An acute intake of plant stanol esters alters immune-related pathways in the jejunum of healthy volunteers.

Plant sterols and stanols inhibit intestinal cholesterol absorption and consequently lower serum LDL-cholesterol (LDL-C) concentrations. The underlying mechanisms are not yet known. In vitro and animal studies have suggested that changes in intestinal sterol metabolism are attributed to the LDL-C-lowering effects of plant stanol esters. However, similar studies in human subjects are lacking. Therefore, we examined the effects of an acute intake of plant stanol esters on gene expression profiles of the upper small intestine in healthy volunteers. In a double-blind cross-over design, fourteen healthy subjects (eight female and six male; age 21-55 years), with a BMI ranging from 21 to 29 kg/m², received in random order a shake with or without plant stanol esters (4 g). At 5 h after consumption of the shake, biopsies were taken from the duodenum (around the papilla of Vater) and from the jejunum (20 cm distal from the papilla of Vater). Microarray analysis showed that the expression profiles of genes involved in sterol metabolism were not altered. Surprisingly, the pathways involved in T-cell functions were down-regulated in the jejunum. Furthermore, immunohistochemical analysis showed that the number of CD3 (cluster of differentiation number 3), CD4 (cluster of differentiation number 4) and Foxp3⁺ (forkhead box P3-positive) cells was reduced in the plant stanol ester condition compared with the control condition, which is in line with the microarray data. The physiological and functional consequences of the plant stanol ester-induced reduction of intestinal T-cell-based immune activity in healthy subjects deserve further investigation.

Chromosome 3p microsatellite allelotyping in neuroblastoma: a report on the technical hurdles.

Pinpointing critical regions of recurrent loss may help localize tumor suppressor genes. To determine the regions of loss on chromosome 3p in neuroblastoma, we performed loss of heterozygosity analysis using 16 microsatellite markers in a series of 65 primary tumors and 29 neuroblastoma cell lines. In this study, we report the results and discuss the technical hurdles that we encountered during data generation and interpretation that are of relevance for current studies or tests employing microsatellites. To provide functional support for the implication of 3p tumor suppressor genes in this childhood malignancy, we performed a microcell-mediated chromosome 3 transfer in neuroblastoma cells.

Effect of strain at low-frequency loading on peri-implant bone (re)modelling: a guinea-pig experimental study.

OBJECTIVES: To investigate whether controlled early loading enhances peri-implant bone mass and bone-to-implant contact. Low-frequency stimulation (3 Hz) and varying force amplitudes, causing varying strains, were applied in three guinea-pig series. MATERIAL AND METHODS: Three series of guinea-pigs received percutaneous TiO(2)-blasted implants in both tibiae. One week after implant installation, one implant was stimulated with a sinusoidally varying bending moment while the contra-lateral implant served as an unloaded control. Force amplitudes of 0.5, 1 and 2 N were applied on a 20-mm-long cantilever, resulting in strains of 133, 267 and 533 muepsilon, respectively, measured by a strain gauge bonded on the surface of the tibial bone at 1.3 mm from the implant's distal surface. Implant stability was followed by means of resonance frequency analysis. Bone-to-implant contact and bone mass [BM (%) bone occupied area fraction] were analysed histomorphometrically. RESULTS: A significant positive effect on the difference in bone mass at the stimulated vs. at the control side was observed in the distal half peri-implant marrow cavity for early mechanical stimulation at a frequency of 3 Hz (P<0.0001). An optimum was reached for the applied load, which causes a strain of approximately 267 muepsilon 1.3 mm from the implant. Implant stability gradually increased in time; no significant effect of early stimulation could be measured. CONCLUSIONS: The effect of early controlled mechanical stimulation on the peri-implant bone, in this cortical bone model, is strongly dependent on force amplitude/strain at low-frequency stimulation.

A rapid and simple HPLC method for the analysis of propofol in biological fluids.

A selective and sensitive high-performance liquid chromatographic method for the analysis of propofol in biological samples was developed. Propofol and thymol (internal standard) were analysed on a Purospher RP-18 endcapped (75 mmx4 mm, 3 microm) stationary phase using acetonitrile and water (65:35, v/v) as eluents at a flow rate of 0.6 mL/min. The excitation and emission wavelengths were 276 and 310 nm, respectively. Sample treatment consisted of deproteinization by acetonitrile containing the internal standard and direct injection of the supernatant. Mean analytical recovery were 105% (CV 2.0%) at concentrations ranging from 0.05 to 10 mg/L. The quantification limit was 3 ng/mL for a 500 microL sample plasma volume and 5 ng/mL for a 500 microL blood sample. The intra-day and inter-day precisions were lower than 5.5% for three concentrations assessed (0.05, 1.0 and 10.0 mg/L). Considering the column size and the flow rate, the separation was achieved with an analysis time less than 6 min with a reduced consumption of solvent. This rapid HPLC method using a simple treatment procedure is sensitive enough for monitoring propofol in human biological samples.

Timing of loading--immediate, early, or delayed--in the outcome of implants in the edentulous mandible: a prospective clinical trial.

PURPOSE: To report on the implant outcome of delayed, early, and immediate loading of implants in the edentulous mandible in a prospective controlled study. MATERIALS AND METHODS: On a consecutive basis, the first 10 patients received an overdenture retained by 2 ball attachments 4 months after implant insertion (delayed), and the next 10 patients received an overdenture 1 week after implant surgery (early). The next 10 patients were treated with a fixed prosthesis on 3 implants (Br6nemark Novum) either the day of or the day after surgery (immediate). All patients were followed for 1 year; half were followed for 2 years. Measures of assessment for this prospective clinical trial included monitoring of loading at prosthesis level (bite fork) as well as at the abutment level (strain gauges), 3-dimensional imaging of marginal bone remodeling, and implant stability. RESULTS AND DISCUSSION: One patient in each OD group lost both implants. The losses occurred 6 months after loading in the delayed group and 1 month after loading in the early group. In the immediate group, 1 patient lost both distal implants 5 months after loading. In 2 other patients, 1 distal implant failed after 1 year of loading. Maximal bite forces increased over time for all groups. Marginal bone loss was the highest for the immediate group, while no differences in implants stability were observed between the 3 groups after 1 year of loading. CONCLUSIONS: According to this prospective controlled clinical trial, the results achieved with implants loaded early were comparable to those achieved with implants loaded after a delay. Distal implants are at higher risk for failure in the immediate loaded protocol.

Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples.

Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593-0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73-2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83-7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice.

Dietary plant stanol ester consumption improves immune function in asthma patients: results of a randomized, double-blind clinical trial.

BACKGROUND: In vitro and ex vivo studies have suggested that plant sterols and stanols can shift the T helper (Th) 1/Th2 balance toward a Th1-type immune response, which may be beneficial in Th2-dominant conditions such as asthma and allergies. OBJECTIVE: We evaluated in vivo whether plant stanol esters affect the immune response in asthma patients. DESIGN: Fifty-eight asthma patients participated in a randomized, double-blind, placebo-controlled intervention study. All subjects started with a 2-wk run-in period in which they consumed 150 mL control soy-based yogurt without added plant stanol esters/d. Next, an 8-wk experimental period was started in which one-half of the participants received plant stanol enriched soy-based yogurts (4.0 g plant stanols/d), whereas the other one-half of subjects continued the consumption of control yogurts. After 4 wk of daily plant stanol consumption, all participants were vaccinated against hepatitis A virus (HAV), and the increase of antibody titres was monitored weekly until 4 wk after vaccination. RESULTS: Asthma patients in the plant stanol ester group showed higher antibody titres against HAV 3 and 4 wk after vaccination [19% (P = 0.037) and 22% (P = 0.030), respectively]. Also, substantial reductions in plasma total immunoglobulin E, interleukin (IL)-1ß, and tumor necrosis factor-α were shown in the plant stanol ester group. The increase in serum plant stanol concentrations was correlated significantly with the decrease in IL-13 concentrations and the Th1 switch in the Th1/Th2 balance. However, no absolute differences in cytokine production between the plant stanol ester group and the control group were shown. CONCLUSION: To the best of our knowledge, we are among the first authors to show that plant stanol ester consumption improves the immune function in vivo in asthma patients. This trial was registered at clinicaltrials.gov as NCT01715675.

Identification of an unbalanced X-autosome translocation by array CGH in a boy with a syndromic form of chondrodysplasia punctata brachytelephalangic type.

Screening of a large series of patients with unexplained mental retardation with a 1 Mb BAC array resulted in the detection of several cryptic chromosomal imbalances. In this paper we present the findings of array CGH screening in a 14-year-old boy with the brachytelephalangic type of chondrodysplasia punctata, mental retardation and obesity. On several occasions, cytogenetic analysis of this boy revealed a normal karyotype. Subsequent screening with array CGH resulted in the detection of a distal 9p trisomy and distal Xp nullisomy caused by an unbalanced X;9 translocation: 46,Y,der(X)t(X;9)(p22.32;p23). The identification of this de novo chromosomal rearrangement not only made accurate genetic counselling possible but also explained most of the phenotypic abnormalities observed in this patient. This study confirms the power of array CGH in the detection of subtle or submicroscopic chromosomal changes.

Acute intake of plant stanol esters induces changes in lipid and lipoprotein metabolism-related gene expression in the liver and intestines of mice.

The kinetics of plant stanol uptake and routing in 8-week-old C57BL/6J mice were determined after a plant stanol ester gavage. In addition, acute changes in intestinal and hepatic gene expression were investigated. Mice were fed a plant sterol/stanol poor diet from weaning. At the age of 8 weeks, they received an oral gavage consisting of 0.25 mg cholesterol + 50 mg plant stanol esters dissolved in olive oil. Animals were euthanized at different time points. In a second comparable set-up, mesenteric lymph-cannulated versus sham-operated mice received the same oral gavage, which was now deuterium labeled. Intestinal and hepatic sitostanol concentrations increased within 15 min post-gavage. This rapid hepatic appearance was absent in lymph-cannulated mice, suggesting a very fast lymph-mediated uptake. Hepatic mRNA expression of SREBP2 and its target genes rapidly decreased, whereas expression of LXR target genes increased. The intestinal SREBP2 pathway was increased, whereas the expression of LXR target genes hardly changed. The fivefold and sixfold increased expression of intestinal LDLr and PCSK9 is suggestive of TICE activation. We conclude that in C57BL/6J mice plant stanol kinetics are fast, and affect intestinal and hepatic gene expression within 15 min postprandial after lymph-mediated uptake.

Application of laser capture microdissection in genetic analysis of neuroblastoma and neuroblastoma precursor cells.

Recently developed quantitative and high-throughput technologies that allow automated and rapid screening of the whole genome, transcriptome and proteome have revolutionized the field of cancer genetics. At the same time, new challenges are met, e.g. the need for improved data analysis and standardization of tumor sample handling. Even if these issues are resolved, an 'old' problem in genetic tumor analysis remains, i.e. contamination of tumor samples by stromal and surrounding normal cells. To overcome this obstacle, laser capture microdissection (LCM) has been developed in order to procure the cells of interest from stained tissue sections with retention of morphology. In this review we describe the possible down-stream applications of LCM in the genetic analysis of neuroblastoma (NB). Special focus is given to MYCN copy number determination using real-time quantitative polymerase chain reaction (Q-PCR), analysis of 1p-, 3p- and 11q-deletions using loss of heterozygosity analysis and Q-PCR expression analysis of microdissected normal neuroblast cells and NB cells.

Protective role of plant sterol and stanol esters in liver inflammation: insights from mice and humans.

The inflammatory component of non-alcoholic steatohepatitis (NASH) can lead to irreversible liver damage. Therefore there is an urgent need to identify novel interventions to combat hepatic inflammation. In mice, omitting cholesterol from the diet reduced hepatic inflammation. Considering the effects of plant sterol/stanol esters on cholesterol metabolism, we hypothesized that plant sterol/stanol esters reduces hepatic inflammation. Indeed, adding plant sterol/stanol esters to a high-fat-diet reduced hepatic inflammation as indicated by immunohistochemical stainings and gene expression for inflammatory markers. Finally, adding sterol/stanol esters lowered hepatic concentrations of cholesterol precursors lathosterol and desmosterol in mice, which were highly elevated in the HFD group similarly as observed in severely obese patients with NASH. In vitro, in isolated LPS stimulated bone marrow derived macrophages desmosterol activated cholesterol efflux whereas sitostanol reduced inflammation. This highly interesting observation that plant sterol/stanol ester consumption leads to complete inhibition of HFD-induced liver inflammation opens new venues in the treatment and prevention of hepatic inflammation.

Constant strain rate and peri-implant bone modeling: an in vivo longitudinal micro-CT analysis.

BACKGROUND: Strain, frequency, loading time, and strain rate, among others, determine mechanical parameters in osteogenic loading. We showed a significant osteogenic effect on bone mass (BM) by daily peri-implant loading at 1.600µÎµ.s(-1) after 4 weeks. PURPOSE: To study the peri-implant osteogenic effect of frequency and strain in the guinea pig tibia by in vivo longitudinal micro-computed tomography (CT) analysis. MATERIAL AND METHODS: One week after implant installation in both hind limb tibiae, one implant was loaded daily for 10' during 4 weeks, while the other served as control. Frequencies (3, 10, and 30Hz) and strains varied alike in the three series to keep the strain rate constant at 1.600µÎµ.s(-1) . In vivo micro-CT scans were taken of both tibiae: 1 week after implantation but before loading (v1) and after 2 (v2) and 4 weeks (v3) of loading as well as postmortem (pm). BM (BM (%) bone-occupied area fraction) was calculated as well as the difference between test and control sides (delta BM) RESULTS: All implants (n=78) were clinically stable at 4 weeks. Significant increase in BM was measured between v1 and v2 (p<.0001) and between v1 and v3 (p<.0001). A significant positive effect of loading on delta BM was observed in the distal peri-implant marrow 500 Region of Interest already 2 weeks after loading (p=.01) and was significantly larger (11%) in series 1 compared with series 2 (p=.006) and 3 (p=.016). CONCLUSIONS: Within the constraints of constant loading time and strain rate, the effect of early implant loading on the peri-implant bone is strongly dependent on strain and frequency. This cortical bone model has shown to be most sensitive for high force loading at low frequency.

Effects of plant sterols and stanols on intestinal cholesterol metabolism: suggested mechanisms from past to present.

Plant sterols and stanols are natural food ingredients found in plants. It was already shown in 1950 that they lower serum low-density lipoprotein cholesterol (LDL-C) concentrations. Meta-analysis has reported that a daily intake of 2.5 g plant sterols/stanols reduced serum LDL-C concentrations up to 10%. Despite many studies, the underlying mechanism remains to be elucidated. Therefore, the proposed mechanisms that have been presented over the past decades will be described and discussed in the context of the current knowledge. In the early days, it was suggested that plant sterols/stanols compete with intestinal cholesterol for incorporation into mixed micelles as well as into chylomicrons. Next, the focus shifted toward cellular processes. In particular, a role for sterol transporters localized in the membranes of enterocytes was suggested. All these processes ultimately lowered intestinal cholesterol absorption. More recently, the existence of a direct secretion of cholesterol from the circulation into the intestinal lumen was described. First results in animal studies suggested that plant sterols/stanols activate this pathway, which also explains the increased fecal neutral sterol content and as such could explain the cholesterol-lowering activity of plant sterols/stanols.

RNA pre-amplification enables large-scale RT-qPCR gene-expression studies on limiting sample amounts.

BACKGROUND: The quantitative polymerase chain reaction (qPCR) is a widely utilized method for gene-expression analysis. However, insufficient material often compromises large-scale gene-expression studies. The aim of this study is to evaluate an RNA pre-amplification method to produce micrograms of cDNA as input for qPCR. FINDINGS: The linear isothermal Ribo-SPIA pre-amplification method (WT-Ovation; NuGEN) was first evaluated by measuring the expression of 20 genes in RNA samples from six neuroblastoma cell lines and of 194 genes in two commercially available reference RNA samples before and after pre-amplification, and subsequently applied on a large panel of 738 RNA samples extracted from neuroblastoma tumours. All RNA samples were evaluated for RNA integrity and purity. Starting from 5 to 50 nanograms of total RNA the sample pre-amplification method was applied, generating approximately 5 microgams of cDNA, sufficient to measure more than 1000 target genes. The results obtained from this study show a constant yield of pre-amplified cDNA independent of the amount of input RNA; preservation of differential gene-expression after pre-amplification without introduction of substantial bias; no co-amplification of contaminating genomic DNA; no necessity to purify the pre-amplified material; and finally the importance of good RNA quality to enable pre-amplification. CONCLUSION: Application of this unbiased and easy to use sample pre-amplification technology offers great advantage to generate sufficient material for diagnostic and prognostic work-up and enables large-scale qPCR gene-expression studies using limited amounts of sample material.

A constitutional translocation t(1;17)(p36.2;q11.2) in a neuroblastoma patient disrupts the human NBPF1 and ACCN1 genes.

The human 1p36 region is deleted in many different types of tumors, and so it probably harbors one or more tumor suppressor genes. In a Belgian neuroblastoma patient, a constitutional balanced translocation t(1;17)(p36.2;q11.2) may have led to the development of the tumor by disrupting or activating a gene. Here, we report the cloning of both translocation breakpoints and the identification of a novel gene that is disrupted by this translocation. This gene, named NBPF1 for Neuroblastoma BreakPoint Family member 1, belongs to a recently described gene family encoding highly similar proteins, the functions of which are unknown. The translocation truncates NBPF1 and gives rise to two chimeric transcripts of NBPF1 sequences fused to sequences derived from chromosome 17. On chromosome 17, the translocation disrupts one of the isoforms of ACCN1, a potential glioma tumor suppressor gene. Expression of the NBPF family in neuroblastoma cell lines is highly variable, but it is decreased in cell lines that have a deletion of chromosome 1p. More importantly, expression profiling of the NBPF1 gene showed that its expression is significantly lower in cell lines with heterozygous NBPF1 loss than in cell lines with a normal 1p chromosome. Meta-analysis of the expression of NBPF and ACCN1 in neuroblastoma tumors indicates a role for the NBPF genes and for ACCN1 in tumor aggressiveness. Additionally, DLD1 cells with inducible NBPF1 expression showed a marked decrease of clonal growth in a soft agar assay. The disruption of both NBPF1 and ACCN1 genes in this neuroblastoma patient indicates that these genes might suppress development of neuroblastoma and possibly other tumor types.

High resolution tiling-path BAC array deletion mapping suggests commonly involved 3p21-p22 tumor suppressor genes in neuroblastoma and more frequent tumors.

The recurrent loss of 3p segments in neuroblastoma suggests the implication of 1 or more tumor suppressor genes but thus far few efforts have been made to pinpoint their detailed chromosomal position. To achieve this goal, array-based comparative genomic hybridization was performed on a panel of 23 neuroblastoma cell lines and 75 primary tumors using a tiling-path bacterial artificial chromosome array for chromosome 3p. A total of 45 chromosome 3 losses were detected, including whole chromosome losses, large terminal deletions and interstitial deletions. The latter, observed in cell lines as well as a number of distal deletions detected in primary tumors, allowed us to demarcate 3 minimal regions of loss of 3.6 Mb [3p21.31-p21.2, shortest regions of overlap (SRO)1], 1.4 Mb (3p22.3-3p22.2, SRO2) and 3.8 Mb (3p25.3-p25.1, SRO3) in size. The present data significantly extend previous findings and now firmly establish critical regions on 3p implicated in neuroblastoma. Interestingly, the 2 proximal regions coincide with previously defined SROs on 3p21.3 in more frequent tumors including lung and breast cancer. As such, similar tumor suppressor genes may play a critical role in development or progression of a variety of neoplasms, including neuroblastoma.

Effect of controlled early implant loading on bone healing and bone mass in guinea pigs, as assessed by micro-CT and histology.

Without controlled loading, the failure of early loaded oral implants is higher than in delayed loading, unless loading regimens can be identified that stimulate bone formation. The purpose of this study was to investigate whether controlled early loading optimizes osseointegration. Six series of guinea pigs received percutaneous implants in both tibiae. One implant was stimulated, the contra-lateral served as the control. The strain rate amplitude varied from 1,620 to 12,000 microstrain s(-1). In vivo microfocus computed tomography (micro-CT) was used to study the peri-implant bone at three time points: 1 wk after implantation, but before starting stimulation (V1); 2 wk after stimulation (V2); and 4 wk after stimulation, after the guinea pigs were killed (PM). Bone implant contact and bone mass [BM (%) bone occupied area fraction] were analyzed. The implant failure was 5.9% (six control/one test). Although bone implant contact did not significantly differ, bone mass in the distal half peri-implant marrow cavity was significantly higher around test implants. Strain rate amplitude and the difference in bone mass between test and control implants were inversely correlated. A strain rate amplitude of 1,620 microstrain s(-1) in the cortical bone at a distance of 1.3 mm from the implant showed the highest effect. Based on these results, early loading did not negatively affect the implant outcome. On the contrary, an improved bone reaction in the marrow cavity around early loaded implants was achieved.

Translocation-excision-deletion-amplification mechanism leading to nonsyntenic coamplification of MYC and ATBF1.

Despite oncogene amplification being a characteristic of many tumor types, the mechanisms leading to amplicon formation have remained largely unresolved. In this study, we used a combinatorial approach of fluorescence in situ hybridization and single-nucleotide polymorphism chip gene copy number analyses to unravel the mechanism leading to nonsyntenic coamplification of MYC and ATBF1 in SJNB-12 cells. To explain our findings, we propose a complex series of events consisting of multiple double-strand breaks, accompanied (or triggered) by the formation of a reciprocal translocation t(8;16), as well as excisions and deletions near the translocation breakpoints. This study provides evidence for a translocation-excision-deletion-amplification sequence of events rather than a breakage-fusion-bridge model, which has been more frequently proposed to explain proto-oncogene amplification. Furthermore, it illustrates the power of presently available tools for detailed analysis of the complex rearrangements that accompany amplicon formation.

The von Hippel-Lindau tumor suppressor gene expression level has prognostic value in neuroblastoma.

Deletions of the short arm of chromosome 3 are often observed in a specific subset of aggressive neuroblastomas (NBs) with loss of distal 11q and without MYCN amplification. The critical deleted region encompasses the locus of the von Hippel-Lindau gene (VHL, 3p25). Constitutional loss of function mutations in the VHL gene are responsible for the VHL syndrome, a dominantly inherited familial cancer syndrome predisposing to a variety of neoplasms, including pheochromocytoma. Pheochromocytomas are, like NB, derived from neural crest cells, but, unlike NB, consist of more mature chromaffin cells instead of immature neuroblasts. Further arguments for a putative role of VHL in NB are its function as oxygen sensitizer and the reported relation between hypoxia and dedifferentiation of NB cells, leading to a more aggressive phenotype. To test the possible involvement of VHL in NB, we did mRNA expression analysis and sought evidence for VHL gene inactivation. Although no evidence for a classic tumor suppressor role for VHL in NB could be obtained, a strong correlation was observed between reduced levels of VHL mRNA and low patient survival probability (p=0.013). Furthermore, VHL appears to have predictive power in NTRK1 (TRKA) positive tumor samples with presumed favorable prognosis, which makes it a potentially valuable marker for more accurate risk assessment in this subgroup of patients. The significance of the reduced VHL expression levels in relation to NB tumor biology remains unexplained, as functional analysis demonstrated no clear effect of the reduction in VHL mRNA expression on protein stability of its downstream target hypoxia-inducible factor alpha.

Small-molecule MDM2 antagonists as a new therapy concept for neuroblastoma.

Circumvention of the p53 tumor suppressor barrier in neuroblastoma is rarely caused by TP53 mutation but might arise from inappropriately increased activity of its principal negative regulator MDM2. We show here that targeted disruption of the p53-MDM2 interaction by the small-molecule MDM2 antagonist nutlin-3 stabilizes p53 and selectively activates the p53 pathway in neuroblastoma cells with wild-type p53, resulting in a pronounced antiproliferative and cytotoxic effect through induction of G(1) cell cycle arrest and apoptosis. A nutlin-3 response was observed regardless of MYCN amplification status. Remarkably, surviving SK-N-SH cells adopted a senescence-like phenotype, whereas CLB-GA and NGP cells underwent neuronal differentiation. p53 dependence of these alternative outcomes of nutlin-3 treatment was evidenced by abrogation of the effects when p53 was knocked down by lentiviral-mediated short hairpin RNA interference. The diversity of cellular responses reveals pleiotropic mechanisms of nutlins to disable neuroblastoma cells and exemplifies the feasibility of exploiting, by a single targeted intervention, the multiplicity of anticancer activities exerted by a key tumor suppressor as p53. The observed treatment effects without the need of imposing a genotoxic burden suggest that selective MDM2 antagonists might be beneficial for treatment of neuroblastoma patients with and without MYCN amplification.