Genotypic interaction and gender specificity of common genetic variants in the p53/mdm2 network in Crohn's disease.

BACKGROUND/AIMS: Defective p53-mediated apoptosis and cell cycle control have been implicated in the immunopathogenesis of Crohn's disease (CD). Since common functional variants of p53 (SNP72 G/C) and its key negative regulator mdm2 (SNP309 T/G) have been reported to affect cellular apoptotic and cell cycle arrest capacities, we assessed the effects of these variants on CD susceptibility and their relationship to NOD2/CARD15 as a well-established genetic CD risk factor. METHODS: The variants SNP72 G/C and SNP309 T/G were genotyped in 149 European CD patients and 478 healthy controls. Subgroup analysis was performed in relation to NOD2/CARD15 status and to demographic/clinical characteristics. RESULTS: The p53 SNP72 CC genotype tended to be less frequent in CD. This reached statistical significance only in the male cohort (0 vs. 7.3%; p = 0.037). Genotype and allele frequencies of both single-nucleotide polymorphisms (SNPs) were otherwise not significantly different. In the combined genotypic analysis, the genotype p53 SNP72 CC was significantly underrepresented in mdm2 SNP309 TT homozygotes (0 vs. 9.7%; p = 0.034). No association was observed between NOD2/CARD15 and the respective SNPs. CONCLUSION: We report on a gender-specific protective effect of the low-apoptotic SNP72 CC genotype, and a gender-unrestricted genotypic interaction between SNP309 TT and SNP72 CC, which, for the first time, links sequence variation of the p53/mdm2 network to CD, independent of NOD2/CARD15.

Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter.

Gene expression was visualized in single living mammalian cells with beta-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each enzyme molecule changed the fluorescence of many substrate molecules from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual cells containing less than 100 beta-lactamase molecules. The robust change in emission ratio reveals quantitative heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.

Association of rheumatoid arthritis with Mdm2 SNP309 and genetic evidence for an allele-specific interaction between MDM2 and p53 P72R variants: a case control study.

OBJECTIVE: This study examines two common, functional, single nucleotide polymorphisms (SNP) in the genes coding the human homolog of murine-double-minute-2 (MDM2) and p53 in patients with rheumatoid arthritis (RA) based on the hypothesis that p53 may be an important negative regulator of the pro-inflammatory transcription factor nuclear factor kappa b (NFKappaB). METHODS: Genomic DNA was obtained from 221 patients with RA who fulfilled at least 4 ACR criteria and from 521 healthy controls. Mdm2 SNP309 and p53 P72R were genotyped by polymerase chain reaction and restriction enzyme analysis. RESULTS: In RA patients the frequencies of the mdm2 SNP309 G allele and both G-containing genotypes were significantly reduced (G allele: OR: 0.75, 95% CI: 0.59-0.95, p=0.016; genotype TG: OR: 0.71, 95% CI: 0.50-1.00; genotype GG: OR. 0.58, 95% CI: 0.34-0.99; both: p=0.049). Concerning p53 P72R, no differences in allele or genotype frequencies were detected. A combined analysis of both polymorphisms revealed a significant interaction between them (p=0.046). In individuals carrying >1 p53 72R allele, MDM2 had a protective effect, whereas in individuals homozygous for p53 72P, MDM2 had the opposite effect. CONCLUSION: The function of MDM2 depends on the p53 P72R genotype, resulting in either an increased or reduced risk for RA. We suggest that in most cases MDM2 stabilizes the conformation of p53, whereas in p53 PP-positive subjects MDM2 supports the degradation of p53.

Endogenous retroviruses and cancer.

The genomes of vertebrates contain sequences that are similar to present-day exogenous retroviruses. Such sequences, called endogenous retroviruses (ERVs), have resulted from ancestral germ line infections by exogenous retroviruses which have thereafter been transmitted in a Mendelian fashion. By analogy to exogenous tumorigenic retroviruses, ERVs have been implicated in the pathogenesis of cancer. Cumulative evidence from animal models indicates that ERVs may participate in the process of malignant transformation or promote tumor growth, e.g. through insertional mutagenesis or via counteracting tumor immunosurveillance. Here, we review the role of ERVs in tumorigenesis with focus on human ERVs (HERVs) in human cancer. Although available data suggest a potential role of HERVs in human cancers, in particular germ cell tumors, the contributions of HERVs to human tumorigenesis warrant further elucidation. (Part of a multi-author review).

The p53 G72C and MDM2 T309G single nucleotide polymorphisms in patients with Wegener's granulomatosis.

OBJECTIVE: Wegener's granulomatosis (WG) is a rare disease with unknown aetiology, but there is evidence for a complex genetic background. The tumor suppressor p53 and its most important negative regulator, MDM2, are positioned in the centre of a pathway that eliminates damaged cells through apoptosis. Furthermore, p53 is one of the most important negative regulators of the pro-inflammatory transcription factor nuclear factor kappa b (NFkappaB). In this respect the investigation of polymorphisms in the p53-network could be a promising approach contributing susceptibility of WG and its course of disease. METHODS: A case control study with 132 patients with WG and 512 healthy blood donors was conducted to evaluate an association of p53-SNP G72C or MDM2-SNP T309G with WG. SNPs were genotyped by polymerase chain reaction (PCR) and subsequent differential enzymatic restriction. All patients showed the clinical pathological findings of WG according to the ACR classification criteria of 1990. RESULTS: The p53 G72C and MDM2 T309G polymorphisms did not show any difference between WG patients and controls. The subgroup analysis of gender differences and earlier onset of WG (younger than median age of 51 years at diagnosis) did not show any differences in allelic or genotype frequencies of p53 G72C or MDM2 T309G SNP between WG patients and the control group. CONCLUSIONS: Our study showed no association between the p53 SNP G72C and the MDM2 SNP T309G with susceptibility or course of disease in patients with WG. The data presented do not suggest that alterations in the p53-network play a key role in the pathogenesis of WG.

Specificity of antibodies directed against Env protein of human endogenous retroviruses in patients with germ cell tumors.

We report here that 85% of the patients with germ cell tumors (GCTs) produce antibodies directed against Env protein of human endogenous retroviruses. Individuals that received antitumor treatment showed a decrease with time in their antibody titers. Importantly, of the rare cases of non-GCT individuals with Env-antibodies (n= 15, 0.8%), none produced antibodies directed against the transmembrane domain (TM), whereas all tested Env-positive GCT patients (n= 49) generated such antibodies at high titers. TM is required for Env to be expressed at the cell surface. Thus, anti-TM antibodies constitute highly specific markers for GCT and may hint at a function of Env during tumorigenesis.

Characterization of the microbunch time structure of proton pencil beams at a clinical treatment facility.

Proton therapy is an advantageous treatment modality compared to conventional radiotherapy. In contrast to photons, charged particles have a finite range and can thus spare organs at risk. Additionally, the increased ionization density in the so-called Bragg peak close to the particle range can be utilized for maximum dose deposition in the tumour volume. Unfortunately, the accuracy of the therapy can be affected by range uncertainties, which have to be covered by additional safety margins around the treatment volume. A real-time range and dose verification is therefore highly desired and would be key to exploit the major advantages of proton therapy. Prompt gamma rays, produced in nuclear reactions between projectile and target nuclei, can be used to measure the proton's range. The prompt gamma-ray timing (PGT) method aims at obtaining this information by determining the gamma-ray emission time along the proton path using a conventional time-of-flight detector setup. First tests at a clinical accelerator have shown the feasibility to observe range shifts of about 5 mm at clinically relevant doses. However, PGT spectra are smeared out by the bunch time spread. Additionally, accelerator related proton bunch drifts against the radio frequency have been detected, preventing a potential range verification. At OncoRay, first experiments using a proton bunch monitor (PBM) at a clinical pencil beam have been conducted. Elastic proton scattering at a hydrogen-containing foil could be utilized to create a coincident proton-proton signal in two identical PBMs. The selection of coincident events helped to suppress uncorrelated background. The PBM setup was used as time reference for a PGT detector to correct for potential bunch drifts. Furthermore, the corrected PGT data were used to image an inhomogeneous phantom. In a further systematic measurement campaign, the bunch time spread and the proton transmission rate were measured for several beam energies between 69 and 225 MeV as well as for variable momentum limiting slit openings. We conclude that the usage of a PBM increases the robustness of the PGT method in clinical conditions and that the obtained data will help to create reliable range verification procedures in clinical routine.

Mutant p53 can provoke apoptosis in p53-deficient Hep3B cells with delayed kinetics relative to wild-type p53.

Wild-type (wt) p53 frequently induces apoptosis when expressed in tumor cells whereas mutant p53 acts as an oncoprotein and consequently, stimulates cell proliferation. We report here exceptions to that rule. p53 conformational mutant 175H and DNA contact mutant 273H provoke apoptosis in human p53-deficient Hep3B hepatoma cells with delayed kinetics relative to wt p53. Similarly, c-Myc strongly stimulates apoptosis in these cells. In contrast, viral oncoproteins E1A and E7, and the cellular oncoprotein MDM-2, fail to elicit cytocidal responses. Efficient apoptotic cell death by mutant p53 requires oligomerization as 175H and 273H with deletions between amino acid residues 326 and 347 of the oligomerization domain are nontoxic. Apoptosis by mutant or wt p53 was significantly inhibited by the serine protease inhibitor AEBSF but not by the inactive analog AEBSA. Together, these results suggest that a wt p53-independent control mechanism is operational in Hep3B cells that eliminates cells upon sensing illegitimate proliferation signals originating from certain oncoproteins, including mutant p53 and Myc. We suggest that some tumor cell types lack p53 altogether because they tolerate neither wild-type nor mutant forms of the protein.

c-Abl tyrosine kinase can mediate tumor cell apoptosis independently of the Rb and p53 tumor suppressors.

Tumor cells frequently lack the p53 tumor suppressor because p53 mediates apoptosis in these cells. We report here that c-Abl, and to a greater extent a c-Abl mutant defective for DNA-binding, can provoke programmed cell death in p53-deficient tumor cells. Tyrosine kinase mutant K290R is less cytotoxic. In contrast, a C-terminal deletion mutant that lacks the RNA polymerase 11 (PolII)/actin interaction domain, fails to mediate apoptosis unless expressed to very high levels. Cytotoxicity is overcome by coexpression of the apoptosis antagonist E1B 19K protein, and partially overcome by full-length retinoblastoma protein (Rb) or the C pocket fragment of Rb (SEA) that associates with c-Abl. c-Abl is also highly toxic to Saos-2 cells that are deficient for both Rb and p53, indicating that cell death is not the result of inhibition through c-Abl of the anti-apoptotic function of Rb. Finally, p53 and c-Abl combined induce apoptosis stronger than either protein alone. Unlike c-Abl-mediated cell death, apoptosis by p53 is antagonized efficiently only by full-length Rb with intact A/B pocket but not by SEA. Mutant p53 inhibits apoptosis by p53 but not c-Abl. Thus, c-Abl with intact kinase and PolII/ actin-binding domains can affect tumor cell survival independently of Rb and p53.

p53 transactivation domain mutant Q22, S23 is impaired for repression of promoters and mediation of apoptosis.

p53 is multifunctional. To assess exactly what function is critical for the prevention of neoplastic transformation has proven difficult. Mutants with substitutions at positions 22 and 23 promised to address the relevance of transcription transactivation since they seemed to be defective specifically for this function. We report here that p53 mutant Q22, S23 [p53 (22,23)] is not only impaired for transactivation but for the repression of the fos promoter and SV40 early promoter. Furthermore, whereas p53 (22,23) fails to efficiently transactivate reporter genes in two p53-negative cell lines, it stimulates reporters and suppresses proliferation in two wild-type (wt) p53-positive cell lines strongly above the levels induced by the transfection procedure alone. This transactivation is refractory to inhibition by MDM-2. Finally, p53 (22,23) expressed from large plasmid quantity (1 microg) is crippled for the mediation of apoptosis in p53-negative Hep3B hepatocarcinoma cells. Nevertheless, at a quantity of only 10 ng, both mutant and wt p53 plasmids but not control plasmid, are able to induce some cell death which is not inhibitable by MDM-2. Thus, a correlation exists between p53's functions to regulate promoters and to efficiently mediate apoptosis in Hep3B cells.

p21 Waf1/Cip1 can protect human colon carcinoma cells against p53-dependent and p53-independent apoptosis induced by natural chemopreventive and therapeutic agents.

The molecular basis for the sensitivity of tumor cells to chemopreventive natural food compounds and commonly used chemotherapeutic agents is not well understood, not least because studies are frequently confounded by the diversity among cell lines or rely on experimental protein overexpression. Here we investigated the effects of n-butyrate, a cancer-preventive short-chain fatty acid produced by anaerobic bacteria in the gastrointestinal tract, on the human wild-type p53 and p21 expressing HCT116 colon carcinoma cell line and on HCT116 cells with either p53 or p21 alleles inactivated by homologous recombination. The effects of n-butyrate were then compared with those elicited by cytotoxic drugs and the natural chemopreventive phytoalexin of wine and grapes, resveratrol. We document that physiological concentrations of n-butyrate stimulate p21 expression and induce apoptosis independently of p53, and that the absence of p21 increases apoptosis drastically. The apoptosis is mediated through the mitochondria and is accompanied by mitochondrial proliferation and membrane potential changes. Adriamycin, etoposide, cisplatinum, colcemid and resveratrol induce distinct cellular responses; however, absence of p21 favors apoptosis-induction by adriamycin, etoposide and colcemid. Thus, control of p21 expression may support chemoprevention and certain tumor therapies.

Human endogenous retrovirus protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein.

Human endogenous retrovirus sequences (HERVs) reside in the genomes of primates and humans for several million years. The majority of HERVs is non-coding but a limited set is intact and can express proteins. We have recently identified an almost intact HERV-K(HML-2) provirus on chromosome 7 and have documented that most patients with germ cell tumors (GCTs) display antibodies directed against proteins of HERV-K(HML-2). To address whether these proteins merely represent tumor markers or contribute to neoplastic transformation, we examined the transforming potential of various HERV sequences and studied physical interactions between HERV and cellular proteins by yeast two-hybrid and biochemical assays. cORF, a protein encoded by the C-terminal open reading frame within the env gene, supports tumor growth in nude mice and associates with the promyelocytic leukemia zinc finger protein (PLZF). The interaction domains map between amino acid residues 21 and 87 of cORF, and between residues 245 and 543 of PLZF. PLZF is critical for spermatogenesis in mice. Abnormal spermatogenesis or maturation of gonocytes is thought to predispose humans to the development of germ cell tumors. Thus, cORF of human endogenous retroviruses may contribute to tumor development by interfering with processes during spermatogenesis that involve PLZF.

Inhibition of p53 protects liver tissue against endotoxin-induced apoptotic and necrotic cell death.

There is increasing evidence that apoptotic and necrotic hepatocyte death following endotoxin-induced liver injury act as signals for leukocyte sequestration in the liver vasculature. p53 has been implicated to promote apoptosis through trans-activation and down-regulation of specific pro- and anti-apoptotic genes. Here, we report that inhibition of p53 decreases apoptotic and necrotic tissue injury as well as inflammatory cell response. Sprague-Dawley rats were injected intraperitoneally with 2.2 mg/kg pifithrin-alpha (PFT), a p53-inactivating agent, or the vehicle DMSO 30 min before intravenous exposure to lipopolysaccharide (LPS). In vehicle-pretreated animals, LPS induced significant apoptosis and necrosis of hepatocytes, which was associated with intrahepatic leukocyte recruitment, microvascular dysfunction, and enzyme release. Inhibition of p53 effectively attenuated (P<0.05) hepatocellular apoptosis and necrosis, but also reduced leukocyte recruitment and microvascular dysfunction. Western blot analysis revealed that PFT lowered the nuclear-to-cytoplasmic p53 ratio and reduced both activation of NF-kappaB and cleavage of procaspase 3 (P<0.05). In parallel, immunohistochemistry of PFT-pretreated, but not vehicle-pretreated, endotoxic animals exhibited nuclear p53 exclusion and reduced NF-kappaB p65 staining. This indicates that p53 mediates, at least in part, LPS-associated apoptosis and contributes to inflammatory endotoxic tissue injury through leukocyte activation and intraorgan sequestration.

Simulation and experimental verification of prompt gamma-ray emissions during proton irradiation.

Irradiation with protons and light ions offers new possibilities for tumor therapy but has a strong need for novel imaging modalities for treatment verification. The development of new detector systems, which can provide an in vivo range assessment or dosimetry, requires an accurate knowledge of the secondary radiation field and reliable Monte Carlo simulations. This paper presents multiple measurements to characterize the prompt γ-ray emissions during proton irradiation and benchmarks the latest Geant4 code against the experimental findings. Within the scope of this work, the total photon yield for different target materials, the energy spectra as well as the γ-ray depth profile were assessed. Experiments were performed at the superconducting AGOR cyclotron at KVI-CART, University of Groningen. Properties of the γ-ray emissions were experimentally determined. The prompt γ-ray emissions were measured utilizing a conventional HPGe detector system (Clover) and quantitatively compared to simulations. With the selected physics list QGSP_BIC_HP, Geant4 strongly overestimates the photon yield in most cases, sometimes up to 50%. The shape of the spectrum and qualitative occurrence of discrete γ lines is reproduced accurately. A sliced phantom was designed to determine the depth profile of the photons. The position of the distal fall-off in the simulations agrees with the measurements, albeit the peak height is also overestimated. Hence, Geant4 simulations of prompt γ-ray emissions from irradiation with protons are currently far less reliable as compared to simulations of the electromagnetic processes. Deviations from experimental findings were observed and quantified. Although there has been a constant improvement of Geant4 in the hadronic sector, there is still a gap to close.

Cell type-specific regulation of calmodulin 2 expression by mutant p53.

To identify genes that are stimulated by oncogenic forms of mutant p53, we studied, by microarray analysis and PCR-select subtractive hybridization, gene expression changes in human wild-type (wt) p53-negative immortal 041 fibroblasts infected to stably express p53 mutant 175H. In contrast to the wt p53 transactivator, 175H induced only few and weak, gene expression changes. We report here the stimulation of calmodulin 2 (CaM 2), but not CaM 1 or 3, gene expression specifically in 041 cells. The stimulation of the CaM 2 promoter required the 5' untranslated sequences as well as the integrity of the transactivation domain of 175H. However, direct binding of 175H to the 5'UT in vitro could not be demonstrated.

Identification of a putative p53 binding sequence within the human mitochondrial genome.

A small fraction of the total cellular amount of nuclear transcription factor p53 seems to be located at and within mitochondria. Transcription factors of the steroid receptor superfamily that, like p53, lack a classical mitochondrial leader sequence are nonetheless imported into mitochondria where they regulate mtDNA transcription through binding to specific recognition sequences. Here, we examined seven candidate sequences from the human mitochondrial genome with similarity to the consensus p53 binding motif. Two imperfect half-sites at coordinate 1553 with homology to the nuclear IGF-BP3 box A binding sequence are demonstrated to confer responsivity to p53 and the p53 relatives p73alpha and beta in the context of the cell nucleus. Mitochondrial p53 may thus bind directly to mtDNA and, perhaps, be involved in the regulation of mitochondrial transcription/replication.

Transduction of foreign regulatory sequences by a replication-defective herpes simplex virus type 1: the rat neuron-specific enolase promoter.

Herpes simplex virus type 1 (HSV-1) can transduce genes into non-proliferating cells such as neurons that are refractory to other means of gene transfer. We have been interested to examine the potential usefulness of HSV-1 as a gene transfer vehicle to analyze neuron-specific regulatory sequences. In this study, we have used a replication-defective HSV-1-based vector deleted for the essential immediate early gene 3 (IE3) to transduce a 1.8 kb promoter fragment from the rat neuron-specific enolase gene (nse) linked to the firefly luciferase reporter gene (luc). It has previously been shown that the same promoter fragment is capable of directing neuron-specific expression of a linked reporter gene in transgenic mice. As an internal control for infection and gene expression, we also inserted the chloramphenicol acetyltransferase (cat) gene driven by the SV40 early promoter/enhancer into the thymidine kinase locus of the same vector. We infected (i) non-neuronal BHK-C13 cells which do not express the endogenous nse gene, (ii) differentiated and non-differentiated pheochromocytoma PC12 cells as well as (iii) N1E-115 neuroblastoma cells, all of which do express endogenous nse. All three cell types produced luciferase upon infection, indicating that the same nse promoter fragment that has previously been shown to be regulated in a cell-specific manner in transgenic mice, was not regulated cell type-specifically in the context of the HSV-1 genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a recombinant line blot for diagnosis of Epstein-Barr Virus compared with ELISA, using immunofluorescence as reference method.

A commercial line blot using recombinant antigens was compared with a commercial ELISA and 'in-house' IFA (reference test). Two panels were evaluated: Panel A was selected to distinguish between primary infections (89), past infections (20) and seronegatives (8) in immunocompetent individuals. In panel B, patients with a high number of reactivations were included: immunosuppressed patients (37), lymphoma (19), nasopharyngeal carcinoma (10), chronic fatigue syndrome (14). Blood donors (43) and cross-reactive sera (29) were added as controls. Line blot and IFA were concordant in 94% of primary infections, 100% of seronegatives and 100% of past infections, similar to ELISA. Results differed significantly with regard to reactivations. When compared with IFA, the incidence of reactivations was overestimated by the blot, 24 and 58% in blood donors and cross-reactive sera, respectively. ELISA showed a similar problems with 21 and 34% indeterminate results, respectively. The line blot is easy to carry out, has a good concordance with the reference IFA for primary infections, and is, therefore, a sufficient choice for distinguishing primary infection from seronegative and past infection. EBV reactivation assessment will require other methods such as EBV viral load.

Motor neuron cell death in a mouse model of FALS is not mediated by the p53 cell survival regulator.

Mutant Cu/Zn superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS) causes selective motor neuron loss through unknown mechanisms of cell damage. Damaged neurons frequently undergo apoptosis mediated by the p53 cell survival regulator. We therefore studied whether motor neuron disease (MND) in mice expressing the human SOD1 mutant G93A is dependent on p53 by crossing G93A mice with p53-knockout mice. Since p53-/- mice's life expectance is usually shorter (160+/-49 days, n=11) than the time at which the G93A mice die from MND (212+/-50 days, n=7), only a few of the G93A/p53-/- double transgenics were expected to live to experience MND. Nevertheless, four of the 22 G93A/p53-/- mice succumbed to MND after 160+/-28 days, as expected under these conditions of competing death risks if the absence of p53 fails to protect from MND. Thus, MND in mice expressing G93A does not require p53. This conclusion is supported by histology: pre-symptomatic G93A mice display disease-associated vacuoles within the dendrites of motor neurons regardless of p53 status.

Artificial sperm reservoir on epididymis using pedicled tunica vaginalis for the management of conductive azoospermia.

In persons with azoospermia due to obstructive or anejaculatory causes, implantation of alloplastic silicone reservoirs onto the epididymis for collection of spermatozoa for artificial insemination, with eventual pregnancy, has become technically feasible, although there have been poor results from the procedure. Our experimental study on dogs indicates that such an epididymal reservoir can easily be constructed from a pedicled tunica vaginalis flap. Observations on the qualitative and quantitative changes in the spermatozoa of these reservoirs and conjecture about possible means of improvement of these parameters are discussed.