Ruolo della Interleuchina-6 nella colite ulcerosa long-standing: regolazione della p53 ed induzione delle cellule mesenchimali epiteliali

L’infiammazione cronica è un fattore di rischio di insorgenza del cancro, e la citochina infiammatoria IL-6 gioca un ruolo importante nella tumorigenesi. In questo studio abbiamo dimostrato che L’IL-6 down-regola l'espressione e l'attività di p53. In linee cellulari umane, IL-6 stimola la trascrizione dell’rRNA mediante espressione della proteina c-myc a livello post-trascrizionale in un meccanismo p38MAPK-dipendente. L'up-regolazione della biogenesi ribosomiale riduce l'espressione di p53 attraverso l'attivazione della via della proteina ribosomale-MDM2. La down-regolazione di p53 produce l’acquisizione di modifiche fenotipiche e funzionali caratteristiche della epitelio mesenchimale di transizione, un processo associato a trasformazione maligna e progressione tumorale. I nostri dati mostrano che questi cambiamenti avvengono anche nelle cellule epiteliali del colon di pazienti affetti da colite ulcerosa, un esempio rappresentativo di una infiammazione cronica soggetta a trasformazione neoplastica, che scompaiono dopo trattamento con farmaci antinfiammatori. Questi risultati svelano un nuovo effetto oncogenico indotto dall’IL-6 che può contribuire notevolmente ad aumentare il rischio di sviluppare il cancro non solo in pazienti con infiammazioni croniche, ma anche in quei pazienti con condizioni patologiche caratterizzate da elevato livello di IL-6 nel plasma, quali l'obesità e e il diabete mellito di tipo 2.

Die Rolle von Ephrin-B2 in der Invasion maligner Gliome

Das Glioblastoma multiforme zählt zu den häufigsten glialen Neoplasien des Menschen und weist zudem unter den Gliomen die höchste Malignität auf. Glioblastompatienten haben trotz aggressiver therapeutischer Ansätze eine mittlere Überlebenszeit von weniger als einem Jahr. Die diffuse Invasion in das umliegende Hirngewebe ist einer der Hauptgründe für die Rezidivbildung und die infauste Prognose von Glioblastompatienten. Neuere Untersuchungen lassen vermuten, dass die starke Invasion auch einer der Gründe für die beobachtete anti-angiogene Resistenz bei der Behandlung von Glioblastomen ist. Das bidirektionale EphB/Ephrin-B-System wurde bei der axonalen Wegfindung als Vermittler repulsiver Signale identifiziert und auch im Zusammenhang der Migration und Invasion von Zellen überprüft. In der vorliegenden Arbeit sollte daher die Funktion der bidirektionalen Eph- und Ephrin-Signaltransduktion in Bezug auf die Glioblastominvasion und Progression untersucht werden. rn Genetische und epigenetische Untersuchungen der EphB/Ephrin-B-Familie in einer Kohorte von Gliompatienten unterschiedlicher Malignitätsgrade identifizierten Ephrin-B2 als mögliches Tumorsuppressorgen. In Übereinstimmung damit führte die Inaktivierung von Ephrin-B2 in einem murinen Gliommodell zu einer verstärkten Invasion und einem erhöhtem Tumorwachstum in vivo. Dies konnte in verschiedenen Invasion-Assays in vitro bestätigt werden. Weiterhin zeigten unsere Untersuchungen, dass Ephrin-B2 transkriptionell durch das hypoxische Mikromilieu HIF-1α-vermittelt reprimiert wird. Da HIF-1α als transkriptioneller Aktivator Ephrin-B2 nicht direkt reprimieren kann, wurden potentielle HIF-1α-regulierte Repressoren untersucht, die für die Ephrin-B2 Herunterregulation verantwortlich sein könnten. Dabei wurde anhand von Ephrin-B2-Promotoranalysen und ChIP-Assays ZEB2 als HIF-1α-induzierbarer Repressor von Ephrin-B2 identifiziert. Zur Bestätigung der Hypothese, dass ZEB2 ein wichtiger Regulator der Tumorinvasion ist, wurden humane ZEB2-Knockdown-Glioblastomzellen generiert und in vitro sowie in vivo untersucht. Im Hinblick auf mögliche therapeutische Anwendungen wurden die ZEB2-Knockdown-Glioblastomzellen zusätzlich im Zusammenhang anti-Angiogenese-induzierter Invasion analysiert. Der Verlust von ZEB2 führte dabei zu einer verringerten Glioblastominvasion und Progression in einem Maus-Xenograft Modell. Die Behandlung der Tumoren mit dem anti-VEGF-Antikörper Avastin resultierte in einer stark erhöhten Invasion, die durch die Inaktivierung von ZEB2 und der dadurch reaktivierten repulsiven Signale von Ephrin-B2 wieder aufgehoben werden konnte. Zusammenfassend konnte in der vorliegenden Arbeit erstmals gezeigt werden, dass Ephrin-B2 als Tumorsuppressor in Gliomen agiert und durch verschiedene Mechanismen wie der genetischen und epigenetischen Kontrolle, aber auch der HIF-1α-vermittelten, ZEB2-abhängigen Repression inaktiviert wird. Dies resultiert in einer Blockade repulsiver Signale, so dass Tumorzellen diffus in das Parenchym und zu den Blutgefäßen migrieren können. Der in dieser Arbeit neu identifizierte Signalweg stellt ein attraktives therapeutisches Ziel zur Inhibition der Tumorzellinvasion dar und ermöglicht darüber hinaus der Ausbildung von Resistenzen gegenüber anti-angiogener Behandlung entgegenzuwirken. rn

Chemopreventive effects of eicosapentaenoic acid free fatty acid in a murine model of colitis-associated colorectal cancer

Inflammatory bowel diseases are associated with increased risk of developing colitis-associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid-free fatty acid (EPA-FFA) reduces polyps formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA-FFA are unknown in CAC. We tested the effectiveness of substituting EPA-FFA, for other dietary fats, in preventing inflammation and cancer in the AOM-DSS model of CAC. The AOM-DSS protocols were designed to evaluate the effect of EPA-FFA on both initiation and promotion of carcinogenesis. We found that EPA-FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA-FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear β-catenin expression, whilst it increased apoptosis. In both arms, EPA-FFA treatment led to increased membrane switch from ω-6 to ω-3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA-FFA treated arms and AOM-DSS controls. Importantly, we found that EPA-FFA treatment restored the loss of Notch signaling found in the AOM-DSS control, resulted in the enrichment of Lactobacillus species in the gut microbiota and led to tumor suppressor miR34-a induction. In conclusion, our data suggest that EPA-FFA is an effective chemopreventive agent in CAC.

An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein

The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking.

Anticancer activity of natural cytokinins: a structure-activity relationship study

Cytokinin ribosides (N(6)-substituted adenosine derivatives) have been shown to have anticancer activity both in vitro and in vivo. This study presents the first systematic analysis of the relationship between the chemical structure of cytokinins and their cytotoxic effects against a panel of human cancer cell lines with diverse histopathological origins. The results confirm the cytotoxic activity of N(6)-isopentenyladenosine, kinetin riboside, and N(6)-benzyladenosine and show that the spectrum of cell lines that are sensitive to these compounds and their tissues of origin are wider than previously reported. The first evidence that the hydroxylated aromatic cytokinins (ortho-, meta-, para-topolin riboside) and the isoprenoid cytokinin cis-zeatin riboside have cytotoxic activities is presented. Most cell lines in the panel showed greatest sensitivity to ortho-topolin riboside (IC(50)=0.5-11.6 microM). Cytokinin nucleotides, some synthesized for the first time in this study, were usually active in a similar concentration range to the corresponding ribosides. However, cytokinin free bases, 2-methylthio derivatives and both O- and N-glucosides showed little or no toxicity. Overall the study shows that structural requirements for cytotoxic activity of cytokinins against human cancer cell lines differ from the requirements for their activity in plant bioassays. The potent anticancer activity of ortho-topolin riboside (GI(50)=0.07-84.60 microM, 1st quartile=0.33 microM, median=0.65 microM, 3rd quartile=1.94 microM) was confirmed using NCI(60), a standard panel of 59 cell lines, originating from nine different tissues. Further, the activity pattern of oTR was distinctly different from those of standard anticancer drugs, suggesting that it has a unique mechanism of activity. In comparison with standard drugs, oTR showed exceptional cytotoxic activity against NCI(60) cell lines with a mutated p53 tumour suppressor gene. oTR also exhibited significant anticancer activity against several tumour models in in vivo hollow fibre assays.

Antagonistic TSC22D1 variants control BRAF(E600)-induced senescence

Oncogene-induced cellular senescence (OIS) is an increasingly recognized tumour suppressor mechanism that confines the outgrowth of neoplastic cells in vivo. It relies on a complex signalling network, but only few components have been identified so far. Gene-expression profiling revealed a >100-fold increase in the levels of the transcription factor and putative tumour suppressor gene TGFβ-stimulated clone 22 (TSC22D1) in BRAF(E600)-induced senescence, in both human fibroblasts and melanocytes. Only the short TSC22D1 transcript was upregulated, whereas the abundance of the large protein variant was suppressed by proteasomal degradation. The TSC22D1 protein variants, in complex with their dimerization partner TSC22 homologue gene 1 (THG1), exerted opposing functions, as selective depletion of the short form, or conversely, overexpression of the large variant, resulted in abrogation of OIS. This was accompanied by the suppression of several inflammatory factors and p15(INK4B), with TSC22D1 acting as a critical effector of C/EBPβ. Our results demonstrate that the differential regulation of antagonistic TSC22D1 variants is required for the establishment of OIS and suggest distinct contributions of TSC22 family members to the progression of BRAF(E600)-driven neoplasia.

Genetic evidence supporting the association of protease and protease inhibitor genes with inflammatory bowel disease: a systematic review

As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs) in inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC]) to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors) were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD) on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH), dystroglycan (DAG1), macrophage-stimulating protein (MST1) and ubiquitin-specific peptidase 4 (USP4), all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4 protease inhibitors), with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family.

Hit proteins, mitochondria and cancer

The histidine triad (HIT) superfamily comprises proteins that share the histidine triad motif, His-ϕ-His-ϕ-His-ϕ-ϕ, where ϕ is a hydrophobic amino acid. HIT proteins are ubiquitous in prokaryotes and eukaryotes. HIT proteins bind nucleotides and exert dinucleotidyl hydrolase, nucleotidylyl transferase or phosphoramidate hydrolase enzymatic activity. In humans, 5 families of HIT proteins are recognized. The accumulated epidemiological and experimental evidence indicates that two branches of the superfamily, the HINT (Histidine Triad Nucleotide Binding) members and FHIT (Fragile Histidine Triad), have tumor suppressor properties but a conclusive physiological role can still not be assigned to these proteins. Aprataxin forms another discrete branch of the HIT superfamily, is implicated in DNA repair mechanisms and unlike the HINT and FHIT members, a defective protein can be conclusively linked to a disease, ataxia with oculomotor apraxia type 1. The scavenger mRNA decapping enzyme, DcpS, forms a fourth branch of the HIT superfamily. Finally, the GalT enzymes, which exert specific nucleoside monophosphate transferase activity, form a fifth branch that is not implicated in tumorigenesis. The molecular mechanisms by which the HINT and FHIT proteins participate in bioenergetics of cancer are just beginning to be unraveled. Their purported actions as tumor suppressors are highlighted in this review.

Phosphatase and tensin homolog regulates stability and activity of EphB1 receptor

Deregulation of receptor tyrosine kinases (RTKs) is linked to a broad range of cancers, stressing the necessity of studying their regulatory pathways. We and others demonstrated previously that c-Cbl is necessary for the lysosomal degradation of erythropoietin-producing hepatocellular B1 (EphB1) carcinoma and epidermal growth factor receptor (EGFR) RTKs. Moreover, the tumor suppressor phosphatase and tensin homolog (PTEN) was shown to modulate c-Cbl-dependent EGFR degradation. We therefore investigated the involvement of PTEN in EphB1 signaling and degradation. We used PTEN mutants, PTEN, and NHERF1 small interfering RNA in CHO-EphB1 and SW480 cells endogenously expressing EphB1 to delineate EphB1-PTEN interactions. PTEN was constitutively associated with c-Cbl, protecting it from degradation. EphB1 stimulation triggered ∼50% serine-threonine PTEN dephosphorylation and PTEN-Cbl complex disruption, a process requiring PTEN protein phosphatase activity. Both proteins independently translocated to EphB1, with PTEN in association with the scaffold protein NHERF1. Biologically, PTEN lipid phosphatase activity impairs EphB1-dependent cell adhesion and chemotaxis. This study demonstrates for the first time in mammalian cells that the Eph receptor and PTEN associate and influence their signaling. Moreover, it contributes to the emerging concept that PTEN regulates expression of RTKs through modulation of their degradation. Finally, it reveals a new role for PTEN protein phosphatase activity involved in this process.

The mitochondrial uncoupling protein-2 promotes chemoresistance in cancer cells

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. This survival process is facilitated through efficient control of oxidative stress originating from mitochondria that typically initiates programmed cell death. We show this critical adaptive response in cancer cells to be linked to uncoupling protein-2 (UCP2), a mitochondrial suppressor of reactive oxygen species (ROS). UCP2 is present in drug-resistant lines of various cancer cells and in human colon cancer. Overexpression of UCP2 in HCT116 human colon cancer cells inhibits ROS accumulation and apoptosis after exposure to chemotherapeutic agents. Tumor xenografts of UCP2-overexpressing HCT116 cells retain growth in nude mice receiving chemotherapy. Augmented cancer cell survival is accompanied by altered NH(2)-terminal phosphorylation of the pivotal tumor suppressor p53 and induction of the glycolytic phenotype (Warburg effect). These findings link UCP2 with molecular mechanisms of chemoresistance. Targeting UCP2 may be considered a novel treatment strategy for cancer.

Intracellular localization of the BCL-2 family member BOK and functional implications

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.

Possible role of Cdx2 in the serrated pathway of colorectal cancer characterized by BRAF mutation, high-level CpG Island methylator phenotype and mismatch repair-deficiency

Colorectal cancer is a heterogeneous disease at the histomorphological, clinical and molecular level. Approximately 20% of cases may progress through the "serrated" pathway characterized by BRAF mutation and high-level CpG Island Methylator Phenotype (CIMP). A large subgroup are additionally microsatellite instable (MSI) and demonstrate significant loss of tumor suppressor Cdx2. The aim of this study is to determine the specificity of Cdx2 protein expression and CpG promoter hypermethylation for BRAF(V600E) and high-level CIMP in colorectal cancer. Cdx2, Mlh1, Msh2, Msh6, and Pms2 were analyzed by immunohistochemistry using a multi-punch tissue microarray (TMA; n = 220 patients). KRAS and BRAF(V600E) mutation analysis, CDX2 methylation and CIMP were investigated. Loss of Cdx2 was correlated with larger tumor size (P = 0.0154), right-sided location (P = 0.0014), higher tumor grade (P < 0.0001), more advanced pT (P = 0.0234) and lymphatic invasion (P = 0.0351). Specificity was 100% for mismatch repair (MMR)-deficiency (P < 0.0001), 92.2% (P < 0.0001) for BRAF(V600E) and 91.8% for CIMP-high. Combined analysis of BRAF(V600E) /CIMP identified Cdx2 loss as sensitive (80%) and specific (91.5%) for mutation/high status. These results were validated on eight well-established colorectal cancer cell lines. CDX2 methylation correlated with BRAF(V600E) (P = 0.0184) and with Cdx2 protein loss (P = 0.0028). These results seem to indicate that Cdx2 may play a role in the serrated pathway to colorectal cancer as underlined by strong relationships with BRAF(V600E) , CIMP-high and MMR-deficiency. Whether this protein can only be used as a "surrogate" marker, or is functionally involved in the progression of these tumors remains to be elucidated.

THE CONSEQUENCES OF DISRUPTING THE MDM2-P53 BALANCE IN HEMATOPOIESIS

The bone marrow accommodates hematopoietic stem cells and progenitors. These cells provide an indispensible resource for replenishing the blood constituents throughout an organism’s life. A tissue with such a high turn-over rate mandates intact cycling checkpoint and apoptotic pathways to avoid inappropriate cell proliferation and ultimately the development of leukemias. p53, a major tumor suppressor, is a transcription factor that regulates cell cycle, and induces apoptosis and senescence. Mice inheriting a hypomorphic p53 allele in the absence of Mdm2, a p53 inhibitor, have elevated p53 cell cycle activity and die by postnatal day 13 due to hematopoietic failure. Hematopoiesis progresses normally during embryogenesis until it moves to the bone marrow in late development. Increased oxidative stress in the bone marrow compartment postnatally is the impediment for normal hematopoiesis via activation of p53. p53 in turn stimulates the generation of more reactive oxygen species and depletes bone marrow cellularity. Also, p53 exerts various defects on the hematopoietic niche by increasing mesenchymal lineage populations and their differentiation. Hematopoietic defects are rescued with antioxidants or when cells are cultured at low oxygen levels. Deletion of p16 partially rescues bone marrow cellularity and progenitors via a p53-independent pathway. Thus, although p53 is required to inhibit tumorigenesis, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis and survival during homeostasis.

Loss of DNA polymerase zeta enhances spontaneous tumorigenesis.

Mammalian genomes encode at least 15 distinct DNA polymerases, functioning as specialists in DNA replication, DNA repair, recombination, or bypass of DNA damage. Although the DNA polymerase zeta (polzeta) catalytic subunit REV3L is important in defense against genotoxins, little is known of its biological function. This is because REV3L is essential during embryogenesis, unlike other translesion DNA polymerases. Outstanding questions include whether any adult cells are viable in the absence of polzeta and whether polzeta status influences tumorigenesis. REV3L-deficient cells have properties that could influence the development of neoplasia in opposing ways: markedly reduced damage-induced point mutagenesis and extensive chromosome instability. To answer these questions, Rev3L was conditionally deleted from tissues of adult mice using MMTV-Cre. Loss of REV3L was tolerated in epithelial tissues but not in the hematopoietic lineage. Thymic lymphomas in Tp53(-/-) Rev3L conditional mice occurred with decreased latency and higher incidence. The lymphomas were populated predominantly by Rev3L-null T cells, showing that loss of Rev3L can promote tumorigenesis. Remarkably, the tumors were frequently oligoclonal, consistent with accelerated genetic changes in the absence of Rev3L. Mammary tumors could also arise from Rev3L-deleted cells in both Tp53(+/+) and Tp53(+/-) backgrounds. Mammary tumors in Tp53(+/-) mice deleting Rev3L formed months earlier than mammary tumors in Tp53(+/-) control mice. Prominent preneoplastic changes in glandular tissue adjacent to these tumors occurred only in mice deleting Rev3L and were associated with increased tumor multiplicity. Polzeta is the only specialized DNA polymerase yet identified that inhibits spontaneous tumor development.

A NOVEL FUNCTION FOR AURORA B KINASE IN THE REGULATION OF P53 BY PHOSPHORYLATION

The mitotic kinase Aurora B plays a pivotal role in mitosis and cytokinesis and governs the spindle assembly checkpoint which ensures correct chromosome segregation and normal progression through mitosis. Aurora B is overexpressed in breast and other cancers and may be an important molecular target for chemotherapy. Tumor suppressor p53 is the guardian of the genome and an important negative regulator of the cell cycle. Previously, it was unknown whether Aurora B and p53 had mutual regulation during the cell cycle. A small molecule specific inhibitor of Aurora B, AZD1152, gave us an indication that Aurora B negatively impacted p53 during interphase and mitosis. Here, we show the antineoplastic activity of AZD1152 in six human breast cancer cell lines, three of which overexpress HER2. AZD1152 specifically inhibited Aurora B kinase activity, thereby causing mitotic catastrophe, polyploidy and apoptosis, which in turn led to apoptotic death. Further, AZD1152 administration efficiently suppressed tumor growth in orthotopic and metastatic breast cancer cell xenograft models. Notably, it was found that the protein level of Aurora B kinase declined after inhibition of Aurora B kinase activity. Investigation of the underlying mechanism suggested that AZD1152 accelerated the protein turnover of Aurora B by enhancing its ubiquitination. As a consequence of inhibition of Aurora B, p53 levels were increased in tissue culture and murine models. This hinted at a possible direct interaction between p53 and Aurora B. Indeed, it was found that p53 and Aurora B exist in complex and interact directly during interphase and at the centromere in mitosis. Further, Aurora B was shown to phosphorylate p53 at several serine/threonine residues in the DNA binding domain and these events caused downregulation of p53 levels via ubiquitination mediated by Mdm2. Importantly, phosphorylation of threonine 211 was shown to reduce p53’s transcriptional activity while other phosphorylation sites did not. On a functional level, Aurora B was shown to reduce p53’s capacity to mediate apoptosis in response to the DNA damaging agent, cisplatin. These results define a novel mechanism for p53 inactivation by Aurora B and imply that oncogenic hyperactivation or overexpression of Aurora B may compromise p53’s tumor suppressor function.