stress to restrict cell survival, proliferation, and tumor formation. When RPRM is silenced as in human pituitary tumors, unrestrained growth and tumor progression may occur.

Wig-1, a novel regulator of N-Myc mRNA and N-Myc-driven tumor growth

Wig-1 is a transcriptional target of the p53 tumor suppressor and encodes an mRNA stability-regulating protein. We show here that Wig-1 knockdown causes a dramatic inhibition of N-Myc expression and triggers differentiation in neuroblastoma cells carrying amplified N-Myc. Transient Wig-1 knockdown significantly delays development of N-Myc-driven tumors in mice. We also show that N-Myc expression is induced upon moderate p53-activating stress, suggesting a role of the p53-Wig-1-N-Myc axis in promoting cell cycle re-entry upon p53-induced cell cycle arrest and DNA repair. Moreover, our findings raise possibilities for the improved treatment of poor prognosis neuroblastomas that carry amplified N-Myc.

Inhibition of tumor suppressor protein p53-dependent transcription by a tetramerization domain peptide via hetero-oligomerization

Tumor suppressor protein p53 induces cell cycle arrest, apoptosis, and senescence in response to cellular stresses. The p53 tetramer formation is essential for its functions. Despite of these crucial functions of p53 for integrity of genome, activation of the p53 signal pathway causes low induced pluripotent stem (iPS) cell generation efficiency. In this study, we report transient inhibition of p53-dependent transcription using a p53 tetramerization domain peptide that contains cell penetrating and nuclear localization signals. The peptide was efficiently introduced into cells and inhibited p21 expression via hetero-tetramerization with endogenous p53 protein. This method can be applied towards safe and efficient iPS cell generation.

B-MYB Positively Regulates Serine-Threonine Kinase Receptor-associated Protein (STRAP) Activity through Direct Interaction

Serine-threonine kinase receptor-associated protein (STRAP) functions as a regulator of both TGF-β and p53 signaling. However, the regulatory mechanism of STRAP activity is not understood. In this study, we report that B-MYB is a new STRAP-interacting protein, and that an amino-terminal DNA-binding domain and an area (amino acids 373-468) between the acidic and conserved regions of B-MYB mediate the B-MYB·STRAP interaction. Functionally, B-MYB enhances STRAP-mediated inhibition of TGF-β signaling pathways, such as apoptosis and growth inhibition, by modulating complex formation between the TGF-β receptor and SMAD3 or SMAD7. Furthermore, coexpression of B-MYB results in a dose-dependent increase in STRAP-mediated stimulation of p53-induced apoptosis and cell cycle arrest via direct interaction. Confocal microscopy showed that B-MYB prevents the normal translocation of SMAD3 in response to TGF-β1 and stimulates p53 nuclear translocation. These results suggest that B-MYB acts as a positive regulator of STRAP.

Decision making of the p53 network: Death by integration

The tumor suppressor protein p53 plays a central role in the multiple response pathways activated by DNA damage. In particular, p53 is involved in both the pro-survival response of cell cycle arrest and DNA repair, and the pro-death response of apoptosis. How does the p53 network coordinate the different pathways that lead to the opposite cell fates and what is its strategy in making the life-death decisions? To address these questions, we develop an integrated mathematical model that embraces three key modules of the p53 network: p53 core regulation, p53-induced cell cycle arrest and p53-dependent apoptosis initiation. Our analyses reveal that different aspects of the nuclear p53 dynamic profile are being used to differentially regulate the pro-survival and the pro-death modules. While the activation of the pro-survival module is dependent on the current or recent status of the DNA damage, the activation of the pro-death module relies on the accumulation or integration of the damage level over time. Thus, the cell will take the death fate if it cannot recover from the damage within a time period that is inversely proportional to the damage level. This “adaptive timer” strategy is likely to be adopted in other stress response systems.

Deletion of Puma and p21Waf1 In Mice Deactivates p53-Induced Cell Death and Cell Cycle Arrest, but Protects Mice From Irradiation-Induced Lymphomagenesis by a Mechanism Involving Hemopoietic Stem Cell Quiescence

AbstractAbstract 90 Introduction:The p53 gene is non-functional in >50% of human tumors. In mice deletion of p53 leads to a high incidence of tumors and to a significant acceleration of tumorigenesis induced by repeated gamma-irradiation. While a large number of effects have been described for p53, current concepts of p53-mediated tumor suppression discuss the roles of p53 in regulation of cell cycle and apoptosis as being essential. Two main targets have been identified in this respect: p21Waf1 as an essential regulator of cell cycle arrest downstream of p53 and Puma as the largest single contribution towards p53 induced cell death. Methods:We have generated p21Waf1/Puma doubly deficient (i.e. double-knockout – DKO) mice on a pure C57BL/6 background to investigate the effects on tumorigenesis. Results:In ex vivo irradiation studies DKO thymocytes expectedly showed reduced cell death and loss of a G1/S arrest upon irradiation. When following a cohort of mice for spontaneous tumor development, the DKO mice did not differ from wild-type (WT) controls. Since this may be explained by additional p53 down-stream effectors essential for tumor suppression, we set out to challenge the mice with an established repeated irradiation protocol (4 × 1.75 Gy over 4 weeks) in order to increase the likelihood of uncovering a defect in tumor suppression not apparent in unchallenged mice. While irradiated WT mice developed thymic lymphomas at an expected rate and p53 deficiency accelerated the lymphoma formation as published, irradiated DKO mice did not develop any thymic lymphoma at all. During the irradiation protocol WT mice followed a series of depletion and regrowth cycles in thymic cellularity with a high rate of cell death early post irradiations in TUNEL assays and a surge of proliferation on day 5 after irradiations detected by in vivo BrdU labeling. By contrast in DKO mice thymic cellularity dropped only slightly during the first irradiation cycle. This was followed by a slow and steady decline in cellularity over the following 3 cycles of irradiation. No late apoptotic wave or loss of proliferative capacity of remaining thymocytes could explain the loss of cellularity, nor could senescence of thymocytes be detected by SA-β-Gal staining in situ, suggesting that thymic influx was defective. It had previously been reported for the repeat-irradiation lymphomagenesis model, that the irradiation of hemopoietic precursor cells was essential for tumorigenesis. In contrast to thymic cellularity, DKO LSK numbers stayed relatively stable over the course of the 4 irradiations. By comparison WT LSK numbers dropped to about 50% by the time 4 irradiations were completed. Indeed, short-term repopulating (ST) cells dropped significantly, while long-term repopulating (LT) and multipotent progenitor (MPP) cell populations stayed more stable. In DKO marrows the relative content of LT, ST and MPP cells proved very stable across the irradiation schedule. In vivo BrdU labelling showed that WT LSK had a higher fraction of labelled cells at baseline and a >100% increase in the proliferative fraction during irradiation, while in DKO LSK the proliferation index was lower and stayed stably low over time, compatible with the replenishment defect observed in the thymus. DKO stem cells were only slightly more efficient (1.6-fold) than WT in bone marrow reconstitution experiments without challenge. However, when mixed chimeras were then subjected to the irradiation protocol with 4 × 1.75 Gy a clear advantage of the DKO cells became apparent (28-fold). Moreover, when reconstituting lethally irradiated mice with a mixture of WT and DKO marrow taken from repeatedly irradiated donors the efficacy ratio was 1:152. Conclusion:Our data contrast observations made in cell lines, where loss of Puma and p21Waf1 led to a p53-resistant outgrowth of cells. We present in an animal model that loss of Puma and p21Waf1 is not tumorigenic and in fact protects mice from irradiation carcinogenesis. Together with our recently published findings in irradiated Puma singly-deficient mice (Labi G&D 2010), our data suggest that tumorigenesis in irradiated DKO mice is inhibited by effects on hemopoietic stem cell reactivity to DNA damage. A combination of lack of generation of free niche space through protection of hemopoietic stem cells from cell death and a stem cell quiescence state retained in DKO stem cells after irradiation seems responsible for the phenotype. Disclosures:No relevant conflicts of interest to declare.

Paradoxical suppression of cellular senescence by p53

The tumor suppressor p53 is a canonical inducer of cellular senescence (irreversible loss of proliferative potential and senescent morphology). p53 can also cause reversible arrest without senescent morphology, which has usually been interpreted as failure of p53 to induce senescence. Here we demonstrate that p53-induced quiescence actually results from suppression of senescence by p53. In previous studies, suppression of senescence by p53 was masked by p53-induced cell cycle arrest. Here, we separated these two activities by inducing senescence through overexpression of p21 and then testing the effect of p53 on senescence. We found that in p21-arrested cells, p53 converted senescence into quiescence. Suppression of senescence by p53 required its transactivation function. Like rapamycin, which is known to suppress senescence, p53 inhibited the mTOR pathway. We suggest that, while inducing cell cycle arrest, p53 may simultaneously suppress the senescence program, thus causing quiescence and that suppression of senescence and induction of cell cycle arrest are distinct functions of p53. Thus, in spite of its ability to induce cell cycle arrest, p53 can act as a suppressor of cellular senescence.

P53 regulates the transcription of its Δ133p53 isoform through specific response elements contained within the TP53 P2 internal promoter

The tumor suppressor p53 protein is activated by genotoxic stress and regulates genes involved in senescence, apoptosis and cell-cycle arrest. Nine p53 isoforms have been described that may modulate suppressive functions of the canonical p53 protein. Among them, Delta133p53 lacks the 132 proximal residues and has been shown to modulate p53-induced apoptosis and cell-cycle arrest. Delta133p53 is expressed from a specific mRNA, p53I4, driven by an alternative promoter P2 located between intron 1 and exon 5 of TP53 gene. Here, we report that the P2 promoter is regulated in a p53-dependent manner. Delta133p53 expression is increased in response to DNA damage by doxorubicin in p53 wild-type cell lines, but not in p53-mutated cells. Chromatin immunoprecipitation and luciferase assays using P2 promoter deletion constructs indicate that p53 binds functional response elements located within the P2 promoter. We also show that Delta133p53 does not bind specifically to p53 consensus DNA sequence in vitro, but competes with wild-type p53 in specific DNA-binding assays. Finally, we report that Delta133p53 counteracts p53-dependent growth suppression in clonogenic assays. These observations indicate that Delta133p53 is a novel target of p53 that may participate in a negative feedback loop controlling p53 function.

Further Preclinical Studies with APR-246, a Novel Anticancer Compound Currently in Clinical Trials for Hematological Malignancies and Prostate Cancer.

Abstract 3773Poster Board III-709 IntroductionThe tumor suppressor protein p53 induces cell cycle arrest and/or apoptosis in response to various forms of cellular stress, through transcriptional regulation of a large number of down stream target genes. p53 is frequently mutated in cancer, and cancer cells carrying defects in the p53 protein are often more resistant to conventional chemotherapy. Thus, restoration of the wild type function to mutant p53 appears to be a new attractive strategy for cancer therapy. APR-246 is a novel small molecule quinuclidinone compound that has been shown to reactivate non-functional p53 and induce apoptosis. Although the exact molecular mechanism remains to be determined, recent results suggest that an active metabolite of APR-246 alkylates thiol groups in the core domain of p53, which promotes correct folding of p53 and induces apoptosis (Lambert et al., Cancer Cell 15, 2009). Currently, APR-246 is in Phase I/IIa clinical trials for hematological malignancies and prostate cancer. In the present abstract results from in vitro, ex vivo and in vivo preclinical studies with APR-246 are presented. ResultsThe lead compound of APR-246, PRIMA-1 (p53 reactivation and induction of massive apoptosis), was originally identified by a cellular screening of the NCI library for low molecular weight compounds (Bykov et al., Nat. Med., 8, 2002). Further development and optimization of PRIMA-1 led to the discovery of the structural analog APR-246 (PRIMA-1MET), with improved drug like and preclinical characteristics. In in vitro experiments APR-246 reduced cell viability (WST-1 assay) in a large number of human cancer cell lines with various p53 status, including several leukemia (CCRF-CEM, CEM/VM-1, KBM3), lymphoma (U-937 GTP, U-937-vcr), and myeloma (RPMI 8226/S, 8226/dox40, 8226/LR5) cell lines, as well as many solid cancer cell lines, including osteosarcoma (SaOS-2, SaOS-2-His273,U-2OS), prostate (PC3, PC3-His175, 22Rv1), breast (BT474, MCF-7, MDA-MB-231), lung (H1299, H1299-His175) and colon cancer (HT-29). In human osteosarcoma cell lines APR-246 reduced cell viability and induced apoptosis (FLICA caspase assay) in a concentration dependent manner being more potent in the p53 mutant (SaOS-2-His273) than in the parental p53 null (SaOS-2) cells. The IC50 values (WST-1 assay) were 14 ± 3 and 27 ± 5 μM, respectively (n=35). In in vivo subcutaneous xenograft studies in SCID (severe combined immunodeficiency) mice APR-246 reduced growth of p53 mutant SaOS-2-His273 cells in a dose-dependent manner, when injected i.v. twice daily with 20 -100 mg/kg (64 – 76% inhibition). An in vivo anticancer effect of APR-246 was also observed in hollow-fiber test with NMRI mice using the acute myeloid leukemia (AML) cell line MV-4-11. An ex vivo cytotoxic effect of APR-246 and/or its lead compound PRIMA-1 has also been shown in primary cells from AML and CLL (chronic lymphocytic leukemia) patients, harbouring both hemizygously deleted p53 as well as normal karyotype (Nahi et al., Br. J. Haematol., 127, 2004; Nahi et al., Br. J. Haematol., 132, 2005; Jonsson-Videsater et al., abstract at this meeting). APR-246 was also tested in a FMCA (fluorometric microculture assay) test using normal healthy lymphocytes (PBMC) and cancer lymphocytes (CLL). It was 4-8 fold more potent in killing cancer cells than normal cells, indicating a favorable therapeutic index. This is in contrast to conventional cytostatics that often show negative ratio in this test. Furthermore, when tested in a well-defined panel of 10 human cancer cell lines consisting of both hematological and solid cancer cell lines, the cytotoxicity profile/activity pattern of APR-246 differed from common chemotherapeutic drugs (correlation coefficient less than 0.4), suggesting a different mechanism of action. ConclusionIn relevant in vitro, in vivo and ex vivo cancer models, APR-246 showed unique pharmacological properties in comparison with conventional cytostatics, by being effective also in cancer cells with p53 mutations and by demonstrating tumor specificity. Moreover, in experimental safety/toxicology models required to start clinical trials, APR-246 was non toxic at the predicted therapeutic plasma concentrations. Thus, APR-246 appears to be a promising novel anticancer compound that may specifically target cancer cells in patients with genetic abnormality associated with poor prognosis. Disclosures:Mohell:Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Wiman:Aprea AB: Co-founder, shareholder, and member of the board. Uhlin:Aprea AB: Employment.

Differential regulation of p53 function by protein kinase C isoforms revealed by a yeast cell system

The complexity of the mammalian p53 pathway and protein kinase C (PKC) family has hampered the discrimination of the effect of PKC isoforms on p53 activity. Using yeasts co-expressing the human wild-type p53 and a mammalian PKC-α, -δ, -ε or -ζ, we showed a differential regulation of p53 activity and phosphorylation state by PKC isoforms. Whereas PKC-α reduced the p53-induced yeast growth inhibition and cell cycle arrest, PKC-δ and -ε enhanced the p53 activity through p53 phosphorylation, and PKC-ζ had no effect on p53. This work identified positive and negative p53 regulators which represent promising pharmacological targets in anti-cancer therapy.

Insight into the Structural Basis of Pro- and Antiapoptotic p53 Modulation by ASPP Proteins

p53-dependent apoptosis is modulated by the ASPP family of proteins (apoptosis-stimulating proteins of p53; also called ankyrin repeat-, Src homology 3 domain-, and Pro-rich region-containing proteins). Its three known members, ASPP1, ASPP2, and iASPP, were previously found to interact with p53, influencing the apoptotic response of cells without affecting p53-induced cell cycle arrest. More specifically, the bona fide tumor suppressors, ASPP1 and ASPP2, bind to the core domain of p53 and stimulate transcription of apoptotic genes, whereas oncogenic iASPP also binds to the p53 core domain but inhibits p53-dependent apoptosis. Although the general interaction regions are known, details of the interfaces for each p53-ASPP complex have not been evaluated. We undertook a comprehensive biophysical characterization of ASPP-p53 complex formation and mapped the binding interfaces by NMR. We found that the interaction interface on p53 for the proapoptotic protein ASPP2 is distinct from that for the antiapoptotic iASPP. ASPP2 primarily binds to the core domain of p53, whereas iASPP predominantly interacts with a linker region adjacent to the core domain. Our detailed structural analyses of the ASPP-p53 interactions provide insight into the structural basis of the differential behavior of pro- and antiapoptotic ASPP family members.

Enhanced Therapeutic Efficacy by Simultaneously Targeting Two Genetic Defects in Tumors

Targeting tumor-specific gene abnormalities has become an attractive approach in developing therapeutics to treat cancer. Overexpression of Bcl2 and mutations of p53 represent two of the most common molecular defects in tumors. In the nucleus, p53 induces cell cycle arrest, while it interacts with Bcl2 outside of the nucleus to regulate signal pathways involved in apoptosis. To potentiate antitumor activity, we tested a "double target" approach to antitumor therapy by combining H101, a recombinant oncolytic adenovirus that targets the inactive p53 in tumors, with a small interfering RNA (siBCL2) that targets Bcl2. In cell culture, the combined treatment significantly enhanced apoptosis and cytotoxicity as compared with treatment with either H101 or siBCL2 alone. In animals carrying tumor xenographs, combined H101 and siBCL2 treatment significantly inhibited tumor growth and prolonged survival. At the end of the study, all animals in the combined therapy group survived and two of the five animals showed complete eradication of their tumors. Interestingly, siBCL2 treatment increased H101 viral replication in both treated cells and tumor tissues. Simultaneously targeting two tumor-specific gene abnormalities using an oncolytic adenovirus and siRNA potentiates total antitumor activity.

Direct Interaction between NM23-H1 and Macrophage Migration Inhibitory Factor (MIF) Is Critical for Alleviation of MIF-mediated Suppression of p53 Activity

Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that is involved in host immune and inflammatory responses, as well as tumorigenesis. However, the regulatory mechanism of MIF function is unclear. Here we report that the NM23-H1 interacts with MIF in cells, as demonstrated by cotransfection and coimmunoprecipitation experiments. Analysis of cysteine (Cys) to serine (Ser) substitution mutants of NM23-H1 (C4S, C109S, and C145S) and MIF (C57S, C60S, and C81S) revealed that Cys(145) of NM23-H1 and Cys(60) of MIF are responsible for complex formation. NM23-H1-MIF complexes were dependent on reducing conditions, such as the presence of dithiothreitol or beta-mercaptoethanol, but not H(2)O(2). NM23-H1 alleviated the MIF-mediated suppression of p53-induced apoptosis and cell cycle arrest by promoting the dissociation of MIF from MIF-p53 complexes. In addition, NM23-H1 significantly inhibited the MIF-induced proliferation of quiescent NIH 3T3 cells through a direct interaction with MIF, and decreased the MIF-induced activation of phosphatidylinositol 3-kinase/PDK1 and p44/p42 extracellular signal-regulated (ERK) mitogen-activated protein kinase. The results of the current study suggest that the NM23-H1 functions as a negative regulator of MIF.