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Sample records for 99tcm labeling mdm2

  1. Imaging inflammation with 99Tcm-labeled chemotactic peptides: analogues with reduced neutropenia.

    PubMed

    Pollak, A; Goodbody, A E; Ballinger, J R; Duncan, G S; Tran, L L; Dunn-Dufault, R; Meghji, K; Lau, F; Andrey, T W; Boxen, I; Sumner-Smith, M

    1996-02-01

    Two 99Tcm-labelled analogues of the chemotactic peptide ForMLF were evaluated as potential agents for imaging inflammation and infection, in the hope that they would be simple to use and would give diagnostically useful images shortly after injection. The peptides differed in the chelation site for 99Tcm and the presence of a hydrophilic spacer. The sequences of RP050 and RP056 were ForNleLFNleYK(G)G-C(Acm)-GPic and ForNleLFNleYKK(DG)GC(Acm)SPic respectively, where Pic is picolinic acid. In in vitro tests of binding to the ForMLF receptor on polymorphonuclear neutrophils and potency for release of myeloperoxidase, RP056 was similar in potency to ForMLF, whereas RP050 was 10 times more potent. When administered in 5-nmol doses to rats, RP050 produced less extensive neutropenia than ForMLF, whereas RP056 produced very little neutropenia. Following labelling by ligand exchange from tartrate or glucoheptonate at 100 degrees C and purification using a C-18 solid-phase extraction cartridge, 4-MBq doses were administered to rats bearing infectious (Escherichia coli) or sterile (zymosan) inflammation sites in the thigh. The inflammation-to-normal muscle ratios at 30 min after injection were 3.9 +/- 0.4 for RP050 and 4.7 +/- 0.3 for RP056 (mean +/- S.E.M., n = 4), and the ratios were maintained for up to 3 h. These peptides are promising agents for imaging inflammation and infection.

  2. Functional assessment of colonic interposition with 99Tcm-labeled milk.

    PubMed

    Sutton, R; Sutton, H; Ackery, D M; Freeman, N V

    1989-09-01

    A prospective study was performed to evaluate the use of radionuclide imaging in the assessment of patients who have undergone esophageal reconstruction. Dynamic radionuclide imaging was performed on ten patients aged 11 months to 11 years who had undergone colon interposition via the normal esophageal route for esophageal atresia. Patients were considered clinically unsatisfactory if at the time of imaging feeding was troubled, or if weight gain had fallen to below the third centile before operation and remained below after operation. Each patient underwent erect imaging with a small milk feed labeled with 10 to 20 MBq (250 to 500 muCi) 99Tcm DTPA adjusted according to body surface area; supine studies were subsequently performed on five patients. All five clinically unsatisfactory patients showed conduit emptying delayed beyond 45 minutes and/or spontaneous reflux, significantly different from the clinically satisfactory patients (X2 = 6.4, P less than .02). Conduit complications were subsequently identified in three of the five clinically unsatisfactory patients. Radionuclide imaging with radiolabeled milk was found to be well tolerated, and obtained results that were clinically useful. These results suggest that dynamic radionuclide imaging can be more widely applied in the assessment of esophageal substitutes.

  3. 99Tcm-HIG accumulates in the synovial tissue of rats with adjuvant arthritis by binding to extracellular matrix proteins.

    PubMed

    de Bois, M H; Welling, M; Verwey, C L; de Vries, E; Pauwels, E K; Breedveld, F C; Tak, P P

    1996-01-01

    Our objective was to investigate the mechanism of accumulation of 99Tcm-labelled non-specific polyclonal human immunoglobulin (99Tcm-HIG) in inflamed synovial tissue (ST) in an experimental animal model of arthritis. Following 99Tcm-HIG scintigraphy, the in vivo localization of 99Tcm-HIG in the ST of knee joints of rats with adjuvant arthritis was studied using immunohistochemical techniques. In addition, the in vitro binding of 99Tcm-HIG to extracellular matrix proteins was analysed by means of immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). After 99Tcm-HIG scintigraphy, 99Tcm-HI was detected in the ST of rats with adjuvant arthritis. 99Tcm-HIG was diffusely distributed and not bound to cells. In vitro incubation of 99Tcm-HIG on the ST of rats with adjuvant arthritis revealed binding of 99Tcm-HIG to inflamed, but not to non-inflamed, ST. In addition, specific binding of 99Tcm-HIG to fibronectin, fibrin, collagen type I and III was demonstrated by ELISA. We conclude that the accumulation of 99Tcm-HIG in inflamed ST can be explained by the binding of 99Tcm-HIG to extracellular matrix proteins.

  4. Clinical evaluation of metastases of malignant melanoma imaging with 99Tcm-glutathione and 99Tcm-anti-melanoma antibody: a comparative study.

    PubMed

    Duman, Y; Burak, Z; Ercan, M T; Dirlik, A; Bilkay, B C; Akin, Y; Taner, M; Bekdik, C F

    1995-11-01

    The aim of this investigation was to test for the scintigraphic detection of metastases of malignant melanoma with a new radiopharmaceutical, 99Tcm-glutathione (99Tcm-GSH), in comparison with 99Tcm-anti-melanoma antibody (99Tcm-AMAb). Glutathione was labelled with 99Tcm by a Sn2+ reduction method with an efficiency of > 99% as determined by instant thin layer chromatography (ITLC). Anti-melanoma antibody was obtained as a kit from SORIN (Italy) and labelled with 99TcmO-4. Forty-three patients with a total of 55 biopsy-proven metastatic melanoma foci, 1 ocular melanoma and 20 benign pathologic foci, also confirmed by ultrasound, computed tomography and magnetic resonance imaging, were included in the study after giving their informed consent. Following the intravenous (i.v.) injection of 500 MBq 99Tcm-AMAb, scintigraphic images of the involved areas were obtained 6 h post-injection. Three days later, the same patients were given 500 MBq 99Tcm-GSH i.v. and images were obtained 6 and 24 h post-injection. The images were classified as positive (focal abnormal accumulation) or negative. Quantitative evaluation was also applied. Regions of interest were drawn over the involved areas and nearby soft tissues and the target-to-nontarget (T/NT) ratios obtained with 99Tcm-AMAb (T/NT: 1.92 +/- 0.2) and 99Tcm-GSH (T/NT: 1.84 +/- 0.2) were compared (0.1 < P < or = 0.3). The sensitivity (and specificity) of 99Tcm-AMAb and 99Tcm-GSH in the detection of malignant melanoma metastases were 91% (95%) and 84% (90%), respectively. Compared with 99Tcm-AMAb, the advantages of 99Tcm-GSH are lower levels of blood radioactivity, lower costs and easy in-house preparation. In conclusion, our results show that 99Tcm-GSH is a potentially useful radiopharmaceutical for the detection of metastases of malignant melanoma.

  5. Radiolabelled mixed leukocytes and pure granulocytes with stabilized 99Tcm-exametazime.

    PubMed

    Hung, J C; Chowdhury, S; Mahoney, D W; Mullan, B P

    1998-10-01

    Although the methylene blue stabilizer extends the shelf life of 99Tcm-exametazime to 4-6 h after reconstitution, the dark blue appearance of the mixture of stabilized 99Tcm-exametazime and blood components makes it impossible to separate out the leukocyte button. The aim of this study was to assess the feasibility of using stabilized 99Tcm-exametazime to radiolabel mixed leukocytes separated by Volex sedimentation with hypotonic lysis (VL) and pure granulocytes isolated by a single-density Ficoll-Hypaque gradient with hypotonic lysis (FL). Isolated cells from 40-ml and 80-ml donor blood samples were mixed with 0.5 ml stabilized 99Tcm-exametazime (approximately 925 MBq 99Tcm and 62.5 micrograms exametazime) and incubated at room temperature for 15 min. After incubation, two dilution steps with 3 ml and 9 ml of 12.6% ACD/NS (anticoagulant citrate dextrose, solution A, USP, mixed with 0.9% NaCl, v/v) were conducted to dilute the dark blue mixture and to remove any unbound 99Tcm activity. With the addition of 9 ml of 12.6% ACD/NS solution to the 1-ml bottom portion from the first dilution, the supernatant of the centrifuged preparation was clear enough to be withdrawn. The overall labelling efficiency (LE) of labelled leukocytes and granulocytes was 87.1 +/- 4.9% and 87.7 +/- 6.2%, respectively (n = 12 each). Overall, radiolabelled cells (n = 12) from the 80-ml blood samples (LE = 90.3 +/- 2.8%) had an approximately 6% higher labelling efficiency than from the 40-ml blood samples (LE = 84.5 +/- 6.0%) and also had a slightly better in vitro stability compared to the 40-ml samples. The in vitro stability studies showed that only approximately 2% (n = 48) 99Tcm activity was eluted each hour from the radiolabelled leukocytes or granulocytes for the 40-ml or 80-ml blood samples during the 6-h evaluation period. Cell viability of all labelled leukocyte samples was confirmed by the trypan blue staining technique. In conclusion, mixed leukocytes separated by the VL method and

  6. Comparison between scintigraphy with polyclonal immunoglobulin (99Tcm-HIG) and physical examination in polyarthritic disease.

    PubMed

    Vesterskiöld, L; Schnell, P O; Jacobsson, H

    1996-01-01

    99Tcm-labelled polyclonal human immunoglobulin 99Tcm-HIG scintigraphy has been suggested as a technique to detect joint inflammation in arthritic disorders. Scintigraphy was performed in fifteen patients with active polyarthritis. All joints except the hips were scored clinically for swelling and pain by a rheumatologist and scintigraphic images were obtained at 30 minutes and four hours after injection of 350MBq 99Tcm-HIG. The images were assessed by a nuclear medicine physician according to a four grade scale. The images were easy to assess. There was a highly significant correlation between swelling and scan score, but no correlation between pain and scan score. The mechanism for the accumulation of activity to inflamed synovial tissue remains unclear. The mean values of the scan score however increased significantly between 30 minutes and 4 hours, indicating an active binding mechanism. The method has a potential as an objective tool in monitoring rheumatic diseases.

  7. Evaluation of 99Tcm-HIG as a lymphoscintigraphic agent in rabbits.

    PubMed

    Ergün, E L; Ercan, M T; Aşansü, A; Unsal, I S

    1998-07-01

    In this pilot study, 99Tcm-labelled human immunoglobulin G (99Tcm-HIG) was evaluated as a lymphoscintigraphic agent in five rabbits. It was injected intradermally into the web space of the hind legs of the rabbits (37 MBq/0.1 ml). Sequential scintigrams were obtained using a gamma camera for 120 min. The injection site and the hind legs were massaged post-injection. Blood samples were obtained at 5, 15, 30, 60, 90 and 120 min. Two of the rabbits were killed after 2 h. Their organs were weighed and tissue specimens were obtained, weighed and counted against a standard using a gamma counter. The lymph channels and the lymph nodes were well visualized on the scintigrams. The background activity was very low, making interpretation easier. About 30% of the injected dose migrated from the injection site by 2 h. The mean popliteal lymph node uptake was 5.71 +/- 4.62% per gram of tissue. The lymph node to other tissue concentration ratios were very high, ranging from 63:1 for the kidneys to 1099:1 for the heart. We conclude that 99Tcm-HIG is a promising new agent for the visualization of the lymphatic system due to its easy labelling procedure, the stability of the label, its widespread availability and good image quality. It may potentially be useful in detecting and evaluating inflammatory lymph nodes.

  8. The MDM2 gene family.

    PubMed

    Mendoza, Michael; Mandani, Garni; Momand, Jamil

    2014-03-01

    MDM2 is an oncoprotein that blocks p53 tumor suppressor-mediated transcriptional transactivation, escorts p53 from the cell nucleus to the cytoplasm, and polyubiquitylates p53. Polyubiquitylated p53 is rapidly degraded in the cytoplasm by the 26S proteasome. MDM2 is abnormally upregulated in several types of cancers, especially those of mesenchymal origin. MDM4 is a homolog of MDM2 that also inhibits p53 by blocking p53-mediated transactivation. MDM4 is required for MDM2-mediated polyubiquitylated of p53 and is abnormally upregulated in several cancer types. MDM2 and MDM4 genes have been detected in all vertebrates to date and only a single gene homolog, named MDM, has been detected in some invertebrates. MDM2, MDM4, and MDM have similar gene structures, suggesting that MDM2 and MDM4 arose through a duplication event more than 440 million years ago. All members of this small MDM2 gene family contain a single really interesting new gene (RING) domain (with the possible exception of lancelet MDM) which places them in the RING-domain superfamily. Similar to MDM2, the vast majority of proteins with RING domains are E3 ubiquitin ligases. Other RING domain E3 ubiquitin ligases that target p53 are COP1, Pirh2, and MSL2. In this report, we present evidence that COP1, Pirh2, and MSL2 evolved independently of MDM2 and MDM4. We also show, through structure homology models of invertebrate MDM RING domains, that MDM2 is more evolutionarily conserved than MDM4. PMID:25372739

  9. Differential renal function measured by 99Tcm-DTPA and 99Tcm-DMSA in a complete unilateral renal obstruction rat model.

    PubMed

    van de Wiele, C; Everaert, K; van der Eecken, H; van Haelst, J P; Simons, M; Dierckx, R A

    1997-11-01

    We performed a prospective study to determine whether 99Tcm-DTPA differential renal function (DRF) in the case of acute obstruction in a unipapillary kidney rat model shows a similar pattern of results as 99Tcm-DMSA in a multipapillary kidney model, and to exclude recirculation or a block of tubular transport of DMSA which might explain the discrepancy reported by Kelleher et al. In 15 male and 4 female Wistar rats (weight 300-350 g), 99Tcm-DMSA renal uptake was measured 24 h before complete obstruction of the right ureter, which served as reference values. Twenty-four hours after obstruction, 99Tcm-DTPA (18.5 MBq) renography and 99Tcm-DMSA (111 MBq) non-depth-corrected renal uptake measurements were performed in the 15 male rats; in the 4 female rats, 740 MBq of 99Tcm-DMSA were injected and absolute, non-depth-corrected renal uptake measured at 24, 48 and 72 h. 99Tcm-DMSA DRF of the right kidney ranged from 48 to 55% (mu: 51%; sigma: 2%) before obstruction and from 14 to 35% (mu: 24%; sigma: 6%) after obstruction, whereas 99Tcm-DTPA DRF ranged from 16 to 30% (mu: 25%; sigma: 4%). No significant differences were found between DRF measured by 99Tcm-DMSA and 99Tcm-DTPA (P = 0.18), or between DRF of the obstructed kidney as measured by 99Tcm-DMSA at 24, 48 and 72 h (P > 0.2). Hypothetically, the discrepancy between our findings and those of Kelleher et al. may be due to intratubular pressure. In conclusion, the present findings and those of Kelleher et al. suggest the differences in DRF following complete unilateral renal obstruction, as determined by DTPA and DMSA, are probably species-specific. Furthermore, recirculation and block of tubular transport are unlikely to contribute significantly to differences in DRF as measured by the two radiopharmaceuticals.

  10. 99Tcm-LL1: a potential new bone marrow imaging agent.

    PubMed

    Juweid, M; Dunn, R M; Sharkey, R M; Rubin, A D; Hansen, H J; Goldenberg, D M

    1997-02-01

    LL1, a monoclonal antibody (MAb) to HLA Class-II-like antigen (li determinant) on the surface of B-lymphocytes, monocytes and histiocytes, was evaluated as an agent for bone marrow imaging. Six patients with diverse diseases (non-Hodgkin's lymphoma, n = 2; multiple myeloma, n = 1; polycythaemia vera, n = 1; lung cancer, n = 1; breast cancer, n = 1) were given low protein doses (< 1 mg) of 99Tcm (30 mCi) labelled Fab' of LL1. 99Tcm-sulphur colloid (SC) imaging was performed in three patients for comparison. Both planar and single photon emission tomographic images were acquired using Sopha gamma cameras. As early as 2 h post-MAb injection, excellent bone marrow images were achieved in all patients, demonstrating both normal or hyperproliferative marrow, as well as 'cold' bone marrow abnormalities such as radiation defects or cancer metastases. Similar to SC, relatively high uptake of LL1 was found in the liver and spleen. However, the bone marrow-to-liver and -spleen uptake ratios were approximately 19-fold higher (0.75 vs 0.04) and 6-fold higher (1.23 vs 0.22), respectively, with LL1 than with SC. The higher bone marrow uptake allowed clearly superior visualization of the thoracic spine when compared to SC. The mean T1/2 of blood and whole-body clearance were 0.4 and 66 h, respectively. The highest radiation absorbed doses (in cGy mCi-1) were observed in the spleen (0.47 +/- 0.24), kidneys (0.25 +/- 0.09) and liver (0.14 +/- 0.04). The bone marrow dose was only 0.05 +/- 0.02 cGy mCi-1. These results indicate that bone marrow imaging with 99Tcm-LL1 is feasible, and that LL1 may be a suitable alternative to SC because of better visualization of the lower thoracic spine. Potential applications include the improved detection of bone marrow metastases of solid tumours and the assessment of haematological disorders. PMID:9076770

  11. The MDM2 gene amplification database.

    PubMed Central

    Momand, J; Jung, D; Wilczynski, S; Niland, J

    1998-01-01

    The p53 tumor suppressor gene is inactivated in human tumors by several distinct mechanisms. The best characterized inactivation mechanisms are: (i) gene mutation; (ii) p53 protein association with viral proteins; (iii) p53 protein association with the MDM2 cellular oncoprotein. The MDM2 gene has been shown to be abnormally up-regulated in human tumors and tumor cell lines by gene amplification, increased transcript levels and enhanced translation. This communication presents a brief review of the spectrum of MDM2 abnormalities in human tumors and compares the tissue distribution of MDM2 amplification and p53 mutation frequencies. In this study, 3889 samples from tumors or xenografts from 28 tumor types were examined for MDM2 amplification from previously published sources. The overall frequency of MDM2 amplification in these human tumors was 7%. Gene amplification was observed in 19 tumor types, with the highest frequency observed in soft tissue tumors (20%), osteosarcomas (16%) and esophageal carcinomas (13%). Tumors which showed a higher incidence of MDM2 amplification than p53 mutation were soft tissue tumors, testicular germ cell cancers and neuro-blastomas. Data from studies where both MDM2 amplification and p53 mutations were analyzed within the same samples showed that mutations in these two genes do not generally occur within the same tumor. In these studies, 29 out of a total of 33 MDM2 amplification-positive tumors had wild-type p53. We hypothesize that heretofore uncharacterized carcinogens favor MDM2 amplification over p53 mutations in certain tumor types. A database listing the MDM2 gene amplifications is available on the World Wide Web at http://www. infosci.coh.org/mdm2 . Charts of MDM2 amplification frequencies and comparisons with p53 genetic alterations are also available at this Web site. PMID:9671804

  12. Chemosensitization by antisense oligonucleotides targeting MDM2.

    PubMed

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  13. Early myocardial damage assessment in dystrophinopathies using 99Tcm-MIBI gated myocardial perfusion imaging

    PubMed Central

    Zhang, Li; Liu, Zhe; Hu, Ke-You; Tian, Qing-Bao; Wei, Ling-Ge; Zhao, Zhe; Shen, Hong-Rui; Hu, Jing

    2015-01-01

    Background Early detection of muscular dystrophy (MD)-associated cardiomyopathy is important because early medical treatment may slow cardiac remodeling and attenuate symptoms of cardiac dysfunction; however, no sensitive and standard diagnostic method for MD at an earlier stage has been well-recognized. Thus, the aim of this study was to test the early diagnostic value of technetium 99m-methoxyisobutylisonitrile (99Tcm-MIBI) gated myocardial perfusion imaging (G-MPI) for MD. Methods and results Ninety-one patients underwent 99Tcm-MIBI G-MPI examinations when they were diagnosed with Duchenne muscular dystrophy (DMD) (n=77) or Becker muscular dystrophy (BMD; n=14). 99Tcm-MIBI G-MPI examinations were repeated in 43 DMD patients who received steroid treatments for 2 years as a follow-up examination. Myocardial defects were observed in nearly every segment of the left ventricular wall in both DMD and BMD patients compared with controls, especially in the inferior walls and the apices by using 99Tcm-MIBI G-MPI. Cardiac wall movement impairment significantly correlated with age in the DMD and BMD groups (rs=0.534 [P<0.05] and rs=0.784 [P<0.05], respectively). Intermittent intravenous doses of glucocorticoids and continuation with oral steroid treatments significantly improved myocardial function in DMD patients (P<0.05), but not in BMD patients. Conclusion 99Tcm-MIBI G-MPI is a sensitive and safe approach for early evaluation of cardiomyopathy in patients with DMD or BMD, and can serve as a candidate method for the evaluation of progression, prognosis, and assessment of the effect of glucocorticoid treatment in these patients. PMID:26677332

  14. Is computer-aided interpretation of 99Tcm-HMPAO leukocyte scans better than the naked eye?

    PubMed

    Almer, S; Peters, A M; Ekberg, S; Franzén, L; Granerus, G; Ström, M

    1995-04-01

    In order to compare visual interpretation of inflammation detected by leukocyte scintigraphy with that of different computer-aided quantification methods, 34 patients (25 with ulcerative colitis and 9 with endoscopically verified non-inflamed colonic mucosa), were investigated using 99Tcm-hexamethylpropyleneamine oxime (99Tcm-HMPAO) leukocyte scintigraphy and colonoscopy with biopsies. Scintigrams were obtained 45 min and 4 h after the injection of labelled cells. Computer-generated grading of seven colon segments using four different methods was performed on each scintigram for each patient. The same segments were graded independently using a 4-point visual scale. Endoscopic and histological inflammation were scored on 4-point scales. At 45 min, a positive correlation was found between endoscopic and scan gradings in individual colon segments when using visual grading and three of the four computer-aided methods (Spearman's rs = 0.30-0.64, P < 0.001). Histological grading correlated with visual grading and with two of the four computer-aided methods at 45 min (rs = 0.42-0.54, P < 0.001). At 4 h, all grading methods correlated positively with both endoscopic and histological assessment. The correlation coefficients were, in all but one instance, highest for the visual grading. As an inter-observer comparison to assess agreement between the visual gradings of two nuclear physicians, 14 additional patients (9 ulcerative colitis, 5 infectious enterocolitis) underwent leukocyte scintigraphy. Agreement assessed using kappa statistics was 0.54 at 45 min (P < 0.001). Separate data concerning the presence/absence of active inflammation showed a high kappa value (0.74, P < 0.001). Our results showed that a simple scintigraphic scoring system based on assessment using the human eye reflects colonic inflammation at least as well as computer-aided grading, and that highly correlated results can be achieved between different investigators.

  15. 99Tcm-MAG3 renogram deconvolution in normal subjects and in normal functioning kidney grafts.

    PubMed

    González, A; Puchal, R; Bajén, M T; Mairal, L; Prat, L; Martin-Comin, J

    1994-09-01

    This study provides values of transit times obtained by 99Tcm- mercaptoacetyl triglycine (99Tcm-MAG3) renogram deconvolution for both normal subjects and kidney graft recipients. The analysis included 50 healthy kidney units from 25 volunteers and 28 normal functioning kidney grafts. The parameters calculated for the whole kidney (WK) and for the renal parenchyma (P) were: mean transit time (MTT) and times at 20% (T20) and 80% (T80) of renal retention function initial height. For healthy kidneys the WK MTT was 174 +/- 27 s and P MTT 148 +/- 22 s. The WK T20 values were 230 +/- 33 s and P T20 231 +/- 34 s. The WK T80 was 108 +/- 19 s and P T80 106 +/- 12 s. Whole kidney and parenchymal values of transit times for normal functioning kidney grafts do not present significant differences with respect to healthy kidneys. PMID:7816379

  16. Regulation of p53 and MDM2 Activity by MTBP

    PubMed Central

    Brady, Mark; Vlatković, Nikolina; Boyd, Mark T.

    2005-01-01

    p53 is a critical coordinator of a wide range of stress responses. To facilitate a rapid response to stress, p53 is produced constitutively but is negatively regulated by MDM2. MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain, inhibiting p53 acetylation, promoting nuclear export, and probably most importantly by promoting proteasomal degradation of p53. The latter is achieved via MDM2's E3 ubiquitin ligase activity harbored within the MDM2 RING finger domain. We have discovered that MTBP promotes MDM2-mediated ubiquitination and degradation of p53 and also MDM2 stabilization in an MDM2 RING finger-dependent manner. Moreover, using small interfering RNA to down-regulate endogenous MTBP in unstressed cells, we have found that MTBP significantly contributes to MDM2-mediated regulation of p53 levels and activity. However, following exposure of cells to UV, but not γ-irradiation, MTBP is destabilized as part of the coordinated cellular response. Our findings suggest that MTBP differentially regulates the E3 ubiquitin ligase activity of MDM2 towards two of its most critical targets (itself and p53) and in doing so significantly contributes to MDM2-dependent p53 homeostasis in unstressed cells. PMID:15632057

  17. Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein-protein interactions by the MDM2 ligand nutlin-3.

    PubMed

    Way, Luke; Faktor, Jakub; Dvorakova, Petra; Nicholson, Judith; Vojtesek, Borek; Graham, Duncan; Ball, Kathryn L; Hupp, Ted

    2016-09-01

    Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53-independent MDM2-drug responsive-binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin-3 responsive MDM2-binding proteins that are perturbed independent of cell density using SWATH-MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin-3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin-3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin-3 treated cells. Mitotracker confirmed that Nutlin-3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin-3 treatment. Proximity ligation identified rearrangements of cellular protein-protein complexes in situ. In response to Nutlin-3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein-protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein-protein complexes in drug-treated cells. PMID:27273042

  18. Rearrangement of mitochondrial pyruvate dehydrogenase subunit dihydrolipoamide dehydrogenase protein–protein interactions by the MDM2 ligand nutlin‐3

    PubMed Central

    Way, Luke; Faktor, Jakub; Dvorakova, Petra; Nicholson, Judith; Vojtesek, Borek; Graham, Duncan; Ball, Kathryn L.

    2016-01-01

    Drugs targeting MDM2's hydrophobic pocket activate p53. However, these agents act allosterically and have agonist effects on MDM2's protein interaction landscape. Dominant p53‐independent MDM2‐drug responsive‐binding proteins have not been stratified. We used as a variable the differential expression of MDM2 protein as a function of cell density to identify Nutlin‐3 responsive MDM2‐binding proteins that are perturbed independent of cell density using SWATH‐MS. Dihydrolipoamide dehydrogenase, the E3 subunit of the mitochondrial pyruvate dehydrogenase complex, was one of two Nutlin‐3 perturbed proteins identified fours hour posttreatment at two cell densities. Immunoblotting confirmed that dihydrolipoamide dehydrogenase was induced by Nutlin‐3. Depletion of MDM2 using siRNA also elevated dihydrolipoamide dehydrogenase in Nutlin‐3 treated cells. Mitotracker confirmed that Nutlin‐3 inhibits mitochondrial activity. Enrichment of mitochondria using TOM22+ immunobeads and TMT labeling defined key changes in the mitochondrial proteome after Nutlin‐3 treatment. Proximity ligation identified rearrangements of cellular protein–protein complexes in situ. In response to Nutlin‐3, a reduction of dihydrolipoamide dehydrogenase/dihydrolipoamide acetyltransferase protein complexes highlighted a disruption of the pyruvate dehydrogenase complex. This coincides with an increase in MDM2/dihydrolipoamide dehydrogenase complexes in the nucleus that was further enhanced by the nuclear export inhibitor Leptomycin B. The data suggest one therapeutic impact of MDM2 drugs might be on the early perturbation of specific protein–protein interactions within the mitochondria. This methodology forms a blueprint for biomarker discovery that can identify rearrangements of MDM2 protein–protein complexes in drug‐treated cells. PMID:27273042

  19. Clinical Value of 99Tcm-MDP SPECT Bone Scintigraphy in the Diagnosis of Unilateral Condylar Hyperplasia

    PubMed Central

    Wen, Bing; Shen, Ying; Wang, Chang-Yin

    2014-01-01

    Purpose. To investigate the clinical value of 99Tcm-MDP SPECT for the diagnosis of unilateral condylar hyperplasia (UCH). Methods. One hundred forty-nine patients who underwent mandibular 99Tcm-MDP SPECT between January 2009 and December 2012 were studied, including 105 cases that were clinically suspected of UCH and 44 comparable cases without UCH as a control group. Results. Increased bone activity was observed in the affected condyles for all UCH patients. In the UCH group, the relative percentage uptake on the affected side was 59% (SD ± 4.3%), significantly higher than the 41% (SD ± 4.1%) uptake on the contralateral side (P<0.001). Similarly, the condyle/skull ratio was significantly higher for the affected side (1.66 ± 0.63) than for the contralateral side (1.34 ± 0.34, P < 0.01. No significant difference was found in the control group between the left and right condyles. Values for the sensitivity (95%), specificity (61%), positive (84.4%) and negative (84.6%) predictive values, and accuracy (84.5%) for 99Tcm-MDP SPECT in the diagnosis of UCH were calculated. However, for the hyperplastic condyle, no correlation was observed between the thickness of each cartilage layer and the relative uptake in the SPECT image. Conclusion. 99Tcm-MDP SPECT is accurate for diagnosing UCH and can provide a reference for treatment options. PMID:24901015

  20. The RP-Mdm2-p53 Pathway and Tumorigenesis

    PubMed Central

    Miliani de Marval, Paula L.; Zhang, Yanping

    2011-01-01

    The dynamic processes of cell growth and division are under constant surveillance. As one of the primary “gatekeepers” of the cell, the p53 tumor suppressor plays a major role in sensing and responding to a variety of stressors to maintain cellular homeostasis. Recent studies have shown that inhibition of ribosomal biogenesis can activate p53 through ribosomal protein (RP)-mediated suppression of Mdm2 E3 ligase activity. Mutations in Mdm2 that disrupt RP binding have been detected in human cancers; however, the physiological significance of the RP-Mdm2 interaction is not completely understood. We generated mice carrying a single cysteine-to-phenylalanine substitution in the central zinc finger of Mdm2 (Mdm2C305F) that disrupts Mdm2’s binding to RPL11 and RPL5. Despite being developmentally normal and maintaining an intact p53 response to DNA damage, the Mdm2C305F mice demonstrate a diminished p53 response to perturbations in ribosomal biogenesis, providing the first in vivo evidence for an RP-Mdm2-p53 signaling pathway. Here we review some recent studies about RP-Mdm2-p53 signaling and speculate on the relevance of this pathway to human cancer. PMID:21406728

  1. Renal vascular transit time and tubular transit time dispersion for 99Tcm-MAG3.

    PubMed

    Russell, C D; Japanwalla, M; Khan, S; Scott, J W; Dubovsky, E V

    1997-09-01

    Renal transit time usually refers to tubular transit time, as introduced by Taplin, but other measures of renal transit have been proposed. Here we examine the vascular transit time (VTT, following Rutland) and the standard deviation of tubular transit time (SDTT, following Britton) in a group of 30 patients having baseline and ACE-inhibitor 99Tcm-MAG3 renography prior to arteriography. A same-day, low-dose/high-dose protocol was used for renography; only the post-captopril dose was high enough to measure VTT. Pre-captopril, the Spearman rank correlation coefficient for SDTT was rho = 0.52 (n = 53 kidneys; P < 0.0002); post-captopril, rho = 0.54 (n = 49 kidneys; P < 0.0002). For VTT, the post-captopril value was rho = 0.24 (n = 30 kidneys; N.S.). For comparison, the same statistics were calculated for Taplin's original measure of transit time: the time from injection to maximum count rate (peak time). Pre-captopril, for peak time, rho was 0.47 (n = 53 kidneys; P < 0.001); post-captopril, rho was 0.39 (n = 50 kidneys, P < 0.01). These findings confirm the diagnostic value of SDTT but not of VTT. SDTT correlated better than peak time with the arteriographic findings.

  2. Extending the life of a 99Tcm generator: a simple and convenient method for concentrating generator eluate for clinical use.

    PubMed

    Blower, P J

    1993-11-01

    99Tcm-pertechnetate can conveniently be concentrated 5-20-fold by passing up to 20 ml through a sequence of two (or three) disposable columns, one (or two) to remove chloride and one to concentrate the pertechnetate for subsequent elution with a small volume of saline. This procedure is useful for utilizing eluates with low radioactive concentration in applications that require high radioactive concentration.

  3. The routine use of sublingual GTN with resting 99Tcm-tetrofosmin myocardial perfusion imaging.

    PubMed

    Thorley, P J; Sheard, K L; Wright, D J; Sivananthan, U M

    1998-10-01

    Nitrates can be used to improve resting myocardial blood flow in patients with severe coronary artery disease. This may enhance tracer uptake during rest myocardial perfusion imaging. Recent studies using nitrates at rest have shown increased detection of reversible ischaemia in this patient group with the 201Tl and 99Tcm perfusion tracers MIBI and tetrofosmin. However, it is not always possible to assess the severity of coronary artery disease before the rest injection and therefore whether a patient would benefit from nitrate administration. To improve the sensitivity for the detection of reversible ischaemia and to avoid a repeat study with nitrates (especially in patients with 'fixed' defects), a protocol in which all patients routinely receive nitrates prior to the rest injection is required. This prospective study evaluated the effect of nitrate administration prior to rest imaging in a randomly selected group of patients. Thirty patients selected at random from routine referrals had stress, rest and rest + GTN tetrofosmin imaging on three separate days. Changes in reversibility between the rest and rest + GTN images were assessed both visually and using semi-quantitative analysis. Defects at stress were seen in 43 coronary artery territories, 33 of which were reversible at rest and 37 reversible at rest + GTN. Of these 43 defects, 82% demonstrated either increased or the same degree of reversibility at rest + GTN imaging compared to standard rest imaging. All defects with reduced reversibility at rest + GTN imaging (i.e. the remaining 18%) were, however, still reversible compared to the stress images. Some of this reduced reversibility may be due to attenuation artefacts. We conclude that the routine use of GTN with rest tetrofosmin imaging will result in increased detection of ischaemic areas with no loss of sensitivity or specificity.

  4. Potential Landscape and Flux of p53-Mdm2 Oscillator Mediated by Mdm2 Degradation Rate

    NASA Astrophysics Data System (ADS)

    Bi, Yuanhong; Yang, Zhuoqin

    The dynamics of the tumor suppressor p53 can play a crucial role in deciding cell fate after DNA damage. In this paper, we explore the dynamics and stability of p53 mediated by Mdm2 degradation rate in p53-Mdm2 oscillator through bifurcation, the potential landscape and flux. Based on the investigation of the bifurcation, we find that p53 can exhibit rich dynamics including monostability, bistability of two stable steady states and oscillation behaviors as well as bistability between a stable steady state and an oscillatory state. The stability of these states are further validated by the potential landscape. In addition, oscillatory behaviors of p53 are explored by means of the negative gradient of the potential landscape and the probability flux. It is shown that the negative gradient of the potential landscape can attract the system towards the oscillatory path and the flux can drive oscillation along the path. Moreover, the quicker the flux runs, the smaller the period is. Besides, stability and sensitivity of the system are explored by the barrier height and the entropy production rate in a single cell level, and we further compare the potential landscapes at single and population cell levels. Our results may be useful for understanding the regulation of p53 signaling pathways in response to DNA damage.

  5. Amplification of MDM2 inhibits MyoD-mediated myogenesis.

    PubMed Central

    Fiddler, T A; Smith, L; Tapscott, S J; Thayer, M J

    1996-01-01

    One obvious phenotype of tumor cells is the lack of terminal differentiation. We previously classified rhabdomyosarcoma cell lines as having either a recessive or a dominant nondifferentiating phenotype. To study the genetic basis of the dominant nondifferentiating phenotype, we utilized microcell fusion to transfer chromosomes from rhabdomyosarcoma cells into C2C12 myoblasts. Transfer of a derivative chromosome 14 inhibits differentiation. The derivative chromosome 14 contains a DNA amplification. MDM2 is amplified and overexpressed in these nondifferentiating hybrids and in the parental rhabdomyosarcoma. Forced expression of MDM2 inhibits MyoD-dependent transcription. Expression of antisense MDM2 restores MyoD-dependent transcriptional activity. We conclude that amplification and overexpression of MDM2 inhibit MyoD function, resulting in a dominant nondifferentiating phenotype. PMID:8756663

  6. Clinical Overview of MDM2/X-Targeted Therapies

    PubMed Central

    Burgess, Andrew; Chia, Kee Ming; Haupt, Sue; Thomas, David; Haupt, Ygal; Lim, Elgene

    2016-01-01

    MDM2 and MDMX are the primary negative regulators of p53, which under normal conditions maintain low intracellular levels of p53 by targeting it to the proteasome for rapid degradation and inhibiting its transcriptional activity. Both MDM2 and MDMX function as powerful oncogenes and are commonly over-expressed in some cancers, including sarcoma (~20%) and breast cancer (~15%). In contrast to tumors that are p53 mutant, whereby the current therapeutic strategy restores the normal active conformation of p53, MDM2 and MDMX represent logical therapeutic targets in cancer for increasing wild-type (WT) p53 expression and activities. Recent preclinical studies suggest that there may also be situations that MDM2/X inhibitors could be used in p53 mutant tumors. Since the discovery of nutlin-3a, the first in a class of small molecule MDM2 inhibitors that binds to the hydrophobic cleft in the N-terminus of MDM2, preventing its association with p53, there is now an extensive list of related compounds. In addition, a new class of stapled peptides that can target both MDM2 and MDMX have also been developed. Importantly, preclinical modeling, which has demonstrated effective in vitro and in vivo killing of WT p53 cancer cells, has now been translated into early clinical trials allowing better assessment of their biological effects and toxicities in patients. In this overview, we will review the current MDM2- and MDMX-targeted therapies in development, focusing particularly on compounds that have entered into early phase clinical trials. We will highlight the challenges pertaining to predictive biomarkers for and toxicities associated with these compounds, as well as identify potential combinatorial strategies to enhance its anti-cancer efficacy. PMID:26858935

  7. Energetic Landscape of MDM2-p53 Interactions by Computational Mutagenesis of the MDM2-p53 Interaction

    PubMed Central

    Thayer, Kelly M.; Beyer, George A.

    2016-01-01

    The ubiquitin ligase MDM2, a principle regulator of the tumor suppressor p53, plays an integral role in regulating cellular levels of p53 and thus a prominent role in current cancer research. Computational analysis used MUMBO to rotamerize the MDM2-p53 crystal structure 1YCR to obtain an exhaustive search of point mutations, resulting in the calculation of the ΔΔG comprehensive energy landscape for the p53-bound regulator. The results herein have revealed a set of residues R65-E69 on MDM2 proximal to the p53 hydrophobic binding pocket that exhibited an energetic profile deviating significantly from similar residues elsewhere in the protein. In light of the continued search for novel competitive inhibitors for MDM2, we discuss possible implications of our findings on the drug discovery field. PMID:26992014

  8. Energetic Landscape of MDM2-p53 Interactions by Computational Mutagenesis of the MDM2-p53 Interaction.

    PubMed

    Thayer, Kelly M; Beyer, George A

    2016-01-01

    The ubiquitin ligase MDM2, a principle regulator of the tumor suppressor p53, plays an integral role in regulating cellular levels of p53 and thus a prominent role in current cancer research. Computational analysis used MUMBO to rotamerize the MDM2-p53 crystal structure 1YCR to obtain an exhaustive search of point mutations, resulting in the calculation of the ΔΔG comprehensive energy landscape for the p53-bound regulator. The results herein have revealed a set of residues R65-E69 on MDM2 proximal to the p53 hydrophobic binding pocket that exhibited an energetic profile deviating significantly from similar residues elsewhere in the protein. In light of the continued search for novel competitive inhibitors for MDM2, we discuss possible implications of our findings on the drug discovery field. PMID:26992014

  9. Scintigraphic evaluation of the severity of inflammation of the joints with 99TCm-HIG in rheumatoid arthritis.

    PubMed

    Pons, F; Sanmarti, R; Herranz, R; Collado, A; Piera, C; Vidal-Sicart, S; Muñoz-Gomez, J; Setoain, J

    1996-06-01

    A prospective study was carried out to determine the usefulness of 99TCm-human immunoglobulin G (HIG) scintigraphy in the assessment of the severity of joint inflammation. Twenty-four patients with rheumatoid arthritis were studied. The presence or absence of pain and/or swelling was evaluated in 34 joints and a clinical index taking into account the surface area of each joint was calculated. We measured the following biological markers of inflammation activity: erythrocyte sedimentation rate, C-reactive protein, haemoglobin, platelet count, serum levels of IL-6, TNF-alpha and soluble receptors of IL-2. Scintigraphic was performed 4 h after the injection of 740 MBq 99Tcm-HIG. The scans were evaluated by visual and quantitative analysis and the scores in each joint were weighted for joint size. Pathological uptake of the radiopharmaceutical was noted in 46% (24/52) of joints evaluated as painful, 89% (146/164) of swollen joints and 94% (78/83) of both painful and swollen joints. Both the visual and the quantitative scintigraphic indices correlated significantly with the clinical index, the number of painful joints, the number of swollen joints and several biological markers of inflammation. A very high correlation was also found between the visual and the quantitative scintigraphic indices (r = 0.91, P < 0.0001). In conclusion, 99Tcm-HIG scintigraphy is an objective test to detect synovitis and to assess the severity of inflammation. A careful visual analysis of scans is good enough for routine evaluations and computer quantitative analysis should be used when more accurate intra-individual variation is required.

  10. Discovery of Dual Inhibitors of MDM2 and XIAP for Cancer Treatment | Office of Cancer Genomics

    Cancer.gov

    MDM2 and XIAP are mutually regulated. Binding of MDM2 RING protein to the IRES region on XIAP mRNA results in MDM2 protein stabilization and enhanced XIAP translation. In this study, we developed a protein-RNA fluorescence polarization (FP) assay for high-throughput screening (HTS) of chemical libraries. Our FP-HTS identified eight inhibitors that blocked the MDM2 protein-XIAP RNA interaction, leading to MDM2 degradation.

  11. Controlling the Mdm2-Mdmx-p53 Circuit

    PubMed Central

    Waning, David L.; Lehman, Jason A.; Batuello, Christopher N.; Mayo, Lindsey D.

    2010-01-01

    The p53 tumor suppressor is a key protein in maintaining the integrity of the genome by inducing either cell cycle arrest or apoptosis following cellular stress signals. Two human family members, Mdm2 and Mdmx, are primarily responsible for inactivating p53 transcription and targeting p53 protein for ubiquitin-mediated degradation. In response to genotoxic stress, post-translational modifications to p53, Mdm2 and Mdmx stabilize and activate p53. The role that phosphorylation of these molecules plays in the cellular response to genotoxic agents has been extensively studied with respect to cancer biology. In this review, we discuss the main phosphorylation events of p53, Mdm2 and Mdmx in response to DNA damage that are important for p53 stability and activity. In tumors that harbor wild-type p53, reactivation of p53 by modulating both Mdm2 and Mdmx signaling is well suited as a therapeutic strategy. However, the rationale for development of kinase inhibitors that target the Mdm2-Mdmx-p53 axis must be carefully considered since modulation of certain kinase signaling pathways has the potential to destabilize and inactivate p53. PMID:20651945

  12. Quantitative SPET 99Tcm-DMSA uptake by the kidneys: age-related decline in healthy males.

    PubMed

    Groshar, D; Gorenberg, M; Osamah, H

    1998-09-01

    To evaluate if 99Tcm-dimercaptosuccinic acid (99Tcm-DMSA) uptake by the kidneys is related to age and creatinine clearance in males with normal renal function, quantitative single photon emission tomography (SPET) of DMSA uptake by the kidneys was performed in 18 volunteers aged 20-79 years. The quantitative uptake of DMSA in the right kidney was 13.9 +/- 2.9% and in the left kidney 14.2 +/- 3.0%. There was no statistically significant difference between left and right kidney uptake (t = 1.2, N.S.). Global kidney uptake (right + left) was 28.1 +/- 5.9%. There was a statistically significant age-related decline in global DMSA uptake. The estimated DMSA uptake (%) was given by -0.27 x age + 42 (r = -0.87, P < 0.001). A good correlation was found between creatinine clearance and global DMSA uptake (r = 0.87, y = 2.8x + 28.3, P < 0.001). The results suggest that normal values of DMSA uptake by the kidneys are age-dependent.

  13. Novel insights about the MDM2/MDM4 heterodimer.

    PubMed

    Moretti, Fabiola

    2016-03-01

    MDM2 (mouse double minute 2 homolog) and MDM4 (double minute 4 human homolog, also known as MDMX) inhibit the activity of tumor protein p53 (TP53, best known as p53) through their heterodimerization. New evidence indicates that under stress conditions the heterodimer is modified, leading to different activities of the single molecules. In particular, following lethal DNA damage, MDM2 and MDM4 dissociate and MDM4 promotes the stabilization of homeodomain-interacting protein kinase 2 (HIPK2) and the phosphorylation of p53, resulting in transcriptional repression of antiapoptotic targets of p53/HIPK2. PMID:27308591

  14. Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs.

    PubMed

    Mendrysa, S M; McElwee, M K; Perry, M E

    2001-02-01

    The murine double minute 2 (mdm2) gene is essential for embryogenesis in mice that express the p53 tumor suppressor protein. Mdm2 levels must be regulated tightly because overexpression of mdm2 contributes to tumorigenesis. We investigated whether the 5' and 3' untranslated regions (UTRs) of murine mdm2 affect the expression of MDM2 proteins. Induction of mdm2 expression by p53 results in synthesis of an mdm2 mRNA with a short 5' UTR. The long 5' UTR increases internal initiation of translation of a minor MDM2 protein, p76(MDM2), without affecting the efficiency of translation of the full-length p90(MDM2). We discovered two alternative 3' untranslated regions in murine mdm2 mRNA expressed in the testis. The longer 3' UTR contains a consensus instability element, but mdm2 mRNAs containing the long and short 3' UTRs have comparable half-lives. The 3' UTRs do not affect either initiation codon use or translation efficiency. Thus, the murine 5' UTR, but not the 3'UTR, influences the ratio of the two MDM2 proteins but neither UTR affects MDM2 abundance significantly.

  15. Therapeutic considerations for Mdm2: not just a one trick pony

    PubMed Central

    Lehman, Jason A.; Eitel, Jacob A.; Batuello, Christopher N.; Mayo, Lindsey D.

    2008-01-01

    Background The mdm2 proto-oncogene is elevated in numerous late stage cancers. The Mdm2 protein manifests its oncogenic properties in part through inactivation of the tumor suppressor protein p53. Recent efforts in anti-cancer drug design have focused on the identification of small molecules that disrupt the Mdm2-p53 interaction, in hopes of re-engaging the p53 pathway. Objective In addition to binding p53, Mdm2 complexes with numerous proteins involved in DNA repair, translation, metabolic activities, tumor growth and apoptosis. Additional biochemical analysis is required to understand how Mdm2 integrates into all of these cellular processes. Post-translational modifications to Mdm2 can alter its ability to associate with numerous proteins. Changes in protein structure may also affect the ability of small molecule inhibitors to effectively antagonize Mdm2. Conclusion The complexity of Mdm2 modification has been largely neglected during the development of previous Mdm2 inhibitors. Future high-throughput or in silico screening efforts will need to recognize the importance of post-translational modifications to Mdm2. Furthermore, the identification of molecules that target other domains in Mdm2 may provide a tool to prevent other pivotal p53-independent functions of Mdm2. These aims provide a useful roadmap for the discovery of new Mdm2 binding compounds with therapeutic potency that may exceed its predecessors. PMID:19738896

  16. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2

    PubMed Central

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-01-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator. PMID:24413661

  17. Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage

    PubMed Central

    Comiskey, Daniel F.; Jacob, Aishwarya G.; Singh, Ravi K.; Tapia-Santos, Aixa S.; Chandler, Dawn S.

    2015-01-01

    Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation. PMID:25845590

  18. Dobutamine 99Tcm-MIBI SPET myocardial perfusion scintigraphy in the prediction of restenosis after percutaneous transluminal coronary angioplasty in patients unable to perform an exercise stress test.

    PubMed

    Elhendy, A; Geleijnse, M L; Roelandt, J R; van Domburg, R T; Nierop, P R; Bax, J J; Kasprzak, J D; el-Said, G M; Ibrahim, M M; Fioretti, P M

    1997-02-01

    After successful percutaneous transluminal coronary angioplasty (PTCA), restenosis occurs in a relatively high proportion of patients. Exercise thallium scintigraphy is a useful method for the detection of restenosis. In patients unable to exercise, dobutamine perfusion scintigraphy may represent a feasible alternative. However, its diagnostic accuracy in this clinical setting has not been evaluated. We studied 40 patients (29 males, 11 females) aged 57 +/- 9 years, at a mean of 185 +/- 80 days after successful PTCA with a high-dose dobutamine (up to 40 micrograms kg-1 min-1) stress test, in conjunction with 99Tcm-methoxyisobutyl isonitrile (99Tcm-MIBI) single photon emission tomography (SPET). Significant restenosis was defined as > or = 50% luminal diameter stenosis of a coronary segment with previous PTCA and was predicted on the basis of the occurrence of reversible perfusion defects in the corresponding territories. Reversible perfusion defects occurred in 20 of 29 arteries with and in 4 of 17 arteries without restenosis. The sensitivity of dobutamine MIBI for the detection of restenosis in arteries with previous PTCA was 69% (CI = 56-82), the specificity 76% (CI = 64-89), the positive predictive value 83% (CI = 73-94), the negative predictive value 59% (CI = 45-73) and accuracy 72% (CI = 59-85). The overall sensitivity of 99Tcm-MIBI SPET for the diagnosis of significant coronary stenosis (including arteries without previous PTCA) on a patient basis was 79% (CI = 67-92), the specificity 82% (CI = 70-94) and accuracy 80% (CI = 68-92). The sensitivity of 99Tcm-MIBI SPET was significantly higher than that of electrocardiography (79 vs 38%, P < 0.005). It is concluded that dobutamine 99Tcm-MIBI SPET is a useful method for the detection of restenosis after PTCA in patients unable to perform an exercise stress test.

  19. Small-Molecule Inhibitors of the MDM2–p53 Protein–Protein Interaction (MDM2 Inhibitors) in Clinical Trials for Cancer Treatment

    PubMed Central

    2015-01-01

    Design of small-molecule inhibitors (MDM2 inhibitors) to block the MDM2–p53 protein–protein interaction has been pursued as a new cancer therapeutic strategy. In recent years, potent, selective, and efficacious MDM2 inhibitors have been successfully obtained and seven such compounds have been advanced into early phase clinical trials for the treatment of human cancers. Here, we review the design, synthesis, properties, preclinical, and clinical studies of these clinical-stage MDM2 inhibitors. PMID:25396320

  20. The Wip1 Phosphatase acts as a gatekeeper in the p53-Mdm2 autoregulatory loop.

    PubMed

    Lu, Xiongbin; Ma, Ou; Nguyen, Thuy-Ai; Jones, Stephen N; Oren, Moshe; Donehower, Lawrence A

    2007-10-01

    The tumor suppressor p53 is a transcription factor that responds to cellular stresses by initiating cell cycle arrest or apoptosis. One transcriptional target of p53 is Mdm2, an E3 ubiquitin ligase that interacts with p53 to promote its proteasomal degradation in a negative feedback regulatory loop. Here we show that the wild-type p53-induced phosphatase 1 (Wip1), or PPM1D, downregulates p53 protein levels by stabilizing Mdm2 and facilitating its access to p53. Wip1 interacts with and dephosphorylates Mdm2 at serine 395, a site phosphorylated by the ATM kinase. Dephosphorylated Mdm2 has increased stability and affinity for p53, facilitating p53 ubiquitination and degradation. Thus, Wip1 acts as a gatekeeper in the Mdm2-p53 regulatory loop by stabilizing Mdm2 and promoting Mdm2-mediated proteolysis of p53.

  1. Quantitation of renal function with 99Tcm-DMSA. A comparison with creatinine clearance in children with single kidney.

    PubMed

    Baillet, G; Gagnadoux, M F; De Vernejoul, P; Broyer, M

    1985-11-01

    To study the accuracy of renal function quantification with 99Tcm-DMSA we compared DMSA renal uptake and creatinine clearance in 16 cases of children with single kidney. The age of the patients ranged from two months to fourteen years. Creatinine clearance was normalized to 1.73 m2. DMSA uptake was measured 7 h after intravenous injection. Background subtraction was used and soft tissue attenuation was taken into account. The uptake was normalized in percentage of the injected activity. A significant correlation was found between creatinine clearance and DMSA uptake (rt = 0.866, p less than 0.01). Normal creatinine clearance range in children (80 to 120 ml min-1/1.73 m2) allowed determination of normal uptake range (36 to 60%). This study indicates that in case of asymmetrical renal impairment renal uptake will reflect split renal creatinine clearance. Since the former is much easier to measure, DMSA should play an important role in the evaluation of differential renal function.

  2. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    SciTech Connect

    Kasim, Vivi; Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia; Yang, Li; Miyagishi, Makoto; Wu, Shourong

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

  3. Ribosomal protein S7 is both a regulator and a substrate of MDM2.

    PubMed

    Zhu, Yan; Poyurovsky, Masha V; Li, Yingchun; Biderman, Lynn; Stahl, Joachim; Jacq, Xavier; Prives, Carol

    2009-08-14

    MDM2 associates with ribosomal protein S7, and this interaction is required to inhibit MDM2's E3 ligase activity, leading to stabilization of MDM2 and p53. Notably, the MDM2 homolog MDMX facilitates the inhibition of MDM2 E3 ligase activity by S7. Further, ablation of S7 inhibits MDM2 and p53 accumulation induced by different stress signals in some cell types. Thus, ribosomal/nucleolar stress is likely a key integrating event in DNA damage signaling to p53. Interestingly, S7 is itself a substrate for MDM2 E3 ligase activity both in vitro and in vivo. An S7-ubiquitin fusion protein (S7-Ub) selectively inhibits MDM2 degradation of p53 and is unaffected by MDMX. S7-Ub promotes apoptosis to a greater extent than S7 alone. This indicates that MDM2 ubiquitination of S7 is involved in sustaining the p53 response. Thus, S7 functions as both effector and affector of MDM2 to ensure a proper cellular response to different stress signals.

  4. Escape, or Vanish: Control the Fate of p53 through MDM2-Mediated Ubiquitination.

    PubMed

    Wei, Jinlian; Yang, Yingrui; Lu, Mengchen; Xu, Lili; Liu, Fang; Yuan, Zhenwei; Bao, Qichao; Jiang, Zhengyu; Xu, Xiaoli; Guo, Xiaoke; Zhang, Xiaojin; You, Qidong; Sun, Haopeng

    2015-01-01

    p53 protein is a prominent tumor suppressor to induce cell cycle arrest, apoptosis and senescence, which attracts significant interest to cancer treatment. Therefore, it would be particularly important to restore the wild-type p53 that retains latent functions in the approximately 50% of tumors. MDM2 (murine double minute 2), the principal cellular antagonist of p53, has long been believed to suppress p53 activity through two main mechanisms: promoting degradation via its E3 ligase activity and masking p53 transcriptional activation by direct binding. Targeting MDM2 E3 ligase activity is becoming a potential antitumor strategy resulting from MDM2's decisive role in controlling the fate of p53: p53 is going to degradation when entrapped into MDM2-mediated ubiquitination, where p53 can escape by abrogating MDM2 E3 ligase activity using regulators. The intensive focus on regulating MDM2 ubiquitin E3 ligase activity has led to the rapid progress of its inhibitors, which may be possible to help p53 escape from degradation and restore its function to control tumor growth. This review summarizes the current inhibitors of MDM2 E3 ligase in cancer therapy based on the understanding the regulation of MDM2 E3 ubiquitin ligase activity, including post-translational modification, interactions between MDM2 and its cofactors, and regulation of MDM2 stability. PMID:26343143

  5. A dynamic P53-MDM2 model with time delay

    NASA Astrophysics Data System (ADS)

    Mihalaş, Gh. I.; Neamţu, M.; Opriş, D.; Horhat, R. F.

    2006-11-01

    Specific activator and repressor transcription factors which bind to specific regulator DNA sequences, play an important role in gene activity control. Interactions between genes coding such transcription factors should explain the different stable or sometimes oscillatory gene activities characteristic for different tissues. Starting with the model P53-MDM2 described into [6] and the process described into [5] we developed a new model of this interaction. Choosing the delay as a bifurcation parameter we study the direction and stability of the bifurcating periodic solutions. Some numerical examples are finally given for justifying the theoretical results.

  6. A dynamic p53-mdm2 model with distributed delay

    NASA Astrophysics Data System (ADS)

    Horhat, Raluca; Horhat, Raul Florin

    2014-12-01

    Specific activator and repressor transcription factors which bind to specific regulator DNA sequences, play an important role in gene activity control. Interactions between genes coding such transcripion factors should explain the different stable or sometimes oscillatory gene activities characteristic for different tissues. In this paper, the dynamic P53-Mdm2 interaction model with distributed delays is investigated. Both weak and Dirac kernels are taken into consideration. For Dirac case, the Hopf bifurcation is investigated. Some numerical examples are finally given for justifying the theoretical results.

  7. Ubiquitination and degradation of the anti-apoptotic protein ARC by MDM2.

    PubMed

    Foo, Roger S-Y; Chan, Lennard K W; Kitsis, Richard N; Bennett, Martin R

    2007-02-23

    Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2(C464A) mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2. Proteasomal inhibitors rescued both MDM2 and H(2)O(2)-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis.

  8. MDM2 overexpression generates a skin phenotype in both wild type and p53 null mice.

    PubMed

    Alkhalaf, M; Ganguli, G; Messaddeq, N; Le Meur, M; Wasylyk, B

    1999-02-18

    The MDM2 proto-oncogene is overexpressed in human tumours and regulates the activities of the tumour suppressors p53 and pRB. We created mice that overexpress MDM2 under the control of the CMV promoter. These mice did not display an increased tumour incidence, but rather a specific skin phenotype, characterized by desquamation and hyperkeratosis. Transgenic MDM2 was found to be overexpressed in the epidermis, a tissue that normally expresses high levels of MDM2. The phenotype appeared during the first week after birth and then lessened with age, closely following the level of expression of the transgene. MDM2 overexpression was associated with an increase in proliferation in the basal layer, thickening of the epidermis, altered expression of the differentiation markers cytokeratin CK14, CK10 and CK1, and a decrease in the size and the number of granules that contain products of differentiation. Transgenic mice on a p53 null background displayed similar although not identical changes, showing that the effects of MDM2 are to a certain degree p53 independent. The skin is a major site of MDM2 expression in mice, raising the possibility that MDM2 overexpression perturbs the normal pattern of MDM2 expression and inhibits differentiation of the epidermis. PMID:10050879

  9. Iron-Dependent Regulation of MDM2 Influences p53 Activity and Hepatic Carcinogenesis

    PubMed Central

    Dongiovanni, Paola; Fracanzani, Anna Ludovica; Cairo, Gaetano; Megazzini, Chiara Paola; Gatti, Stefano; Rametta, Raffaela; Fargion, Silvia; Valenti, Luca

    2010-01-01

    Iron overload is a risk factor for hepatocarcinoma, but the pathways involved are poorly characterized. Gene expression analysis in immortalized mouse hepatocytes exposed to iron or the iron chelator deferoxamine revealed that iron downregulated, whereas deferoxamine upregulated, mRNA levels of mouse double minute gene 2 (MDM2), the ubiquitin ligase involved in the degradation of the oncosuppressor p53. Regulation of MDM2 by iron status was observed at protein levels in mouse hepatocytes and rat liver, and was associated with specular changes in p53 expression. Iron dependent regulation of MDM2/p53 was confirmed ex-vivo in human monocytes, by manipulation of iron pool and in a genetic model of iron deficiency, leading to modulation of p53 target genes involved in the antioxidant response and apoptosis. Iron status influenced p53 ubiquitination and degradation rate, and the MDM2 inhibitor nutlin increased p53 levels in iron-depleted cells. Furthermore, nutlin enhanced the antiproliferative activity of deferoxamine in HepG2 hepatoblastoma cells. The MDM2 −309T > G promoter polymorphism, determining increased MDM2 and lower p53 activity, was associated with higher risk of hepatocarcinoma in cirrhotic patients with hemochromatosis, and with HFE mutations in patients with hepatocarcinoma without hemochromatosis, suggesting an interaction between MDM2 and iron in the pathogenesis of hepatocarcinoma. In conclusion, iron status influences p53 activity and antioxidant response by modulating MDM2 expression. MDM2 inhibitors may enhance the antiproliferative activity of iron chelators. PMID:20019189

  10. Ribosomal protein S7 as a novel modulator of p53-MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function.

    PubMed

    Chen, D; Zhang, Z; Li, M; Wang, W; Li, Y; Rayburn, E R; Hill, D L; Wang, H; Zhang, R

    2007-08-01

    As a major negative regulator of p53, the MDM2 oncogene plays an important role in carcinogenesis and tumor progression. MDM2 promotes p53 proteasomal degradation and negatively regulates p53 function. The mechanisms by which the MDM2-p53 interaction is regulated are not fully understood, although several MDM2-interacting molecules have recently been identified. To search for novel MDM2-binding partners, we screened a human prostate cDNA library by the yeast two-hybrid assay using full-length MDM2 protein as the bait. Among the candidate proteins, ribosomal protein S7 was identified and confirmed as a novel MDM2-interacting protein. Herein, we demonstrate that S7 binds to MDM2, in vitro and in vivo, and that the interaction between MDM2 and S7 leads to modulation of MDM2-p53 binding by forming a ternary complex among MDM2, p53 and S7. This results in the stabilization of p53 protein through abrogation of MDM2-mediated p53 ubiquitination. Consequently, S7 overexpression increases p53 transactivational activities, induces apoptosis, and inhibits cell proliferation. The identification of S7 as a novel MDM2-interacting partner contributes to elucidation of the complex regulation of the MDM2-p53 interaction and has implications in cancer prevention and therapy.

  11. Flexible lid to the p53-binding domain of human Mdm2: implications for p53 regulation.

    PubMed

    McCoy, Mark A; Gesell, Jennifer J; Senior, Mary M; Wyss, Daniel F

    2003-02-18

    The stabilization of p53 against Mdm2-mediated degradation is an important event in DNA damage response. Initial models of p53 stabilization focused on posttranslational modification of p53 that would disrupt the p53-Mdm2 interaction. The N-terminal regions of both p53 and Mdm2 are modified in vivo in response to cellular stress, suggesting that modifications to Mdm2 also may affect the p53-Mdm2 interaction. Our NMR studies of apo-Mdm2 have found that, in addition to Mdm2 residues 25-109 that form the well ordered p53-binding domain that was observed in the p52-Mdm2 complex, Mdm2 residues 16-24 form a lid that closes over the p53-binding site. The Mdm2 lid, which is strictly conserved in mammals, may help to stabilize apo-Mdm2. It also competes weakly with peptidic and nonpeptidic antagonists. Modifications to the Mdm2 lid may disrupt p53-Mdm2 binding leading to p53 stabilization. Mdm2 and Mdm4 possess nearly identical p53-binding domains but different lids suggesting that lid modifications may select for p53 binding.

  12. Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis

    PubMed Central

    Kamio, Takuya; Gu, Bai-wei; Olson, Timothy S.; Zhang, Yanping; Mason, Philip J.; Bessler, Monica

    2016-01-01

    MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed

  13. The p53–Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans

    PubMed Central

    Coffill, Cynthia R.; Lee, Alison P.; Siau, Jia Wei; Chee, Sharon M.; Joseph, Thomas L.; Tan, Yaw Sing; Madhumalar, Arumugam; Tay, Boon-Hui; Brenner, Sydney; Verma, Chandra S.; Ghadessy, Farid J.; Venkatesh, Byrappa; Lane, David P.

    2016-01-01

    The extant jawless vertebrates, represented by lampreys and hagfish, are the oldest group of vertebrates and provide an interesting genomic evolutionary pivot point between invertebrates and jawed vertebrates. Through genome analysis of one of these jawless vertebrates, the Japanese lamprey (Lethenteron japonicum), we identified all three members of the important p53 transcription factor family—Tp53, Tp63, and Tp73—as well as the Mdm2 and Mdm4 genes. These genes and their products are significant cellular regulators in human cancer, and further examination of their roles in this most distant vertebrate relative sheds light on their origin and coevolution. Their important role in response to DNA damage has been highlighted by the discovery of multiple copies of the Tp53 gene in elephants. Expression of lamprey p53, Mdm2, and Mdm4 proteins in mammalian cells reveals that the p53–Mdm2 interaction and the Mdm2/Mdm4 E3 ligase activity existed in the common ancestor of vertebrates and have been conserved for >500 million years of vertebrate evolution. Lamprey Mdm2 degrades human p53 with great efficiency, but this interaction is not blocked by currently available small molecule inhibitors of the human HDM2 protein, suggesting utility of lamprey Mdm2 in the study of the human p53 signaling pathway. PMID:26798135

  14. The p53-Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans.

    PubMed

    Coffill, Cynthia R; Lee, Alison P; Siau, Jia Wei; Chee, Sharon M; Joseph, Thomas L; Tan, Yaw Sing; Madhumalar, Arumugam; Tay, Boon-Hui; Brenner, Sydney; Verma, Chandra S; Ghadessy, Farid J; Venkatesh, Byrappa; Lane, David P

    2016-02-01

    The extant jawless vertebrates, represented by lampreys and hagfish, are the oldest group of vertebrates and provide an interesting genomic evolutionary pivot point between invertebrates and jawed vertebrates. Through genome analysis of one of these jawless vertebrates, the Japanese lamprey (Lethenteron japonicum), we identified all three members of the important p53 transcription factor family--Tp53, Tp63, and Tp73--as well as the Mdm2 and Mdm4 genes. These genes and their products are significant cellular regulators in human cancer, and further examination of their roles in this most distant vertebrate relative sheds light on their origin and coevolution. Their important role in response to DNA damage has been highlighted by the discovery of multiple copies of the Tp53 gene in elephants. Expression of lamprey p53, Mdm2, and Mdm4 proteins in mammalian cells reveals that the p53-Mdm2 interaction and the Mdm2/Mdm4 E3 ligase activity existed in the common ancestor of vertebrates and have been conserved for >500 million years of vertebrate evolution. Lamprey Mdm2 degrades human p53 with great efficiency, but this interaction is not blocked by currently available small molecule inhibitors of the human HDM2 protein, suggesting utility of lamprey Mdm2 in the study of the human p53 signaling pathway.

  15. MDM2 restrains estrogen-mediated AKT activation by promoting TBK1-dependent HPIP degradation.

    PubMed

    Shostak, K; Patrascu, F; Göktuna, S I; Close, P; Borgs, L; Nguyen, L; Olivier, F; Rammal, A; Brinkhaus, H; Bentires-Alj, M; Marine, J-C; Chariot, A

    2014-05-01

    Restoration of p53 tumor suppressor function through inhibition of its interaction and/or enzymatic activity of its E3 ligase, MDM2, is a promising therapeutic approach to treat cancer. However, because the MDM2 targetome extends beyond p53, MDM2 inhibition may also cause unwanted activation of oncogenic pathways. Accordingly, we identified the microtubule-associated HPIP, a positive regulator of oncogenic AKT signaling, as a novel MDM2 substrate. MDM2-dependent HPIP degradation occurs in breast cancer cells on its phosphorylation by the estrogen-activated kinase TBK1. Importantly, decreasing Mdm2 gene dosage in mouse mammary epithelial cells potentiates estrogen-dependent AKT activation owing to HPIP stabilization. In addition, we identified HPIP as a novel p53 transcriptional target, and pharmacological inhibition of MDM2 causes p53-dependent increase in HPIP transcription and also prevents HPIP degradation by turning off TBK1 activity. Our data indicate that p53 reactivation through MDM2 inhibition may result in ectopic AKT oncogenic activity by maintaining HPIP protein levels. PMID:24488098

  16. Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction.

    PubMed

    Zhang, Rumin; Mayhood, Todd; Lipari, Philip; Wang, Yaolin; Durkin, James; Syto, Rosalinda; Gesell, Jennifer; McNemar, Charles; Windsor, William

    2004-08-01

    MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.

  17. Systems biology analysis reveals role of MDM2 in diabetic nephropathy

    PubMed Central

    Saito, Rintaro; Rocanin-Arjo, Anaïs; You, Young-Hyun; Darshi, Manjula; Van Espen, Benjamin; Miyamoto, Satoshi; Pham, Jessica; Pu, Minya; Romoli, Simone; Natarajan, Loki; Ju, Wenjun; Kretzler, Matthias; Nelson, Robert; Ono, Keiichiro; Thomasova, Dana; Mulay, Shrikant R.; Ideker, Trey; D’Agati, Vivette; Beyret, Ergin; Belmonte, Juan Carlos Izpisua; Anders, Hans Joachim

    2016-01-01

    To derive new insights in diabetic complications, we integrated publicly available human protein-protein interaction (PPI) networks with global metabolic networks using metabolomic data from patients with diabetic nephropathy. We focused on the participating proteins in the network that were computationally predicted to connect the urine metabolites. MDM2 had the highest significant number of PPI connections. As validation, significant downregulation of MDM2 gene expression was found in both glomerular and tubulointerstitial compartments of kidney biopsy tissue from 2 independent cohorts of patients with diabetic nephropathy. In diabetic mice, chemical inhibition of MDM2 with Nutlin-3a led to reduction in the number of podocytes, increased blood urea nitrogen, and increased mortality. Addition of Nutlin-3a decreased WT1+ cells in embryonic kidneys. Both podocyte- and tubule-specific MDM2-knockout mice exhibited severe glomerular and tubular dysfunction, respectively. Interestingly, the only 2 metabolites that were reduced in both podocyte and tubule-specific MDM2-knockout mice were 3-methylcrotonylglycine and uracil, both of which were also reduced in human diabetic kidney disease. Thus, our bioinformatics tool combined with multi-omics studies identified an important functional role for MDM2 in glomeruli and tubules of the diabetic nephropathic kidney and links MDM2 to a reduction in 2 key metabolite biomarkers. PMID:27777973

  18. Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

    PubMed Central

    Ahmed, M. Rafiuddin; Zhan, Xuanzhi; Song, Xiufeng; Kook, Seunghyi; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2011-01-01

    Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmuno-precipitation of endogenous proteins from brain tissue, and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both non-visual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology. PMID:21466165

  19. NORE1A Regulates MDM2 Via β-TrCP

    PubMed Central

    Schmidt, M. Lee; Calvisi, Diego F.; Clark, Geoffrey J.

    2016-01-01

    Mouse Double Minute 2 Homolog (MDM2) is a key negative regulator of the master tumor suppressor p53. MDM2 regulates p53 on multiple levels, including acting as an ubiquitin ligase for the protein, thereby promoting its degradation by the proteasome. MDM2 is oncogenic and is frequently found to be over-expressed in human tumors, suggesting its dysregulation plays an important role in human cancers. We have recently found that the Ras effector and RASSF (Ras Association Domain Family) family member RASSF5/NORE1A enhances the levels of nuclear p53. We have also found that NORE1A (Novel Ras Effector 1A) binds the substrate recognition component of the SCF-ubiquitin ligase complex β-TrCP. Here, we now show that NORE1A regulates MDM2 protein levels by targeting it for ubiquitination by SCF-β-TrCP. We also show the suppression of NORE1A protein levels enhances MDM2 protein expression. Finally, we show that MDM2 can suppress the potent senescence phenotype induced by NORE1A over-expression. Thus, we identify a mechanism by which Ras/NORE1A can modulate p53 protein levels. As MDM2 has several important targets in addition to p53, this finding has broad implications for cancer biology in tumor cells that have lost expression of NORE1A due to promoter methylation. PMID:27023610

  20. Ribosomal protein L4 is a novel regulator of the MDM2-p53 loop

    PubMed Central

    He, Xia; Li, Yuhuang; Dai, Mu-Shui; Sun, Xiao-Xin

    2016-01-01

    A number of ribosomal proteins (RPs) have been shown to play a critical role in coordinating ribosome biogenesis with cell growth and proliferation by suppressing MDM2 to induce p53 activation. While how the MDM2-p53 pathway is regulated by multiple RPs is unclear, it remains to be interesting to identify additional RPs that can regulate this pathway. Here we report that ribosomal protein L4 (RPL4) directly interacts with MDM2 at the central acidic domain and suppresses MDM2-mediated p53 ubiquitination and degradation, leading to p53 stabilization and activation. Interestingly, overexpression of RPL4 promotes the binding of MDM2 to RPL5 and RPL11 and forms a complex with RPL5, RPL11 and MDM2 in cells. Conversely, knockdown of RPL4 also induces p53 levels and p53-dependent cell cycle arrest. This p53-dependent effect requires both RPL5 and RPL11, suggesting that depletion of RPL4 triggers ribosomal stress. Together, our results reveal that balanced levels of RPL4 are critical for normal cell growth and proliferation via regulating the MDM2-p53 loop. PMID:26908445

  1. Transient Protein States in Designing Inhibitors of the MDM2-p53 Interaction

    PubMed Central

    Bista, Michal; Wolf, Siglinde; Khoury, Kareem; Kowalska, Kaja; Huang, Yijun; Wrona, Ewa; Arciniega, Marcelino; Popowicz, Grzegorz M.; Holak, Tad A.; Dömling, Alexander

    2014-01-01

    SUMMARY Reactivation of p53 by release of the functional protein from its inhibition by MDM2 provides an efficient, nongenotoxic approach to a wide variety of cancers. We present the cocrystal structures of two complexes of MDM2 with inhibitors based on 6-chloroindole scaffolds. Both molecules bound to a distinct conformational state of MDM2 with nM-μM affinities. In contrast to other structurally characterized antagonists, which mimic three amino acids of p53 (Phe19, Trp23, and Leu26), the compounds induced an additional hydrophobic pocket on the MDM2 surface and unveiled a four-point binding mode. The enlarged interaction interface of the inhibitors resulted in extension of small molecules binding toward the “lid” segment of MDM2 (residues 19–23)—a nascent element that interferes with p53 binding. As supported by protein engineering and molecular dynamics studies, employing these unstable elements of MDM2 provides an efficient and yet unexplored alternative in development of MDM2-p53 association inhibitors. PMID:24207125

  2. Defects in transcription coupled repair interfere with expression of p90(MDM2) in response to ultraviolet light.

    PubMed

    Michalowski, J; Seavey, S E; Mendrysa, S M; Perry, M E

    2001-09-13

    Ultraviolet (UV) irradiation transiently stabilizes p53 through a mechanism that may require a decrease in the activity of the ubiquitin ligase, p90(MDM2). Conversely, the recovery of low levels of p53 following UV exposure may depend on an increase in p90(MDM2). The level of p90(MDM2) is increased by UV light following the p53-dependent induction of an internal mdm2 promoter, P2. If this induction of mdm2 were critical for the recovery of low levels of p53 following UV exposure, defects in mdm2's transcription would result in a prolonged increase in p53. Cells defective in transcription coupled repair (TCR) maintain high levels of p53 for a prolonged period following UV exposure. Such cells also have defects in general transcription after UV irradiation. We investigated whether TCR-deficient cells express diminished levels of mdm2 mRNA and p90(MDM2) following UV exposure. We found that transcription of mdm2 was reduced in TCR-deficient cells. The uninducible mdm2 promoter, P1, was more sensitive to the inhibitory effects of UV irradiation than the P2 promoter. The decrease in transcription from the P1 promoter was sufficient to reduce the level of p90(MDM2) and correlated with a prolonged increase in p53. Thus, p53-independent transcription of mdm2 appears critical to p53's regulation.

  3. The p53 inhibitor MDM2 facilitates Sonic Hedgehog-mediated tumorigenesis and influences cerebellar foliation.

    PubMed

    Malek, Reem; Matta, Jennifer; Taylor, Natalie; Perry, Mary Ellen; Mendrysa, Susan M

    2011-01-01

    Disruption of cerebellar granular neuronal precursor (GNP) maturation can result in defects in motor coordination and learning, or in medulloblastoma, the most common childhood brain tumor. The Sonic Hedgehog (Shh) pathway is important for GNP proliferation; however, the factors regulating the extent and timing of GNP proliferation, as well as GNP differentiation and migration are poorly understood. The p53 tumor suppressor has been shown to negatively regulate the activity of the Shh effector, Gli1, in neural stem cells; however, the contribution of p53 to the regulation of Shh signaling in GNPs during cerebellar development has not been determined. Here, we exploited a hypomorphic allele of Mdm2 (Mdm2(puro)), which encodes a critical negative regulator of p53, to alter the level of wild-type MDM2 and p53 in vivo. We report that mice with reduced levels of MDM2 and increased levels of p53 have small cerebella with shortened folia, reminiscent of deficient Shh signaling. Indeed, Shh signaling in Mdm2-deficient GNPs is attenuated, concomitant with decreased expression of the Shh transducers, Gli1 and Gli2. We also find that Shh stimulation of GNPs promotes MDM2 accumulation and enhances phosphorylation at serine 166, a modification known to increase MDM2-p53 binding. Significantly, loss of MDM2 in Ptch1(+/-) mice, a model for Shh-mediated human medulloblastoma, impedes cerebellar tumorigenesis. Together, these results place MDM2 at a major nexus between the p53 and Shh signaling pathways in GNPs, with key roles in cerebellar development, GNP survival, cerebellar foliation, and MB tumorigenesis. PMID:21437245

  4. Inhibition of MDM2 by nilotinib contributes to cytotoxicity in both Philadelphia-positive and negative acute lymphoblastic leukemia.

    PubMed

    Zhang, Hailong; Gu, Lubing; Liu, Tao; Chiang, Kuang-Yueh; Zhou, Muxiang

    2014-01-01

    Nilotinib is a selective BCR-ABL tyrosine kinase inhibitor related to imatinib that is more potent than imatinib. Nilotinib is widely used to treat chronic myelogenous leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). The present study identifies Mouse double minute 2 homolog (MDM2) as a target of nilotinib. In studying ALL cell lines, we found that the expression of MDM2 in both Philadelphia positive (Ph+) and Philadelphia negative (Ph-) ALL cells was remarkably inhibited by nilotinib, in a dose- and time-dependent manner. Further studies demonstrated that nilotinib inhibited MDM2 at the post-translational level by inducing MDM2 self-ubiquitination and degradation. Nilotinib-mediated MDM2 downregulation did not result in accumulation and activation of p53. Inhibition of MDM2 in nilotinib-treated ALL cells led to downregulation of the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP), a translational target of MDM2, resulting in activation of caspases. Inhibition of XIAP following nilotinib-mediated downregulation of MDM2 resulted in apoptosis of MDM2-expressing ALL; however, similar nilotinib treatment induced stronger apoptosis in Ph+/MDM2+ ALL than in Ph-/MDM2+ or Ph+/MDM2- ALL. The ALL cells that were Ph-/MDM2- were totally resistant to nilotinib. These results suggested that nilotinib can inhibit MDM2 and induce a p53-independent apoptosis pathway by downregulating XIAP; thus, nilotinib can treat not only Ph+, but also Ph- ALL patients whose cancer cells overexpress MDM2.

  5. Polymorphism of MDM2 promoter 309 (rs 2279744) and the risk of PCOS.

    PubMed

    Chan, Ying; Jiang, Hongguo; Yang, Xiaoling; Li, Dongya; Ma, Lan; Luo, Ying; Tang, Wenru

    2016-01-01

    This study aimed at evaluating possible association between MDM2 SNP309 polymorphism (rs 2279744) and polycystic ovary syndrome (PCOS). One hundred and twenty-five women with PCOS and two hundred and fifty women without PCOS were collected from the department of reproductive medicine of college hospital in this case-control study. Peripheral blood samples were collected from all participants and DNA was extracted, MDM2 SNP309 polymorphism (rs 2279744) was determined from the 125 cases and 250 controls. Women were grouped into PCOS (n = 125) group and control group (n = 250). Odds ratios (OR) and 95% confidence intervals (CI) were used to evaluate the association between MDM2 SNP309 polymorphism (rs 2279744) and PCOS. The distribution of T allele was significant higher in PCOS cases than controls. MDM2 SNP 309 T allele is associated with PCOS.

  6. Mdm2 Phosphorylation Regulates Its Stability and Has Contrasting Effects on Oncogene and Radiation-Induced Tumorigenesis.

    PubMed

    Carr, Michael I; Roderick, Justine E; Gannon, Hugh S; Kelliher, Michelle A; Jones, Stephen N

    2016-09-01

    ATM phosphorylation of Mdm2-S394 is required for robust p53 stabilization and activation in DNA-damaged cells. We have now utilized Mdm2(S394A) knockin mice to determine that phosphorylation of Mdm2-S394 regulates p53 activity and the DNA damage response in lymphatic tissues in vivo by modulating Mdm2 stability. Mdm2-S394 phosphorylation delays lymphomagenesis in Eμ-myc transgenic mice, and preventing Mdm2-S394 phosphorylation obviates the need for p53 mutation in Myc-driven tumorigenesis. However, irradiated Mdm2(S394A) mice also have increased hematopoietic stem and progenitor cell functions, and we observed decreased lymphomagenesis in sub-lethally irradiated Mdm2(S394A) mice. These findings document contrasting effects of ATM-Mdm2 signaling on p53 tumor suppression and reveal that destabilizing Mdm2 by promoting its phosphorylation by ATM would be effective in treating oncogene-induced malignancies, while inhibiting Mdm2-S394 phosphorylation during radiation exposure or chemotherapy would ameliorate bone marrow failure and prevent the development of secondary hematological malignancies. PMID:27568562

  7. Mdm2 is a novel activator of ApoCIII promoter which is antagonized by p53 and SHP inhibition

    SciTech Connect

    Yang, Zhihong; Zhang, Yuxia; Wang, Li

    2012-01-13

    Highlights: Black-Right-Pointing-Pointer Mdm2 enhances HNF4{alpha} activation of the ApoCIII promoter via interaction with HNF4{alpha}. Black-Right-Pointing-Pointer p53 antagonizes the effect of Mdm2 activation of the ApoCIII promoter. Black-Right-Pointing-Pointer SHP strengthens p53 inhibition but abolishes Mdm2 activation of the ApoCIII promoter. Black-Right-Pointing-Pointer Mdm2 alters the enrichment of HNF4{alpha}, p53 and SHP to the ApoCIII promoter. -- Abstract: We examined the effect of Mdm2 on regulation of the ApoCIII promoter and its cross-talk with p53 and nuclear receptor SHP. Overexpression of Mdm2 markedly enhanced ApoCIII promoter activity by HNF4{alpha}. A direct association of Mdm2 protein with the HNF4{alpha} protein was observed by co-immunoprecipitation. Ectopic expression of p53 decreased HNF4{alpha} activation of the ApoCIII promoter and antagonized the effect of Mdm2. Co-expression of SHP further strengthened p53 inhibition and abolished Mdm2 activation of the ApoCIII promoter. Mdm2 inhibited p53-mediated enrichment of HNF4{alpha} to the ApoCIII promoter while simultaneously reducing p53 binding and increasing recruitment of SHP to the ApoCIII promoter. The results from this study implicate a potentially important function of Mdm2 in regulation of lipoprotein metabolism.

  8. Challenging dedifferentiated liposarcoma identified by MDM2-amplification, a report of two cases

    PubMed Central

    2014-01-01

    Background Liposarcoma is the most frequent soft tissue sarcoma. Well differentiated liposarcoma may progress into dedifferentiated liposarcoma with pleomorphic histology. A minority additionally features myogenic, osteo- or chondrosarcomatous heterologous differentiation. Genomic amplification of the Mouse double minute 2 homolog (MDM2) locus is characteristic for well differentiated and dedifferentiated liposarcomas. Detection of MDM2 amplification may supplement histopathology and aid to distinguish liposarcoma from other soft tissue neoplasia. Case presentation Here we present two cases of dedifferentiated liposarcoma with challenging presentation. Case 1 features a myogenic component. As the tumour infiltrated the abdominal muscles and showed immunohistochemical expression of myogenic proteins, rhabdomyosarcoma had to be ruled out. Case 2 has an osteosarcomatous component resembling extraosseous osteosarcoma. The MDM2 status was determined in both cases and helped making the correct diagnosis. Overexpression of MDM2 and co-overexpression of Cyclin-dependent kinase 4 is demonstrated by immunohistochemistry. The underlying MDM2 amplification is shown by fluorescence in situ hybridisation. Since low grade osteosarcoma may also harbour MDM2 amplification it is emphasised that the amplification has to be present in the lipomatous parts of the tumour to distinguish liposarcoma from extraosseous osteosarcoma. Conclusions The two cases exemplify challenges in the diagnoses of dedifferentiated liposarcoma. Liposarcoma often has pleomorphic histology and additionally may feature heterologous components that mimic other soft tissue neoplasms. Amplification of MDM2 is characteristic for well differentiated and dedifferentiated liposarcomas. Determination of the MDM2 status by in situ hybridisation may assist histopathology and help to rule out differential diagnoses. PMID:25126005

  9. MDM2 E3 ligase-mediated ubiquitination and degradation of HDAC1 in vascular calcification

    PubMed Central

    Kwon, Duk-Hwa; Eom, Gwang Hyeon; Ko, Jeong Hyeon; Shin, Sera; Joung, Hosouk; Choe, Nakwon; Nam, Yoon Seok; Min, Hyun-Ki; Kook, Taewon; Yoon, Somy; Kang, Wanseok; Kim, Yong Sook; Kim, Hyung Seok; Choi, Hyuck; Koh, Jeong-Tae; Kim, Nacksung; Ahn, Youngkeun; Cho, Hyun-Jai; Lee, In-Kyu; Park, Dong Ho; Suk, Kyoungho; Seo, Sang Beom; Wissing, Erin R.; Mendrysa, Susan M.; Nam, Kwang-Il; Kook, Hyun

    2016-01-01

    Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC. PMID:26832969

  10. MDM2-MDM4 molecular interaction investigated by atomic force spectroscopy and surface plasmon resonance.

    PubMed

    Moscetti, Ilaria; Teveroni, Emanuela; Moretti, Fabiola; Bizzarri, Anna Rita; Cannistraro, Salvatore

    2016-01-01

    Murine double minute 2 (MDM2) and 4 (MDM4) are known as the main negative regulators of p53, a tumor suppressor. They are able to form heterodimers that are much more effective in the downregulation of p53. Therefore, the MDM2-MDM4 complex could be a target for promising therapeutic restoration of p53 function. To this aim, a deeper understanding of the molecular mechanisms underlining the heterodimerization is needed. The kinetic and thermodynamic characterization of the MDM2-MDM4 complex was performed with two complementary approaches: atomic force spectroscopy and surface plasmon resonance. Both techniques revealed an equilibrium dissociation constant (KD ) in the micromolar range for the MDM2-MDM4 heterodimer, similar to related complexes involved in the p53 network. Furthermore, the MDM2-MDM4 complex is characterized by a relatively high free energy, through a single energy barrier, and by a lifetime in the order of tens of seconds. New insights into the MDM2-MDM4 interaction could be highly important for developing innovative anticancer drugs focused on p53 reactivation. PMID:27621617

  11. Expression of p14ARF, MDM2, and MDM4 in human retinoblastoma.

    PubMed

    Guo, Ying; Pajovic, Sanja; Gallie, Brenda L

    2008-10-10

    It is still not clear whether the p53 pathway is altered in retinoblastoma development. We assessed the expression of the p53 pathway genes p14(ARF), mouse double minute 2 (MDM2), and mouse double minute 4 (MDM4) in human retinoblastoma compared to normal retina. Primary human retinoblastomas, retinoblastoma cell lines and normal retinas were assessed for p14(ARF) and MDM4 mRNA by quantitative RT-PCR. p14(ARF), MDM2, and MDM4 protein were measured by immunoblot and immunohistochemistry. Compared to retina, p14(ARF) mRNA expression was notably increased in retinoblastoma but p14(ARF) protein was undetectable. MDM2 and MDM4 proteins were expressed in 22/22 retinoblastomas. MDM2 was expressed in 3/10 retinas tested, and MDM4 in 10/10 retinas. The expression level of MDM2 protein in retinoblastomas and retina was comparable, while MDM4 protein was overexpressed in one retinoblastoma cell line Y79 and two primary retinoblastomas. We observe that overexpression of MDM2 and MDM4 is not a necessary step in retinoblastoma development. However, loss of detectable p14(ARF) protein and resultant lack of functional inactivation of these p53 inhibitors may contribute to retinoblastoma development by constitutive inhibition of p53. PMID:18644346

  12. Nucleoplasmic Mobilization of Nucleostemin Stabilizes MDM2 and Promotes G2/M Progression and Cell Survival

    PubMed Central

    Meng, Lingjun; Lin, Tao; Tsai, Robert Y.L.

    2016-01-01

    Summary Nucleolar disassembly occurs during mitosis and nucleolar stress, releasing several MDM2-interactive proteins residing in the nucleolus that share the common activity of p53 stabilization. Here, we demonstrated that mobilization of nucleostemin (NS), a cancer and stem cell-enriched nucleolar protein, plays the opposite role by stabilizing MDM2 and suppressing p53 functions. Our results showed that NS increases the protein stability and nucleoplasmic retention of MDM2, and competes with L23 for MDM2 binding. These activities are significantly elevated when NS is released into the nucleoplasm by mutations that abolish its nucleolar localization or by chemotherapeutic agents that disassemble the nucleoli. NS depletion decreases MDM2 protein, increases the transcriptional activities without changing the protein level of p53, and triggers G2/M arrest and cell death in U2OS but not in H1299 cells. This work reveals that nucleoplasmic relocation of NS during nucleolar disassembly safeguards the G2/M transit and survival of continuously dividing cells by MDM2 stabilization and p53 inhibition. PMID:19033382

  13. Multiple murine double minute gene 2 (MDM2) proteins are induced by ultraviolet light.

    PubMed

    Saucedo, L J; Myers, C D; Perry, M E

    1999-03-19

    The mdm2 (murine double minute 2) oncogene encodes several proteins, the largest of which (p90) binds to and inactivates the p53 tumor suppressor protein. Multiple MDM2 proteins have been detected in tumors and in cell lines expressing high levels of mdm2 mRNAs. Here we show that one of these proteins (p76) is expressed, along with p90, in wild-type and p53-null mouse embryo fibroblasts, indicating that it may have an important physiological role in normal cells. Expression of this protein is induced, as is that of p90, by UV light in a p53-dependent manner. The p76 protein is synthesized via translational initiation at AUG codon 50 and thus lacks the N terminus of p90 and does not bind p53. In cells, p90 and p76 can be synthesized from mdm2 mRNAs transcribed from both the P1 (constitutive) and P2 (p53-responsive) promoters. Site-directed mutagenesis reveals that these RNAs give rise to p76 via internal initiation of translation. In addition, mdm2 mRNAs lacking exon 3 give rise to p76 exclusively, and such mRNAs are induced by p53 in response to UV light. These data indicate that p76 may be an important product of the mdm2 gene and a downstream effector of p53.

  14. Melatonin down-regulates MDM2 gene expression and enhances p53 acetylation in MCF-7 cells.

    PubMed

    Proietti, Sara; Cucina, Alessandra; Dobrowolny, Gabriella; D'Anselmi, Fabrizio; Dinicola, Simona; Masiello, Maria Grazia; Pasqualato, Alessia; Palombo, Alessandro; Morini, Veronica; Reiter, Russel J; Bizzarri, Mariano

    2014-08-01

    Compelling evidence demonstrated that melatonin increases p53 activity in cancer cells. p53 undergoes acetylation to be stabilized and activated for driving cells destined for apoptosis/growth inhibition. Over-expression of p300 induces p53 acetylation, leading to cell growth arrest by increasing p21 expression. In turn, p53 activation is mainly regulated in the nucleus by MDM2. MDM2 also acts as E3 ubiquitin ligase, promoting the proteasome-dependent p53 degradation. MDM2 entry into the nucleus is finely tuned by two different modulations: the ribosomal protein L11, acts by sequestering MDM2 in the cytosol, whereas the PI3K-AkT-dependent MDM2 phosphorylation is mandatory for MDM2 translocation across the nuclear membrane. In addition, MDM2-dependent targeting of p53 is regulated in a nonlinear fashion by MDM2/MDMX interplay. Melatonin induces both cell growth inhibition and apoptosis in MCF7 breast cancer cells. We previously reported that this effect is associated with reduced MDM2 levels and increased p53 activity. Herein, we demonstrated that melatonin drastically down-regulates MDM2 gene expression and inhibits MDM2 shuttling into the nucleus, given that melatonin increases L11 and inhibits Akt-PI3K-dependent MDM2 phosphorylation. Melatonin induces a 3-fold increase in both MDMX and p300 levels, decreasing simultaneously Sirt1, a specific inhibitor of p300 activity. Consequently, melatonin-treated cells display significantly higher values of both p53 and acetylated p53. Thus, a 15-fold increase in p21 levels was observed in melatonin-treated cancer cells. Our results provide evidence that melatonin enhances p53 acetylation by modulating the MDM2/MDMX/p300 pathway, disclosing new insights for understanding its anticancer effect. PMID:24920214

  15. MDM2 SNP309 promoter polymorphism confers risk for hereditary melanoma.

    PubMed

    Thunell, Lena K; Bivik, Cecilia; Wäster, Petra; Fredrikson, Mats; Stjernström, Annika; Synnerstad, Ingrid; Rosdahl, Inger; Enerbäck, Charlotta

    2014-06-01

    The p53 pathway regulates stress response, and variations in p53, MDM2, and MDM4 may predispose an individual to tumor development. The aim of this study was to study the impact of genetic variation on sporadic and hereditary melanoma. We have analyzed a combination of three functionally relevant variants of the p53 pathway in 258 individuals with sporadic malignant melanomas, 50 with hereditary malignant melanomas, and 799 healthy controls. Genotyping was performed by PCR-restriction fragment length polymorphism, pyrosequencing, and allelic discrimination. We found an increased risk for hereditary melanoma in MDM2 GG homozygotes, which was more pronounced among women (P=0.035). In the event of pairwise combinations of the single nucleotide polymorphisms, a risk elevation was shown for MDM2 GG homozygotes/p53 wild-type Arg in hereditary melanoma (P=0.01). Individuals with sporadic melanomas of the superficial spreading type, including melanoma in situ, showed a slightly higher frequency of the MDM2 GG genotype compared with those with nodular melanomas (P=0.04). The dysplastic nevus phenotype, present in the majority of our hereditary melanoma cases and also in some sporadic cases, further enhanced the effect of the MDM2 GG genotype on melanoma risk (P=0.005). In conclusion, the results show an association between MDM2 SNP309 and an increased risk for hereditary melanoma, especially among women. Analysis of sporadic melanoma also shows an association between MDM2 and the superficial spreading melanoma subtype, as well as an association with the presence of dysplastic nevi in sporadic melanoma. PMID:24625390

  16. p53 expression and relationship with MDM2 amplification in breast carcinomas.

    PubMed

    Buyukpinarbasili, Nur; Gucin, Zuhal; Ersoy, Yeliz Emine; İlbak, Ayca; Kadioglu, Huseyin; Muslumanoglu, Mahmut

    2016-04-01

    Carcinoma of the breast, like other malignancies, is a genetic disease with multiple genetic events leading to the malignant phenotype. p53 mutations are the most common genetic events in human cancer. Inactivation of p53 can be a result of mutation in gene sequence. One of the main structures that regulate p53 stabilization is MDM2. It suppresses p53 transcriptional activation by recognizing transactivation domain of p53. The loss of MDM2 function on p53 regulation results in deprivation of p53 tumor suppressor ability. Single nucleotide polymorphisms (SNP309 T->G exchange) or MDM2 amplification has been proposed to play a role in this issue. In the present study, our aim is to analyze p53 and MDM2 status and investigate their interactions in human sporadic breast carcinoma. The study groups were separated according to their molecular classifications. In each group, histologic type of the tumor, conventional prognostic parameters, p53, and MDM2 interactions were compared statistically. Tumors are divided into 4 subtypes due to estrogen and progesterone receptor status, HER-2, and Ki-67 proliferation index results. According to this classification, 23 cases are in the luminal A, 32 cases are in the luminal B, 15 cases are in the HER-2 positive, and 22 cases are in the triple-negative group, with a total of 92 cases. p53 expression is low in luminal breast carcinomas than HER-2 and triple-negative subtypes. MDM2 amplification frequency was found to be 5.4% in total. MDM2 gene amplification does not have a significant role in breast carcinogenesis, but other possible mechanisms may play a role in its inactivation. PMID:27040927

  17. Interplay between Mdm2 and HIPK2 in the DNA damage response

    PubMed Central

    Zhang, Xiao-Peng; Liu, Feng; Wang, Wei

    2014-01-01

    The tumour suppressor p53 is activated to induce cell-cycle arrest or apoptosis in the DNA damage response (DDR). p53 phosphorylation at Ser46 by HIPK2 (homeodomain-interacting protein kinase 2) is a critical event in apoptosis induction. Interestingly, HIPK2 is degraded by Mdm2 (a negative regulator of p53), whereas Mdm2 is downregulated by HIPK2 through several mechanisms. Here, we develop a four-module network model for the p53 pathway to clarify the role of interplay between Mdm2 and HIPK2 in the DDR evoked by ultraviolet radiation. By numerical simulations, we reveal that Mdm2-dependent HIPK2 degradation promotes cell survival after mild DNA damage and that inhibition of HIPK2 degradation is sufficient to trigger apoptosis. In response to severe damage, p53 phosphorylation at Ser46 is promoted by the accumulation of HIPK2 due to downregulation of nuclear Mdm2 in the later phase of the response. Meanwhile, the concentration of p53 switches from moderate to high levels, contributing to apoptosis induction. We show that the presence of three mechanisms for Mdm2 downregulation, i.e. repression of mdm2 expression, inhibition of its nuclear entry and HIPK2-induced degradation, guarantees the apoptosis of irreparably damaged cells. Our results agree well with multiple experimental observations, and testable predictions are also made. This work advances our understanding of the regulation of p53 activity in the DDR and suggests that HIPK2 should be a significant target for cancer therapy. PMID:24829283

  18. TRIM25 has a dual function in the p53/Mdm2 circuit.

    PubMed

    Zhang, P; Elabd, S; Hammer, S; Solozobova, V; Yan, H; Bartel, F; Inoue, S; Henrich, T; Wittbrodt, J; Loosli, F; Davidson, G; Blattner, C

    2015-11-12

    P53 is an important tumor suppressor that, upon activation, induces growth arrest and cell death. Control of p53 is thus of prime importance for proliferating cells, but also for cancer therapy, where p53 activity contributes to the eradication of tumors. Mdm2 functionally inhibits p53 and targets the tumor suppressor protein for degradation. In a genetic screen, we identified TRIM25 as a novel regulator of p53 and Mdm2. TRIM25 increased p53 and Mdm2 abundance by inhibiting their ubiquitination and degradation in 26 S proteasomes. TRIM25 co-precipitated with p53 and Mdm2 and interfered with the association of p300 and Mdm2, a critical step for p53 polyubiquitination. Despite the increase in p53 levels, p53 activity was inhibited in the presence of TRIM25. Downregulation of TRIM25 resulted in an increased acetylation of p53 and p53-dependent cell death in HCT116 cells. Upon genotoxic insults, TRIM25 dampened the p53-dependent DNA damage response. The downregulation of TRIM25 furthermore resulted in massive apoptosis during early embryogenesis of medaka, which was rescued by the concomitant downregulation of p53, demonstrating the functional relevance of the regulation of p53 by TRIM25 in an organismal context. PMID:25728675

  19. Autoactivation of the MDM2 E3 Ligase by Intramolecular Interaction

    PubMed Central

    Cheng, Qian; Song, Tanjing; Chen, Lihong

    2014-01-01

    The RING domain ubiquitin E3 ligase MDM2 is a key regulator of p53 degradation and a mediator of signals that stabilize p53. The current understanding of the mechanisms by which MDM2 posttranslational modifications and protein binding cause p53 stabilization remains incomplete. Here we present evidence that the MDM2 central acidic region is critical for activating RING domain E3 ligase activity. A 30-amino-acid minimal region of the acidic domain binds to the RING domain through intramolecular interactions and stimulates the catalytic function of the RING domain in promoting ubiquitin release from charged E2. The minimal activation sequence is also the binding site for the ARF tumor suppressor, which inhibits ubiquitination of p53. The acidic domain-RING domain intramolecular interaction is modulated by ATM-mediated phosphorylation near the RING domain or by binding of ARF. These results suggest that MDM2 phosphorylation and association with protein regulators share a mechanism in inhibiting the E3 ligase function and stabilizing p53 and suggest that targeting the MDM2 autoactivation mechanism may be useful for therapeutic modulation of p53 levels. PMID:24842904

  20. Downregulation of MDM2 expression by RNAi inhibits LoVo human colorectal adenocarcinoma cells growth and the treatment of LoVo cells with mdm2siRNA3 enhances the sensitivity to cisplatin

    SciTech Connect

    Yu Yan . E-mail: gyfyuyan@hotmail.com; Sun Ping . E-mail: sunny19750502@hotmail.com; Sun Lichun; Liu Guoyi; Chen Guohua . E-mail: olivebranch_82@hotmail.com; Shang Lihua . E-mail: leval1000@sina.com; Wu Hongbo . E-mail: whpwl@sina.com; Hu Jing; Li Yue; Mao Yinling; Sui Guangjie; Sun Xiwen

    2006-01-06

    To investigate the biological effect of mdm2 in human colorectal adenocarcinoma LoVo cells, three mdm2siRNA constructions were recombinated and transient transfected into human colorectal adenocarcinoma LoVo cells with low differentiation character in vitro. The results showed that mdm2siRNA3 reduced mRNA level of mdm2 and protein level of mdm2, leading to proliferation inhibition on LoVo cells, and reduced tumor growth in nude mice. It was found that depletion of MDM2 in this pattern promoted apoptosis of LoVo cells and Cisplatin (DDP) treated in the mdm2siRNA3 transfected cell population would result in a substantial decrease by MTT colorimetry. Decreasing the MDM2 protein level in LoVo cells by RNAi could significantly inhibit tumor growth both in vitro and in vivo, which indicated that mdm2 gene played a definite role in the development and aggressiveness of human colon carcinoma. It also could be a therapeutic target in colorectal carcinoma. The synergistic activation of RNAi and cell toxicity agents indicated that the combination of chemotherapy and gene therapy will be a promising approach in the future.

  1. Structure of human MDM2 complexed with RPL11 reveals the molecular basis of p53 activation

    PubMed Central

    Zheng, Jiangge; Lang, Yue; Zhang, Qi; Cui, Di; Sun, Haili; Jiang, Lun; Chen, Zhenhang; Zhang, Rui; Gao, Yina; Tian, Wenli; Wu, Wei; Tang, Jun; Chen, Zhongzhou

    2015-01-01

    The central region of MDM2 is critical for p53 activation and tumor suppression. Upon ribosomal stress, this region is bound by ribosomal proteins, particularly ribosomal protein L11 (RPL11), leading to MDM2 inactivation and subsequent p53 activation. Here, we solved the complex structure of human MDM2–RPL11 at 2.4 Å. MDM2 extensively interacts with RPL11 through an acidic domain and two zinc fingers. Formation of the MDM2–RPL11 complex induces substantial conformational changes in both proteins. RPL11, unable to bind MDM2 mutants, fails to induce the activation of p53 in cells. MDM2 mimics 28S rRNA binding to RPL11. The C4 zinc finger determines RPL11 binding to MDM2 but not its homolog, MDMX. Our results highlight the essential role of the RPL11–MDM2 interaction in p53 activation and tumor suppression and provide a structural basis for potential new anti-tumor drug development. PMID:26220995

  2. Controlled access of p53 to the nucleus regulates its proteasomal degradation by MDM2.

    PubMed

    Davis, James R; Mossalam, Mohanad; Lim, Carol S

    2013-04-01

    The tumor suppressor p53 can be sent to the proteasome for degradation by placing its nucleo-cytoplasmic shuttling under ligand control. Endogenous p53 is ubiquitinated by MDM2 in the nucleus, and controlling the access of p53 to the nuclear compartment regulates its ubiquitination and proteasomal degradation. This was accomplished by the use of a protein switch that places nuclear translocation under the control of externally applied dexamethasone. Fluorescence microscopy revealed that sending protein switch p53 (PS-p53) to the nucleus produces a distinct punctate distribution in both the cytoplasm and nucleus. The nuclear role in accessing the proteasome was investigated by inhibiting classical nuclear export with leptomycin B. Trapping PS-p53 in the nucleus only allows this punctate staining in that compartment, suggesting that PS-p53 must translocate first to the nuclear compartment for cytoplasmic punctate staining to occur. The role of MDM2 binding was explored by inhibiting MDM2/p53 binding with nutlin-3. Inhibition of this interaction blocked both nuclear export and cytoplasmic and nuclear punctate staining, providing evidence that any change in localization after nuclear translocation is due to MDM2 binding. Further, blocking the proteolytic activity of the proteasome maintained the nuclear localization of the construct. Truncations of p53 were made to determine smaller constructs still capable of interacting with MDM2, and their subcellular localization and degradation potential was observed. PS-p53 and a smaller construct containing the two MDM2 binding regions of p53 (Box I + V) were indeed degraded by the proteasome as measured by loss of enhanced green fluorescent protein that was also fused to the construct. The influence of these constructs on p53 gene transactivation function was assessed and revealed that PS-p53 decreased gene transactivation, while PS-p53(Box I + V) did not significantly change baseline gene transactivation.

  3. A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

    PubMed Central

    ElSawy, Karim M; Sim, Adelene; Lane, David P; Verma, Chandra S; Caves, Leo SD

    2015-01-01

    The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development. PMID:25584963

  4. Medicinal Chemistry Strategies to Disrupt the p53-MDM2/MDMX Interaction.

    PubMed

    Lemos, Agostinho; Leão, Mariana; Soares, Joana; Palmeira, Andreia; Pinto, Madalena; Saraiva, Lucília; Sousa, Maria Emília

    2016-09-01

    The growth inhibitory activity of p53 tumor suppressor is tightly regulated by interaction with two negative regulatory proteins, murine double minute 2 (MDM2) and X (MDMX), which are overexpressed in about half of all human tumors. The elucidation of crystallographic structures of MDM2/MDMX complexes with p53 has been pivotal for the identification of several classes of inhibitors of the p53-MDM2/MDMX interaction. The present review provides in silico strategies and screening approaches used in drug discovery as well as an overview of the most relevant classes of small-molecule inhibitors of the p53-MDM2/MDMX interaction, their progress in pipeline, and highlights particularities of each class of inhibitors. Most of the progress made with high-throughput screening has led to the development of inhibitors belonging to the cis-imidazoline, piperidinone, and spiro-oxindole series. However, novel potent and selective classes of inhibitors of the p53-MDM2 interaction with promising antitumor activity are emerging. Even with the discovery of the 3D structure of complex p53-MDMX, only two small molecules were reported as selective p53-MDMX antagonists, WK298 and SJ-172550. Dual inhibition of the p53-MDM2/MDMX interaction has shown to be an alternative approach since it results in full activation of the p53-dependent pathway. The knowledge of structural requirements crucial to the development of small-molecule inhibitors of the p53-MDMs interactions has enabled the identification of novel antitumor agents with improved in vivo efficacy.

  5. Medicinal Chemistry Strategies to Disrupt the p53-MDM2/MDMX Interaction.

    PubMed

    Lemos, Agostinho; Leão, Mariana; Soares, Joana; Palmeira, Andreia; Pinto, Madalena; Saraiva, Lucília; Sousa, Maria Emília

    2016-09-01

    The growth inhibitory activity of p53 tumor suppressor is tightly regulated by interaction with two negative regulatory proteins, murine double minute 2 (MDM2) and X (MDMX), which are overexpressed in about half of all human tumors. The elucidation of crystallographic structures of MDM2/MDMX complexes with p53 has been pivotal for the identification of several classes of inhibitors of the p53-MDM2/MDMX interaction. The present review provides in silico strategies and screening approaches used in drug discovery as well as an overview of the most relevant classes of small-molecule inhibitors of the p53-MDM2/MDMX interaction, their progress in pipeline, and highlights particularities of each class of inhibitors. Most of the progress made with high-throughput screening has led to the development of inhibitors belonging to the cis-imidazoline, piperidinone, and spiro-oxindole series. However, novel potent and selective classes of inhibitors of the p53-MDM2 interaction with promising antitumor activity are emerging. Even with the discovery of the 3D structure of complex p53-MDMX, only two small molecules were reported as selective p53-MDMX antagonists, WK298 and SJ-172550. Dual inhibition of the p53-MDM2/MDMX interaction has shown to be an alternative approach since it results in full activation of the p53-dependent pathway. The knowledge of structural requirements crucial to the development of small-molecule inhibitors of the p53-MDMs interactions has enabled the identification of novel antitumor agents with improved in vivo efficacy. PMID:27302609

  6. Influence of MDM2 polymorphisms on squamous cell carcinoma susceptibility: a meta-analysis

    PubMed Central

    Yu, Huanxin; Li, Haiyan; Zhang, Jinling; Liu, Gang

    2016-01-01

    Purpose Controversial associations between single-nucleotide polymorphisms (rs2279744, rs937283, rs3730485) of the MDM2 gene and the etiology of squamous cell carcinomas (SCCs) have been reported. This merits further comprehensive assessment. Materials and methods We systematically reviewed the available data and conducted an updated meta-analysis to evaluate the genetic effect of MDM2 polymorphisms in SCC susceptibility, using Stata/SE 12.0 software. Results After screening, 7,987 SCC cases and 12,954 controls from 26 eligible case–control studies were enrolled. Overall, compared with the control group, a significantly increased SCC risk was observed for the MDM2 rs2279744 polymorphism in the Asian population (test of association: odds ratio [OR] 1.12, P=0.027 for G vs T; OR 1.26, P=0.016 for GG vs TT; OR 1.25, P<0.001 for GG vs TT + TG; and OR 1.08, P=0.023 for carrier G vs T). In subgroup analysis by SCC type, a similarly increased esophageal SCC risk was detected (OR 1.19, P<0.001 for G vs T; OR 1.46, P<0.001 for GG vs TT; and OR 1.48, P=0.005 for GG vs TT + TG). Furthermore, MDM2–TP53 double mutation was statistically associated with increased SCC susceptibility overall (OR 1.52, P=0.001), especially in the Asian population (OR 1.49, P=0.022). However, no significant difference between the control and case groups was obtained for MDM2 rs937283 or rs3730485 under any genetic model (all P>0.05). Conclusion Our results highlight a positive association between the GG genotype of MDM2 rs2279744 polymorphism and an increased risk of esophageal SCC in the Asian population, which needs to be clarified by more large-scale studies. PMID:27785069

  7. A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2.

    PubMed

    ElSawy, Karim M; Sim, Adelene; Lane, David P; Verma, Chandra S; Caves, Leo Sd

    2015-01-01

    The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development. PMID:25584963

  8. MDM2/MDMX: Master negative regulators for p53 and RB.

    PubMed

    Hu, Linshan; Zhang, Haibo; Bergholz, Johann; Sun, Shengnan; Xiao, Zhi-Xiong Jim

    2016-03-01

    MDM2 (mouse double minute 2 homolog) and MDMX (double minute X human homolog, also known as MDM4) are critical negative regulators of tumor protein p53. Our recent work shows that MDMX binds to and promotes degradation of retinoblastoma protein (RB) in an MDM2-dependent manner. In a xenograft tumor growth mouse model, silencing of MDMX results in inhibition of p53-deficient tumor growth, which can be effectively reversed by concomitant RB silencing. Thus, MDMX exerts its oncogenic activity via suppression of RB.

  9. Quantitative assessment of the p53-Mdm2 feedback loop using protein lysate microarrays.

    PubMed

    Ramalingam, Sundhar; Honkanen, Peter; Young, Lynn; Shimura, Tsutomu; Austin, John; Steeg, Patricia S; Nishizuka, Satoshi

    2007-07-01

    Mathematical simulations of the p53-Mdm2 feedback loop suggest that both proteins will exhibit impulsive expression characteristics in response to high cellular stress levels. However, little quantitative experimental evaluation has been done, particularly of the phosphorylated forms. To evaluate the mathematical models experimentally, we used lysate microarrays from an isogenic pair of gamma-ray-irradiated cell lysates from HCT116 (p53(+/+) and p53(-/-)). Both p53 and Mdm2 proteins showed expected pulses in the wild type, whereas no pulses were seen in the knockout. Based on experimental observations, we determined model parameters and generated an in silico "knockout," reflecting the experimental data, including phosphorylated proteins.

  10. MDM2/MDMX: Master negative regulators for p53 and RB.

    PubMed

    Hu, Linshan; Zhang, Haibo; Bergholz, Johann; Sun, Shengnan; Xiao, Zhi-Xiong Jim

    2016-03-01

    MDM2 (mouse double minute 2 homolog) and MDMX (double minute X human homolog, also known as MDM4) are critical negative regulators of tumor protein p53. Our recent work shows that MDMX binds to and promotes degradation of retinoblastoma protein (RB) in an MDM2-dependent manner. In a xenograft tumor growth mouse model, silencing of MDMX results in inhibition of p53-deficient tumor growth, which can be effectively reversed by concomitant RB silencing. Thus, MDMX exerts its oncogenic activity via suppression of RB. PMID:27308631

  11. The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

    PubMed

    Duan, Yajian; Ma, Gaoen; Huang, Xionggao; D'Amore, Patricia A; Zhang, Feng; Lei, Hetian

    2016-07-29

    The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.

  12. MDM2 T309G polymorphism and esophageal cancer risk: a meta-analysis.

    PubMed

    Lei, Caipeng; Zhang, Weiguo; Fan, Junli; Qiao, Bin; Chen, Qiang; Liu, Qin; Zhao, Chunling

    2015-01-01

    Murine double minute 2 (MDM2) has suggested to play an important role in esophageal cancer. The association between MDM2 T309G polymorphism and esophageal cancer risk was inconclusive. To clarify the possible association, we conducted a meta-analysis. We searched in the PubMed, Embase, and Wanfang databases. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association. A total of 6 studies with 4909 cases and controls were included based on the search criteria. The MDM2 T309G polymorphism was associated with a significantly decreased risk of esophageal cancer (OR=0.88; 95% CI, 0.81-0.96; I(2)=22%). When stratified by type of race, a significantly decreased esophageal cancer risk were observed in Asians (OR=0.85; 95% CI, 0.78-0.93; I(2)=0%). In conclusion, this meta-analysis suggested that MDM2 T309G polymorphism was associated with a significantly decreased risk of esophageal cancer. PMID:26550276

  13. MDM2 T309G polymorphism and esophageal cancer risk: a meta-analysis

    PubMed Central

    Lei, Caipeng; Zhang, Weiguo; Fan, Junli; Qiao, Bin; Chen, Qiang; Liu, Qin; Zhao, Chunling

    2015-01-01

    Murine double minute 2 (MDM2) has suggested to play an important role in esophageal cancer. The association between MDM2 T309G polymorphism and esophageal cancer risk was inconclusive. To clarify the possible association, we conducted a meta-analysis. We searched in the PubMed, Embase, and Wanfang databases. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association. A total of 6 studies with 4909 cases and controls were included based on the search criteria. The MDM2 T309G polymorphism was associated with a significantly decreased risk of esophageal cancer (OR=0.88; 95% CI, 0.81-0.96; I2=22%). When stratified by type of race, a significantly decreased esophageal cancer risk were observed in Asians (OR=0.85; 95% CI, 0.78-0.93; I2=0%). In conclusion, this meta-analysis suggested that MDM2 T309G polymorphism was associated with a significantly decreased risk of esophageal cancer. PMID:26550276

  14. The Mdm2 and p53 genes are conserved in the Arachnids.

    PubMed

    Lane, David P; Cheok, Chit Fang; Brown, Christopher J; Madhumalar, Arumugam; Ghadessy, Farid J; Verma, Chandra

    2010-02-15

    The p53 protein and its negative regulator the ubiquitin E3 ligase Mdm2 have been shown to be conserved from the T. adhaerens to man. In common with D. melanogaster and C. elegans, there is a single copy of the p53 gene in T. adhaerens, while in the vertebrates three p53-like genes can be found: p53, p63 and p73. The Mdm2 gene is not present within the fully sequenced and highly annotated genomes of C. elegans and D. melanogaster. However, it is present in Placazoanand the presence of multiple distinct p53 genes in the Sea anemone N. vectensis led us to examine the genomes of other phyla for p53 and Mdm2-like genes. We report here the discovery of an Mdm2-like gene and two distinct p53-like genes in the Arachnid Ioxodes scapularis (Northern Deer Tick). The two predicted Deer Tick p53 proteins are much more highly related to the human p53 protein in sequence than are the fruit fly and nematode proteins. One of the Deer Tick genes encodes a p53 protein that is initiated within the DNA binding domain of p53 and shows remarkable homology to the newly described N-terminally truncated delta isoforms of human and zebrafish p53. PMID:20160485

  15. KMT Set7/9 affects genotoxic stress response via the Mdm2 axis

    PubMed Central

    Fedorova, Olga; Malikova, Daria; Shuvalov, Oleg; Antonov, Alexey V.; Tentler, Dmitri; Garabadgiu, Alexander V.; Melino, Gerry; Barlev, Nikolai A.

    2015-01-01

    Genotoxic stress inflicted by anti-cancer drugs causes DNA breaks and genome instability. DNA double strand breaks induced by irradiation or pharmacological inhibition of Topoisomerase II activate ATM (ataxia-telangiectasia-mutated) kinase signalling pathway that in turn triggers cell cycle arrest and DNA repair. ATM-dependent gamma-phosphorylation of histone H2Ax and other histone modifications, including ubiquitnylation, promote exchange of histones and recruitment of DNA damage response (DDR) and repair proteins. Signal transduction pathways, besides DDR itself, also control expression of genes whose products cause cell cycle arrest and/or apoptosis thus ultimately affecting the sensitivity of cells to genotoxic stress. In this study, using a number of experimental approaches we provide evidence that lysine-specific methyltransferase (KMT) Set7/9 affects DDR and DNA repair, at least in part, by regulating the expression of an E3 ubiquitin ligase, Mdm2. Furthermore, we show that Set7/9 physically interacts with Mdm2. Several cancer cell lines with inverse expression of Set7/9 and Mdm2 displayed diminished survival in response to genotoxic stress. These findings are signified by our bioinformatics studies suggesting that the unleashed expression of Mdm2 in cancer patients with diminished expression of Set7/9 is associated with poor survival outcome. PMID:26317544

  16. Population distribution and ancestry of the cancer protective MDM2 SNP285 (rs117039649).

    PubMed

    Knappskog, Stian; Gansmo, Liv B; Dibirova, Khadizha; Metspalu, Andres; Cybulski, Cezary; Peterlongo, Paolo; Aaltonen, Lauri; Vatten, Lars; Romundstad, Pål; Hveem, Kristian; Devilee, Peter; Evans, Gareth D; Lin, Dongxin; Van Camp, Guy; Manolopoulos, Vangelis G; Osorio, Ana; Milani, Lili; Ozcelik, Tayfun; Zalloua, Pierre; Mouzaya, Francis; Bliznetz, Elena; Balanovska, Elena; Pocheshkova, Elvira; Kučinskas, Vaidutis; Atramentova, Lubov; Nymadawa, Pagbajabyn; Titov, Konstantin; Lavryashina, Maria; Yusupov, Yuldash; Bogdanova, Natalia; Koshel, Sergey; Zamora, Jorge; Wedge, David C; Charlesworth, Deborah; Dörk, Thilo; Balanovsky, Oleg; Lønning, Per E

    2014-09-30

    The MDM2 promoter SNP285C is located on the SNP309G allele. While SNP309G enhances Sp1 transcription factor binding and MDM2 transcription, SNP285C antagonizes Sp1 binding and reduces the risk of breast-, ovary- and endometrial cancer. Assessing SNP285 and 309 genotypes across 25 different ethnic populations (>10.000 individuals), the incidence of SNP285C was 6-8% across European populations except for Finns (1.2%) and Saami (0.3%). The incidence decreased towards the Middle-East and Eastern Russia, and SNP285C was absent among Han Chinese, Mongolians and African Americans. Interhaplotype variation analyses estimated SNP285C to have originated about 14,700 years ago (95% CI: 8,300 - 33,300). Both this estimate and the geographical distribution suggest SNP285C to have arisen after the separation between Caucasians and modern day East Asians (17,000 - 40,000 years ago). We observed a strong inverse correlation (r = -0.805; p < 0.001) between the percentage of SNP309G alleles harboring SNP285C and the MAF for SNP309G itself across different populations suggesting selection and environmental adaptation with respect to MDM2 expression in recent human evolution. In conclusion, we found SNP285C to be a pan-Caucasian variant. Ethnic variation regarding distribution of SNP285C needs to be taken into account when assessing the impact of MDM2 SNPs on cancer risk.

  17. MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3.

    PubMed

    Hallenborg, P; Siersbæk, M; Barrio-Hernandez, I; Nielsen, R; Kristiansen, K; Mandrup, S; Grøntved, L; Blagoev, B

    2016-01-01

    The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each resulted in abolished induction of a subset of cAMP-stimulated genes, with Cebpd being among the most affected. Moreover, STATs were able to interact with the transcriptional cofactors CRTC2 and CRTC3, hitherto only reported to associate with the cAMP-responsive transcription factor CREB. Last but not least, the binding of CRTC2 to a transcriptional enhancer that interacts with the Cebpd promoter was dramatically decreased upon JAK inhibition. Our data reveal the existence of an unusual functional interplay between STATs and CREB at the onset of adipogenesis through shared CRTC cofactors. PMID:27362806

  18. MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3.

    PubMed

    Hallenborg, P; Siersbæk, M; Barrio-Hernandez, I; Nielsen, R; Kristiansen, K; Mandrup, S; Grøntved, L; Blagoev, B

    2016-06-30

    The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each resulted in abolished induction of a subset of cAMP-stimulated genes, with Cebpd being among the most affected. Moreover, STATs were able to interact with the transcriptional cofactors CRTC2 and CRTC3, hitherto only reported to associate with the cAMP-responsive transcription factor CREB. Last but not least, the binding of CRTC2 to a transcriptional enhancer that interacts with the Cebpd promoter was dramatically decreased upon JAK inhibition. Our data reveal the existence of an unusual functional interplay between STATs and CREB at the onset of adipogenesis through shared CRTC cofactors.

  19. Chlamydia infection depends on a functional MDM2-p53 axis.

    PubMed

    González, Erik; Rother, Marion; Kerr, Markus C; Al-Zeer, Munir A; Abu-Lubad, Mohammad; Kessler, Mirjana; Brinkmann, Volker; Loewer, Alexander; Meyer, Thomas F

    2014-11-13

    Chlamydia, a major human bacterial pathogen, assumes effective strategies to protect infected cells against death-inducing stimuli, thereby ensuring completion of its developmental cycle. Paired with its capacity to cause extensive host DNA damage, this poses a potential risk of malignant transformation, consistent with circumstantial epidemiological evidence. Here we reveal a dramatic depletion of p53, a tumor suppressor deregulated in many cancers, during Chlamydia infection. Using biochemical approaches and live imaging of individual cells, we demonstrate that p53 diminution requires phosphorylation of Murine Double Minute 2 (MDM2; a ubiquitin ligase) and subsequent interaction of phospho-MDM2 with p53 before induced proteasomal degradation. Strikingly, inhibition of the p53-MDM2 interaction is sufficient to disrupt intracellular development of Chlamydia and interferes with the pathogen's anti-apoptotic effect on host cells. This highlights the dependency of the pathogen on a functional MDM2-p53 axis and lends support to a potentially pro-carcinogenic effect of chlamydial infection.

  20. Effects of the MDM2 promoter SNP285 and SNP309 on Sp1 transcription factor binding and cancer risk.

    PubMed

    Knappskog, Stian; Lønning, Per E

    2011-01-01

    The proto-oncogene MDM2 inhibits p53 and plays a key role in cell growth control and apoptosis. Identification of two antagonizing MDM2 polymorphisms, SNP285 and SNP309, affecting cancer risk through modulation of Sp1 transcription factor binding, shed new light on the biological activity and phylogeny of this gene.

  1. Antisense-MDM2 Sensitizes LNCaP Prostate Cancer Cells to Androgen Deprivation, Radiation, and the Combination In Vivo

    SciTech Connect

    Stoyanova, Radka; Hachem, Paul; Hensley, Harvey; Khor, L.-Y.; Mu Zhaomei; Hammond, M. Elizabeth H.; Agrawal, Sudhir; Pollack, Alan . E-mail: Alan.Pollack@fccc.edu

    2007-07-15

    Purpose: To test the effects of antisense (AS)-MDM2 alone and with androgen deprivation (AD), radiotherapy (RT), and AD + RT on wild-type LNCaP cells in an orthotopic in vivo model. Methods: Androgen-sensitive LNCaP cells were grown in the prostates of nude mice. Magnetic resonance imaging-based tumor volume and serum prostate-specific antigen (PSA) measurements were used to assess effects on tumor response. Tumor response was measured by biochemical and tumor volume failure definitions and doubling time estimates from fitted PSA and tumor volume growth curves. Expression of MDM2, p53, p21, and Ki-67 was quantified using immunohistochemical staining and image analysis of formalin-fixed tissue, analogous to methods used clinically. Results: Antisense-MDM2 significantly inhibited the growth of LNCaP tumors over the mismatch controls. The most significant increase in tumor growth delay and tumor doubling time was from AS-MDM2 + AD + RT, although the effect of AS-MDM2 + AD was substantial. Expression of MDM2 was significantly reduced by AS-MDM2 in the setting of RT. Conclusions: This is the first in vivo investigation of the effects of AS-MDM2 in an orthotopic model and the first to demonstrate incremental sensitization when added to AD and AD + RT. The results with AD underscore the potential to affect micrometastatic disease, which is probably responsible for treatment failure in 30-40% of men with high-risk disease.

  2. Targeting the MDM2/MDM4 interaction interface as a promising approach for p53 reactivation therapy.

    PubMed

    Pellegrino, Marsha; Mancini, Francesca; Lucà, Rossella; Coletti, Alice; Giacchè, Nicola; Manni, Isabella; Arisi, Ivan; Florenzano, Fulvio; Teveroni, Emanuela; Buttarelli, Marianna; Fici, Laura; Brandi, Rossella; Bruno, Tiziana; Fanciulli, Maurizio; D'Onofrio, Mara; Piaggio, Giulia; Pellicciari, Roberto; Pontecorvi, Alfredo; Marine, Jean Christophe; Macchiarulo, Antonio; Moretti, Fabiola

    2015-11-01

    Restoration of wild-type p53 tumor suppressor function has emerged as an attractive anticancer strategy. Therapeutics targeting the two p53-negative regulators, MDM2 and MDM4, have been developed, but most agents selectively target the ability of only one of these molecules to interact with p53, leaving the other free to operate. Therefore, we developed a method that targets the activity of MDM2 and MDM4 simultaneously based on recent studies indicating that formation of MDM2/MDM4 heterodimer complexes are required for efficient inactivation of p53 function. Using computational and mutagenesis analyses of the heterodimer binding interface, we identified a peptide that mimics the MDM4 C-terminus, competes with endogenous MDM4 for MDM2 binding, and activates p53 function. This peptide induces p53-dependent apoptosis in vitro and reduces tumor growth in vivo. Interestingly, interfering with the MDM2/MDM4 heterodimer specifically activates a p53-dependent oxidative stress response. Consistently, distinct subcellular pools of MDM2/MDM4 complexes were differentially sensitive to the peptide; nuclear MDM2/MDM4 complexes were particularly highly susceptible to the peptide-displacement activity. Taken together, these data identify the MDM2/MDM4 interaction interface as a valuable molecular target for therapeutic reactivation of p53 oncosuppressive function. PMID:26359458

  3. Human Glioblastoma Multiforme: p53 Reactivation by a Novel MDM2 Inhibitor

    PubMed Central

    Costa, Barbara; Bendinelli, Sara; Gabelloni, Pamela; Da Pozzo, Eleonora; Daniele, Simona; Scatena, Fabrizio; Vanacore, Renato; Campiglia, Pietro; Bertamino, Alessia; Gomez-Monterrey, Isabel; Sorriento, Daniela; Del Giudice, Carmine; Iaccarino, Guido; Novellino, Ettore; Martini, Claudia

    2013-01-01

    Cancer development and chemo-resistance are often due to impaired functioning of the p53 tumor suppressor through genetic mutation or sequestration by other proteins. In glioblastoma multiforme (GBM), p53 availability is frequently reduced because it binds to the Murine Double Minute-2 (MDM2) oncoprotein, which accumulates at high concentrations in tumor cells. The use of MDM2 inhibitors that interfere with the binding of p53 and MDM2 has become a valid approach to inhibit cell growth in a number of cancers; however little is known about the efficacy of these inhibitors in GBM. We report that a new small-molecule inhibitor of MDM2 with a spirooxoindolepyrrolidine core structure, named ISA27, effectively reactivated p53 function and inhibited human GBM cell growth in vitro by inducing cell cycle arrest and apoptosis. In immunoincompetent BALB/c nude mice bearing a human GBM xenograft, the administration of ISA27 in vivo activated p53, inhibited cell proliferation and induced apoptosis in tumor tissue. Significantly, ISA27 was non-toxic in an in vitro normal human cell model and an in vivo mouse model. ISA27 administration in combination with temozolomide (TMZ) produced a synergistic inhibitory effect on GBM cell viability in vitro, suggesting the possibility of lowering the dose of TMZ used in the treatment of GBM. In conclusion, our data show that ISA27 releases the powerful antitumor capacities of p53 in GBM cells. The use of this MDM2 inhibitor could become a novel therapy for the treatment of GBM patients. PMID:23977270

  4. Genome-scale functional analysis of the human genes modulating p53 activity by regulating MDM2 expression in a p53-independent manner.

    PubMed

    Kim, Dong Min; Choi, Seung-Hyun; Yeom, Young Il; Min, Sang-Hyun; Kim, Il-Chul

    2016-09-16

    MDM2, a critical negative regulator of p53, is often overexpressed in leukemia, but few p53 mutations are found, suggesting that p53-independent MDM2 expression occurs due to alterations in MDM2 upstream regulators. In this study, a high MDM2 transcription level was observed (41.17%) regardless of p53 expression in patient with acute myeloid leukemia (AML). Therefore, we performed genome-scale functional screening of the human genes modulating MDM2 expression in a p53-independent manner. We searched co-expression profiles of genes showing a positive or negative pattern with MDM2 expression in a DNA microarray database, selected1089 links, and composed a screening library of 368 genes. Using MDM2 P1 and P2 promoter-reporter systems, we screened clones regulating MDM2 transcriptions in a p53-independent manner by overexpression. Nine clones from the screening library showed enhanced MDM2 promoter activity and MDM2 expression in p53-deficient HCT116 cells. Among them, six clones, including NTRK2, GNA15, SFRS2, EIF5A, ELAVL1, and YWHAB mediated MAPK signaling for expressing MDM2. These results indicate that p53-independent upregulation of MDM2 by increasing selected clones may lead to oncogenesis in AML and that MDM2-modulating genes are novel potential targets for AML treatment. PMID:27524244

  5. Genome-scale functional analysis of the human genes modulating p53 activity by regulating MDM2 expression in a p53-independent manner.

    PubMed

    Kim, Dong Min; Choi, Seung-Hyun; Yeom, Young Il; Min, Sang-Hyun; Kim, Il-Chul

    2016-09-16

    MDM2, a critical negative regulator of p53, is often overexpressed in leukemia, but few p53 mutations are found, suggesting that p53-independent MDM2 expression occurs due to alterations in MDM2 upstream regulators. In this study, a high MDM2 transcription level was observed (41.17%) regardless of p53 expression in patient with acute myeloid leukemia (AML). Therefore, we performed genome-scale functional screening of the human genes modulating MDM2 expression in a p53-independent manner. We searched co-expression profiles of genes showing a positive or negative pattern with MDM2 expression in a DNA microarray database, selected1089 links, and composed a screening library of 368 genes. Using MDM2 P1 and P2 promoter-reporter systems, we screened clones regulating MDM2 transcriptions in a p53-independent manner by overexpression. Nine clones from the screening library showed enhanced MDM2 promoter activity and MDM2 expression in p53-deficient HCT116 cells. Among them, six clones, including NTRK2, GNA15, SFRS2, EIF5A, ELAVL1, and YWHAB mediated MAPK signaling for expressing MDM2. These results indicate that p53-independent upregulation of MDM2 by increasing selected clones may lead to oncogenesis in AML and that MDM2-modulating genes are novel potential targets for AML treatment.

  6. Absolute 24 h quantification of 99Tcm-DMSA uptake in patients with severely reduced kidney function: a comparison with 51Cr-EDTA clearance.

    PubMed

    van de Wiele, C; van den Eeckhaut, A; Verweire, W; van Haelst, J P; Versijpt, J; Dierckx, R A

    1999-09-01

    The aim of this study was to determine whether absolute 24 h DMSA uptake measurements (%DMSA) correlate well with 51Cr-EDTA clearance measurements in patients with severely reduced kidney function (SRKF). Between 1990 and 1997, 55 of 482 patients who underwent EDTA clearance measurements also underwent %DMSA within 1 week. Of these, 31 were women and 24 were men (mean age 60 years; range 19-77 years). EDTA clearance was determined using the slope-intercept method. Absolute depth- and background-corrected %DMSA were determined 24 h following the injection of 185 MBq per 1.73 m2 freshly prepared 99Tcm-DMSA. All patients had EDTA clearance < or = 60 ml.min-1. Eighteen patients (group A: 9 men and 9 women, mean age 55.8 years, range 28-73 years) had EDTA clearance > 20 ml.min-1 (mean +/- S.D. = 30.9 +/- 13.8 ml.min-1), whereas 37 patients (group B: 22 women and 15 men, mean age 62.0 years, range 19-77 years) had EDTA clearance < 20 ml.min-1 (mean +/- S.D. = 10.2 +/- 6.6 ml.min-1). EDTA clearance correlated well with %DMSA for the patients as a whole and for group A (r = 0.87, P = 0.73; r = 0.79, P = 0.0001 respectively). The regression equation suggests that %DMSA is not a marker of early renal dysfunction. In group B, the r-value (r = 0.48, P = 0.004) suggests that %DMSA is reliable as a marker of severe renal dysfunction to the extent that it provides rough information. In conclusion, %DMSA may not be used as a marker of early renal impairment. Additionally, in patients with severely reduced kidney function (EDTA clearance < 20 ml.min-1), it only provides a rough estimate.

  7. MDM2 Associates with Polycomb Repressor Complex 2 and Enhances Stemness-Promoting Chromatin Modifications Independent of p53.

    PubMed

    Wienken, Magdalena; Dickmanns, Antje; Nemajerova, Alice; Kramer, Daniela; Najafova, Zeynab; Weiss, Miriam; Karpiuk, Oleksandra; Kassem, Moustapha; Zhang, Yanping; Lozano, Guillermina; Johnsen, Steven A; Moll, Ute M; Zhang, Xin; Dobbelstein, Matthias

    2016-01-01

    The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53. PMID:26748827

  8. Mdm2, p53, p21 and pAKT protein pathways in benign neoplasms of the salivary gland.

    PubMed

    Marques, Yonara Maria Freire Soares; de Lima, Marina de Deus Moura; de Melo Alves, Sérgio de Melo; Soares, Fernando Augusto; de Araújo, Vera Cavalcanti; Pinto, Décio dos Santos; Mantesso, Andrea

    2008-09-01

    The p53 protein can be altered virtually in all human cancers. In the absence of p53 mutations, p53 inactivation is possible via complex formation with other proteins, such as Mdm2. Previous studies have shown an overexpression of Mdm2 and lack of p53 expression in pleomorphic adenomas. The pAkt protein is closely related to Mdm2, and has not been previously reported in salivary gland tumors. The aim of this study was to analyze the expression of Mdm2, p53, p21 and pAkt proteins in pleomorphic adenomas and myoepitheliomas by immunohistochemistry, Western blotting and immunofluorescence techniques. Overexpression of Mdm2 and pAkt was present in all the cell lines and tumors studied, whereas the expression of p53 and p21 proteins was considered absent. In conclusion, the signaling pathway in benign salivary gland neoplasm showed an important participation of Mdm2 overexpression protein in tumor formation, progression through inactivation of p53 action, or both, and of pAkt overexpression through increased translocation of Mdm2 protein to cellular nuclei.

  9. MDM4 expression as an indicator of TP53 reactivation by combined targeting of MDM2 and MDM4 in cancer cells without TP53 mutation

    PubMed Central

    Hirose, Mitsuaki; Yamato, Kenji; Endo, Shinji; Saito, Rie; Ueno, Takunori; Hirai, Sachiko; Suzuki, Hideo; Abei, Masato; Natori, Yukikazu; Hyodo, Ichinosuke

    2014-01-01

    MDM2 and MDM4, a structurally related MDM2 homolog, negatively regulates expression and functions of TP53 tumor suppressor gene. To explore the precise expression patterns and function of MDM2 and MDM4 in wild-type (wt) TP53 cancer cells, we analyzed 11 various cancer cell lines with wt TP53. All cell lines exhibited deregulated expression of MDM2 and MDM4, and were divided into two distinct types; the one expressing high levels of MDM4 and another expressing low levels of MDM4. The low MDM4 type expressed higher MDM2 levels than the high MDM4 type. In cells with high MDM4 expression, knockdown of MDM4 or MDM2 reactivated TP53, and simultaneous knockdown of MDM2 and MDM4 synergistically reactivated TP53. In contrast, in cells with low MDM4 expression, knockdown of only MDM2 reactivated TP53. These results suggest that both MDM2 and MDM4 are closely involved in TP53 inactivation in cancer cells with high MDM4 expression, whereas only MDM2, and not MDM4, is a regulator of TP53 in cells with low MDM4 expression. MDM4 expression in wt TP53-tumors is a potential indicator for TP53 reactivation cancer therapy by simultaneous targeting of MDM4 and MDM2. Specific knockdown of MDM2 and MDM4 might be applicable for TP53 restoration therapy. PMID:25621298

  10. Mdm2 is required for survival of hematopoietic stem cells/progenitors via dampening of ROS-induced p53 activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mdm2 is an E3 ubiquitin ligase that targets p53 for degradation. p53(515C) (encoding p53R172P) is a hypomorphic allele of p53 that rescues the embryonic lethality of Mdm2(-/-) mice. Mdm2(-/-) p53(515C/515C) mice, however, die by postnatal day 13 resulting from hematopoietic failure. Hematopoietic st...

  11. Rare Aggressive Behavior of MDM2-Amplified Retroperitoneal Dedifferentiated Liposarcoma, with Brain, Lung and Subcutaneous Metastases

    PubMed Central

    Ben Salha, Imen; Zaidi, Shane; Noujaim, Jonathan; Miah, Aisha B.; Fisher, Cyril; Jones, Robin L.; Thway, Khin

    2016-01-01

    Dedifferentiated liposarcoma (DDL) is a histologically pleomorphic sarcoma, traditionally defined as well-differentiated liposarcoma with abrupt transition to high grade, non-lipogenic sarcoma. It can occur as part of recurrent well-differentiated liposarcoma, or may arise de novo. DDL most frequently occurs within the retroperitoneum, and while it is prone to local recurrence, it usually has a lower rate of metastasis than other pleomorphic sarcomas. We describe a case of retroperitoneal dedifferentiated liposarcoma in a 63-year-old male, who showed MDM2 amplification with fluorescence in situ hybridization, which displayed unusually aggressive behavior, with brain, lung and subcutaneous soft tissue metastases. As previous reports of metastatic liposarcoma have largely grouped DDL in with other (genetically and clinically distinct) liposarcoma subtypes, we highlight and discuss the rare occurrence of brain metastasis in MDM2-amplified retroperitoneal liposarcoma. PMID:27746879

  12. The inhibition of calcium carbonate crystal growth by the cysteine-rich Mdm2 peptide.

    PubMed

    Dalas, E; Chalias, A; Gatos, D; Barlos, K

    2006-08-15

    The crystal growth of calcite, the most stable calcium carbonate polymorph, in the presence of the cysteine-rich Mdm2 peptide (containing 48 amino acids in the ring finger configuration), has been investigated by the constant composition technique. Crystallization took place exclusively on well-characterized calcite crystals in solutions supersaturated only with respect to this calcium carbonate salt. The kinetic results indicated a surface diffusion spiral growth mechanism. The presence of the Mdm2 peptide inhibited the crystal growth of calcite by 22-58% in the concentration range tested, through adsorption onto the active growth sites of the calcite crystal surface. The kinetic results favored a Langmuir-type adsorption model, and the value of the calculated affinity constant was k(aff)=147x10(4) dm(3)mol(-1), a(ads)=0.29. PMID:16678843

  13. Assessing the Efficacy of Mdm2/Mdm4-Inhibiting Stapled Peptides Using Cellular Thermal Shift Assays

    PubMed Central

    Xiong Tan, Ban; Brown, Christopher J.; Ferrer, Fernando J.; Yuen, Tsz Ying; Quah, Soo Tng; Chan, Boon Hong; Jansson, Anna E.; Teo, Hsiang Ling; Nordlund, Pär; Lane, David P.

    2015-01-01

    Previous publications on stapled peptide inhibitors against Mdm2/Mdm4-p53 interactions have established that this new class of drugs have the potential to be easily optimised to attain high binding affinity and specificity, but the mechanisms controlling their cellular uptake and target engagement remain elusive and controversial. To aid in understanding the rules of peptide and staple design, and to enable rapid optimisation, we employed the newly-developed cellular thermal shift assay (CETSA). CETSA was able to validate stapled peptide binding to Mdm2 and Mdm4, and the method was also used to determine the extent of cellular uptake, cellular availability, and intracellular binding of the endogenous target proteins in its native environment. Our data suggest that while the stapled peptides engage their targets intracellularly, more work is needed to improve their cellular entry and target engagement efficiency in vivo. CETSA now provides a valuable tool to optimize such in vivo properties of stapled peptides. PMID:26159518

  14. The Ribosomal Protein-Mdm2-p53 Pathway and Energy Metabolism

    PubMed Central

    Deisenroth, Chad; Zhang, Yanping

    2011-01-01

    Cellular growth and division are two fundamental processes that are exquisitely sensitive and responsive to environmental fluctuations. One of the most energetically demanding functions of these processes is ribosome biogenesis, the key component to regulating overall protein synthesis and cell growth. Perturbations to ribosome biogenesis have been demonstrated to induce an acute stress response leading to p53 activation through the inhibition of Mdm2 by a number of ribosomal proteins. The energy status of a cell is a highly dynamic variable that naturally contributes to metabolic fluctuations, which can affect both the rates of ribosome biogenesis and p53 function. This, in turn, determines whether a cell is in an anabolic, growth-promoting state or a catabolic, growth-suppressing state. Here the authors integrate the known functions of p53 to postulate how changes in nutrient availability may induce the ribosomal protein–Mdm2-p53 signaling pathway to modulate p53-dependent metabolic regulation. PMID:21779508

  15. Population distribution and ancestry of the cancer protective MDM2 SNP285 (rs117039649).

    PubMed

    Knappskog, Stian; Gansmo, Liv B; Dibirova, Khadizha; Metspalu, Andres; Cybulski, Cezary; Peterlongo, Paolo; Aaltonen, Lauri; Vatten, Lars; Romundstad, Pål; Hveem, Kristian; Devilee, Peter; Evans, Gareth D; Lin, Dongxin; Van Camp, Guy; Manolopoulos, Vangelis G; Osorio, Ana; Milani, Lili; Ozcelik, Tayfun; Zalloua, Pierre; Mouzaya, Francis; Bliznetz, Elena; Balanovska, Elena; Pocheshkova, Elvira; Kučinskas, Vaidutis; Atramentova, Lubov; Nymadawa, Pagbajabyn; Titov, Konstantin; Lavryashina, Maria; Yusupov, Yuldash; Bogdanova, Natalia; Koshel, Sergey; Zamora, Jorge; Wedge, David C; Charlesworth, Deborah; Dörk, Thilo; Balanovsky, Oleg; Lønning, Per E

    2014-09-30

    The MDM2 promoter SNP285C is located on the SNP309G allele. While SNP309G enhances Sp1 transcription factor binding and MDM2 transcription, SNP285C antagonizes Sp1 binding and reduces the risk of breast-, ovary- and endometrial cancer. Assessing SNP285 and 309 genotypes across 25 different ethnic populations (>10.000 individuals), the incidence of SNP285C was 6-8% across European populations except for Finns (1.2%) and Saami (0.3%). The incidence decreased towards the Middle-East and Eastern Russia, and SNP285C was absent among Han Chinese, Mongolians and African Americans. Interhaplotype variation analyses estimated SNP285C to have originated about 14,700 years ago (95% CI: 8,300 - 33,300). Both this estimate and the geographical distribution suggest SNP285C to have arisen after the separation between Caucasians and modern day East Asians (17,000 - 40,000 years ago). We observed a strong inverse correlation (r = -0.805; p < 0.001) between the percentage of SNP309G alleles harboring SNP285C and the MAF for SNP309G itself across different populations suggesting selection and environmental adaptation with respect to MDM2 expression in recent human evolution. In conclusion, we found SNP285C to be a pan-Caucasian variant. Ethnic variation regarding distribution of SNP285C needs to be taken into account when assessing the impact of MDM2 SNPs on cancer risk. PMID:25327560

  16. High frequency of p53/MDM2/p14ARF pathway abnormalities in relapsed neuroblastoma

    PubMed Central

    Carr-Wilkinson, Jane; O' Toole, Kieran; Wood, Katrina M.; Challen, Christine C.; Baker, Angela G.; Board, Julian R.; Evans, Laura; Cole, Michael; Cheung, Nai-Kong V.; Boos, Joachim; Köhler, Gabriele; Leuschner, Ivo; Pearson, Andrew D.J.; Lunec, John; Tweddle, Deborah A.

    2010-01-01

    Purpose: Most neuroblastomas initially respond to therapy but many relapse with chemoresistant disease. p53 mutations are rare in diagnostic neuroblastomas, but we have previously reported inactivation of the p53/MDM2/p14ARF pathway in 9/17 (53%) neuroblastoma cell lines established at relapse. Hypothesis: Inactivation of the p53/MDM2/p14ARF pathway develops during treatment and contributes to neuroblastoma relapse. Methods: Eighty-four neuroblastomas were studied from 41 patients with relapsed neuroblastoma including 38 paired neuroblastomas at different stages of therapy. p53 mutations were detected by automated sequencing, p14ARF methylation and deletion by methylation-specific PCR and duplex PCR respectively, and MDM2 amplification by fluorescent in-situ hybridisation. Results: Abnormalities in the p53 pathway were identified in 20/41(49%) cases. Downstream defects due to inactivating missense p53 mutations were identified in 6/41 (15%) cases, 5 following chemotherapy and/or at relapse and 1 at diagnosis, post chemotherapy and relapse. The presence of a p53 mutation was independently prognostic for overall survival (hazard ratio 3.4, 95% confidence interval 1.2, 9.9; p = 0.02). Upstream defects were present in 35% cases: MDM2 amplification in 3 cases, all at diagnosis & relapse and p14ARF inactivation in 12/41 (29%) cases: 3 had p14ARF methylation, 2 after chemotherapy, and 9 had homozygous deletions, 8 at diagnosis and relapse. Conclusions: These results show that a high proportion of neuroblastomas which relapse have an abnormality in the p53 pathway. The majority have upstream defects suggesting that agents which reactivate wild-type p53 would be beneficial, in contrast to those with downstream defects where p53 independent therapies are indicated. PMID:20145180

  17. Population distribution and ancestry of the cancer protective MDM2 SNP285 (rs117039649)

    PubMed Central

    Knappskog, Stian; Gansmo, Liv B.; Dibirova, Khadizha; Metspalu, Andres; Cybulski, Cezary; Peterlongo, Paolo; Aaltonen, Lauri; Vatten, Lars; Romundstad, Pål; Hveem, Kristian; Devilee, Peter; Evans, Gareth D.; Lin, Dongxin; Camp, Guy Van; Manolopoulos, Vangelis G.; Osorio, Ana; Milani, Lili; Ozcelik, Tayfun; Zalloua, Pierre; Mouzaya, Francis; Bliznetz, Elena; Balanovska, Elena; Pocheshkova, Elvira; Kučinskas, Vaidutis; Atramentova, Lubov; Nymadawa, Pagbajabyn; Titov, Konstantin; Lavryashina, Maria; Yusupov, Yuldash; Bogdanova, Natalia; Koshel, Sergey; Zamora, Jorge; Wedge, David C.; Charlesworth, Deborah; Dörk, Thilo; Balanovsky, Oleg; Lønning, Per E.

    2014-01-01

    The MDM2 promoter SNP285C is located on the SNP309G allele. While SNP309G enhances Sp1 transcription factor binding and MDM2 transcription, SNP285C antagonizes Sp1 binding and reduces the risk of breast-, ovary- and endometrial cancer. Assessing SNP285 and 309 genotypes across 25 different ethnic populations (>10.000 individuals), the incidence of SNP285C was 6-8% across European populations except for Finns (1.2%) and Saami (0.3%). The incidence decreased towards the Middle-East and Eastern Russia, and SNP285C was absent among Han Chinese, Mongolians and African Americans. Interhaplotype variation analyses estimated SNP285C to have originated about 14,700 years ago (95% CI: 8,300 – 33,300). Both this estimate and the geographical distribution suggest SNP285C to have arisen after the separation between Caucasians and modern day East Asians (17,000 - 40,000 years ago). We observed a strong inverse correlation (r = -0.805; p < 0.001) between the percentage of SNP309G alleles harboring SNP285C and the MAF for SNP309G itself across different populations suggesting selection and environmental adaptation with respect to MDM2 expression in recent human evolution. In conclusion, we found SNP285C to be a pan-Caucasian variant. Ethnic variation regarding distribution of SNP285C needs to be taken into account when assessing the impact of MDM2 SNPs on cancer risk. PMID:25327560

  18. ARF Impedes NPM/B23 Shuttling in an Mdm2-Sensitive Tumor Suppressor Pathway

    PubMed Central

    Brady, Suzanne N.; Yu, Yue; Maggi, Leonard B.; Weber, Jason D.

    2004-01-01

    The ARF tumor suppressor is widely regarded as an upstream activator of p53-dependent growth arrest and apoptosis. However, recent findings indicate that ARF can also regulate the cell cycle in the absence of p53. In search of p53-independent ARF targets, we isolated nucleophosmin (NPM/B23), a protein we show is required for proliferation, as a novel ARF binding protein. In response to hyperproliferative signals, ARF is upregulated, resulting in the nucleolar retention of NPM and concomitant cell cycle arrest. The Mdm2 oncogene outcompetes NPM/B23 for ARF binding, and introduction of Mdm2 reverses ARF's p53-independent properties: in vitro, NPM is released from ARF-containing protein complexes, and in vivo S phase progression ensues. ARF induction by oncogenes or replicative senescence does not alter NPM/B23 protein levels but rather prevents its nucleocytoplasmic shuttling without inhibiting rRNA processing. By actively sequestering NPM in the nucleolus, ARF utilizes an additional mechanism of tumor suppression, one that is readily antagonized by Mdm2. PMID:15485902

  19. Bcl-2/MDM2 Dual Inhibitors Based on Universal Pyramid-Like α-Helical Mimetics.

    PubMed

    Wang, Ziqian; Song, Ting; Feng, Yingang; Guo, Zongwei; Fan, Yudan; Xu, Wenjie; Liu, Lu; Wang, Anhui; Zhang, Zhichao

    2016-04-14

    No α-helical mimetic that exhibits Bcl-2/MDM2 dual inhibition has been rationally designed due to the different helicities of the α-helixes at their binding interfaces. Herein, we extracted a one-turn α-helix-mimicking ortho-triarene unit from o-phenylene foldamers. Linking benzamide substrates with a rotatable C-N bond, we constructed a novel semirigid pyramid-like scaffold that could support its two-turn α-helix mimicry without aromatic stacking interactions and could adopt the different dihedral angles of the key residues of p53 and BH3-only peptides. On the basis of this universal scaffold, a series of substituent groups were installed to capture the key residues of both p53TAD and BimBH3 and balance the differences of the bulks between them. Identified by FP, ITC, and NMR spectroscopy, a compound 6e (zq-1) that directly binds to Mcl-1, Bcl-2, and MDM2 with balanced submicromolar affinities was obtained. Cell-based experiments demonstrated its antitumor ability through Bcl-2/MDM2 dual inhibition simultaneously.

  20. Zinc Oxide Nanoparticles Demoted MDM2 Expression to Suppress TSLP-Induced Mast Cell Proliferation.

    PubMed

    Kim, Min-Ho; Jeong, Hyun-Ja

    2016-03-01

    Activation of murine double minute 2 (MDM2) through thymic stromal lymphopoietin (TSLP)-induced signal transducers and activators of transcription (STAT6) phosphorylation plays a critical role in proliferation and survival of mast cells. Previously, we reported that zinc oxide nanoparticles (ZnO-NP) effectively decrease the mast cell-mediated allergic inflammatory reactions. Here, we evaluated the effect of ZnO-NP on TSLP-induced proliferation of mast cells. ZnO-NP significantly reduced the number of BrdU-incorporating mast cells increased by TSLP. ZnO-NP decreased the expression of MDM2 through the blockade of STAT6 phosphorylation. TSLP increased the production and mRNA expression of interleukin-13 (a growth factor of mast cells), its increase was significantly decreased by ZnO-NP (10 μg/mL). ZnO-NP induced the down-regulation of Bcl2 (an anti-apoptotic factor) and up-regulation of Bax (an apoptotic factor) through the stabilization of p53 protein. However, ZnO-NP has no effect on caspase-3 activation, cytochrome c release into cytosol, and apoptosis-inducing factor translocation into nucleus in TSLP-stimulated cells. The results of the present study demonstrated that ZnO-NP inhibited the proliferation of mast cells through the regulation of MDM2 and p53 protein levels. These finding suggest that ZnO-NP could be improved mast cell-mediated various diseases.

  1. KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction

    SciTech Connect

    Okamoto, Koji . E-mail: kojokamo@gan2.res.ncc.go.jp; Kitabayashi, Issay; Taya, Yoichi . E-mail: ytaya@gan2.res.ncc.go.jp

    2006-12-08

    KAP1 recruits many proteins involved in gene silencing and functions as an integral part of co-repressor complex. KAP1 was identified as Mdm2-binding protein and shown to form a complex with Mdm2 and p53 in vivo. We examined the role of KAP1 in p53 activation after the treatment of cells with different types of external stresses. KAP1 reduction markedly enhanced the induction of p21, a product of the p53 target gene, after treatment with actinomycin D or {gamma}-irradiation, but not with camptothecin. Treatment with actinomycin D, but not with camptothecin, augmented the interaction of p53 with Mdm2 and KAP1. Further, KAP1 reduction in actinomycin D-treated cells facilitated cell cycle arrest and negatively affected clonal cell growth. Thus, the reduction of KAP1 levels promotes p53-dependent p21 induction and inhibits cell proliferation in actinomycin D-treated cells. KAP1 may serve as a therapeutic target against cancer in combination with actinomycin D.

  2. 8-Triazolylpurines: Towards Fluorescent Inhibitors of the MDM2/p53 Interaction.

    PubMed

    Pettersson, Mariell; Bliman, David; Jacobsson, Jimmy; Nilsson, Jesper R; Min, Jaeki; Iconaru, Luigi; Guy, R Kiplin; Kriwacki, Richard W; Andréasson, Joakim; Grøtli, Morten

    2015-01-01

    Small molecule nonpeptidic mimics of α-helices are widely recognised as protein-protein interaction (PPIs) inhibitors. Protein-protein interactions mediate virtually all important regulatory pathways in a cell, and the ability to control and modulate PPIs is therefore of great significance to basic biology, where controlled disruption of protein networks is key to understanding network connectivity and function. We have designed and synthesised two series of 2,6,9-substituted 8-triazolylpurines as α-helix mimetics. The first series was designed based on low energy conformations but did not display any biological activity in a biochemical fluorescence polarisation assay targeting MDM2/p53. Although solution NMR conformation studies demonstrated that such molecules could mimic the topography of an α-helix, docking studies indicated that the same compounds were not optimal as inhibitors for the MDM2/p53 interaction. A new series of 8-triazolylpurines was designed based on a combination of docking studies and analysis of recently published inhibitors. The best compound displayed low micromolar inhibitory activity towards MDM2/p53 in a biochemical fluorescence polarisation assay. In order to evaluate the applicability of these compounds as biologically active and intrinsically fluorescent probes, their absorption/emission properties were measured. The compounds display fluorescent properties with quantum yields up to 50%.

  3. Convergent solid-phase and solution approaches in the synthesis of the cysteine-rich Mdm2 RING finger domain.

    PubMed

    Vasileiou, Zoe; Barlos, Kostas; Gatos, Dimitrios

    2009-12-01

    The RING finger domain of the Mdm2, located at the C-terminus of the protein, is necessary for regulation of p53, a tumor suppressor protein. The 48-residues long Mdm2 peptide is an important target for studying its interaction with small anticancer drug candidates. For the chemical synthesis of the Mdm2 RING finger domain, the fragment condensation on solid-phase and the fragment condensation in solution were studied. The latter method was performed using either protected or free peptides at the C-terminus as the amino component. Best results were achieved using solution condensation where the N-component was applied with the C-terminal carboxyl group left unprotected. The developed method is well suited for large-scale synthesis of Mdm2 RING finger domain, combining the advantages of both solid-phase and solution synthesis. PMID:19824037

  4. MDM2–MDM4 molecular interaction investigated by atomic force spectroscopy and surface plasmon resonance

    PubMed Central

    Moscetti, Ilaria; Teveroni, Emanuela; Moretti, Fabiola; Bizzarri, Anna Rita; Cannistraro, Salvatore

    2016-01-01

    Murine double minute 2 (MDM2) and 4 (MDM4) are known as the main negative regulators of p53, a tumor suppressor. They are able to form heterodimers that are much more effective in the downregulation of p53. Therefore, the MDM2–MDM4 complex could be a target for promising therapeutic restoration of p53 function. To this aim, a deeper understanding of the molecular mechanisms underlining the heterodimerization is needed. The kinetic and thermodynamic characterization of the MDM2–MDM4 complex was performed with two complementary approaches: atomic force spectroscopy and surface plasmon resonance. Both techniques revealed an equilibrium dissociation constant (KD) in the micromolar range for the MDM2–MDM4 heterodimer, similar to related complexes involved in the p53 network. Furthermore, the MDM2–MDM4 complex is characterized by a relatively high free energy, through a single energy barrier, and by a lifetime in the order of tens of seconds. New insights into the MDM2–MDM4 interaction could be highly important for developing innovative anticancer drugs focused on p53 reactivation. PMID:27621617

  5. Opposite regulation of MDM2 and MDMX expression in acquisition of mesenchymal phenotype in benign and cancer cells

    PubMed Central

    Slabáková, Eva; Kharaishvili, Gvantsa; Smějová, Monika; Pernicová, Zuzana; Suchánková, Tereza; Remšík, Ján; Lerch, Stanislav; Straková, Nicol; Bouchal, Jan; Král, Milan; Culig, Zoran; Kozubík, Alois; Souček, Karel

    2015-01-01

    Plasticity of cancer cells, manifested by transitions between epithelial and mesenchymal phenotypes, represents a challenging issue in the treatment of neoplasias. Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are implicated in the processes of metastasis formation and acquisition of stem cell-like properties. Mouse double minute (MDM) 2 and MDMX are important players in cancer progression, as they act as regulators of p53, but their function in EMT and metastasis may be contradictory. Here, we show that the EMT phenotype in multiple cellular models and in clinical prostate and breast cancer samples is associated with a decrease in MDM2 and increase in MDMX expression. Modulation of EMT-accompanying changes in MDM2 expression in benign and transformed prostate epithelial cells influences their migration capacity and sensitivity to docetaxel. Analysis of putative mechanisms of MDM2 expression control demonstrates that in the context of defective p53 function, MDM2 expression is regulated by EMT-inducing transcription factors Slug and Twist. These results provide an alternative context-specific role of MDM2 in EMT, cell migration, metastasis, and therapy resistance. PMID:26416355

  6. MDM2–MDM4 molecular interaction investigated by atomic force spectroscopy and surface plasmon resonance

    PubMed Central

    Moscetti, Ilaria; Teveroni, Emanuela; Moretti, Fabiola; Bizzarri, Anna Rita; Cannistraro, Salvatore

    2016-01-01

    Murine double minute 2 (MDM2) and 4 (MDM4) are known as the main negative regulators of p53, a tumor suppressor. They are able to form heterodimers that are much more effective in the downregulation of p53. Therefore, the MDM2–MDM4 complex could be a target for promising therapeutic restoration of p53 function. To this aim, a deeper understanding of the molecular mechanisms underlining the heterodimerization is needed. The kinetic and thermodynamic characterization of the MDM2–MDM4 complex was performed with two complementary approaches: atomic force spectroscopy and surface plasmon resonance. Both techniques revealed an equilibrium dissociation constant (KD) in the micromolar range for the MDM2–MDM4 heterodimer, similar to related complexes involved in the p53 network. Furthermore, the MDM2–MDM4 complex is characterized by a relatively high free energy, through a single energy barrier, and by a lifetime in the order of tens of seconds. New insights into the MDM2–MDM4 interaction could be highly important for developing innovative anticancer drugs focused on p53 reactivation.

  7. Genetic polymorphisms of MDM2 and TP53 genes are associated with risk of nasopharyngeal carcinoma in a Chinese population

    PubMed Central

    2010-01-01

    Background The tumor suppressor TP53 and its negative regulator MDM2 play crucial roles in carcinogenesis. Previous case-control studies also revealed TP53 72Arg>Pro and MDM2 309T>G polymorphisms contribute to the risk of common cancers. However, the relationship between these two functional polymorphisms and nasopharyngeal carcinoma (NPC) susceptibility has not been explored. Methods In this study, we performed a case-control study between 522 NPC patients and 722 healthy controls in a Chinese population by using PCR-RFLP. Results We found an increased NPC risk associated with the MDM2 GG (odds ratio [OR] = 2.83, 95% confidence interval [CI] = 2.08-3.96) and TG (OR = 1.49, 95% CI = 1.16-2.06) genotypes. An increased risk was also associated with the TP53 Pro/Pro genotype (OR = 2.22, 95% CI = 1.58-3.10) compared to the Arg/Arg genotype. The gene-gene interaction of MDM2 and TP53 polymorphisms increased adult NPC risk in a more than multiplicative manner (OR for the presence of both MDM2 GG and TP53 Pro/Pro genotypes = 7.75, 95% CI = 3.53-17.58). Conclusion The findings suggest that polymorphisms of MDM2 and TP53 genes may be genetic modifier for developing NPC. PMID:20398418

  8. HAUSP-nucleolin interaction is regulated by p53-Mdm2 complex in response to DNA damage response

    PubMed Central

    Lim, Key-Hwan; Park, Jang-Joon; Gu, Bon-Hee; Kim, Jin-Ock; Park, Sang Gyu; Baek, Kwang-Hyun

    2015-01-01

    HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N- and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSP-p53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage. PMID:26238070

  9. HAUSP-nucleolin interaction is regulated by p53-Mdm2 complex in response to DNA damage response.

    PubMed

    Lim, Key-Hwan; Park, Jang-Joon; Gu, Bon-Hee; Kim, Jin-Ock; Park, Sang Gyu; Baek, Kwang-Hyun

    2015-08-04

    HAUSP (herpes virus-associated ubiquitin specific protease, known as ubiquitin specific protease 7), one of DUBs, regulates the dynamics of the p53 and Mdm2 network in response to DNA damage by deubiquitinating both p53 and its E3 ubiquitin ligase, Mdm2. Its concerted action increases the level of functional p53 by preventing proteasome-dependent degradation of p53. However, the protein substrates that are targeted by HAUSP to mediate DNA damage responses in the context of the HAUSP-p53-Mdm2 complex are not fully identified. Here, we identified nucleolin as a new substrate for HAUSP by proteomic analysis. Nucleolin has two HAUSP binding sites in its N- and C-terminal regions, and the mutation of HAUSP interacting peptides on nucleolin disrupts their interaction and it leads to the increased level of nucleolin ubiquitination. In addition, HAUSP regulates the stability of nucleolin by removing ubiquitin from nucleolin. Nucleolin exists as a component of the HAUSP-p53-Mdm2 complex, and both Mdm2 and p53 are required for the interaction between HAUSP and nucleolin. Importantly, the irradiation increases the HAUSP-nucleolin interaction, leading to nucleolin stabilization significantly. Taken together, this study reveals a new component of the HAUSP-p53-Mdm2 complex that governs dynamic cellular responses to DNA damage.

  10. Mdm2 ligase dead mutants did not act in a dominant negative manner to re-activate p53, but promoted tumor cell growth.

    PubMed

    Swaroop, Manju; Sun, Yi

    2003-01-01

    Mdm2 (murine double minute 2) is an oncogene, first identified in BALB/c 3T3 cells. Over-expression and gene amplification of Mdm2 were found in a variety of human cancers. Recently, Mdm2 was found to be an E3 ubiquitin ligase that promotes degradation of p53, which contributes significantly to its oncogenic activity. In this study, we test a hypothesis that Mdm2 ligase dead mutants, which retained p53 binding activity but lost degradation activity, would act in a dominant negative manner to re-activate p53, especially upon stressed conditions. Five Mdm2 constructs expressing wild-type and E3 ligase-dead Mdm2 proteins were generated in a Tet-Off system and transfected into MCF-7 breast cancer cells (p53+/+ with Mdm2 overexpression) as well as MCF10A immortalized breast cells (p53+/+ without Mdm2 overexpression) as a normal control. We found that expression of Mdm2 mutants were tightly regulated by doxycycline. Withdrawal of doxycycline in culture medium triggered overexpression of Mdm2 mutants. However, expression of ligase dead mutants in MCF7 and MCF10A cells did not reactivate p53 as shown by a luciferase-reporter transcription assay and Western blot of p53 and its downstream target p21 under either unstressed condition or after exposure to DNA damaging agents. Biologically, over-expression of Mdm2 mutants had no effect on p53-induced apoptosis following DNA damage. Interestingly, over-expression of Mdm2 mutants promoted growth of MCF7 tumor cells probably via a p53-independent mechanism. Over-expression of Mdm2 mutants, however, had no effect on the growth of normal MCF10A cells and did not cause their transformation. Thus, ligase dead mutants of Mdm2 did not act in a dominant negative manner to reactivate p53 and they are not oncogenes in MCF10A cells.

  11. Is MDM2 SNP309 Variation a Risk Factor for Head and Neck Carcinoma?

    PubMed Central

    Zhuo, Xianlu; Ye, Huiping; Li, Qi; Xiang, Zhaolan; Zhang, Xueyuan

    2016-01-01

    Abstract Murine double minute-2 (MDM2) is a negative regulator of P53, and its T309G polymorphism has been suggested as a risk factor for a variety of cancers. Increasing evidence has shown the association of MDM2 T309G polymorphism with head and neck carcinoma (HNC) risk. However, the results are inconsistent. Thus, we performed a meta-analysis to elucidate the association. The meta-analysis retrieved studies published up to August 2015, and essential information was extracted for analysis. Separate analyses on ethnicity, source of controls, sample size, detection method, and cancer types were also conducted. Odds ratios (ORs) and their 95% confidence intervals (CIs) were used to estimate the association. Pooled data from 16 case–control studies including 4625 cases and 6927 controls failed to indicate a significant association. However, in the subgroup analysis of sample sizes, an increased risk was observed in the largest sample size group (>1000) under a recessive model (OR = 1.52; 95% CI = 1.08–2.13). Increased risks were also found in the nasopharyngeal cancer in the subgroup analysis of cancer types (GG vs TT: OR = 2.07; 95% CI = 1.38–3.12; dominant model: OR = 1.48; 95% CI = 1.13–1.93; recessive model: OR = 1.76; 95% CI = 1.17–2.65). The results suggest that homozygote GG alleles of MDM2 SNP309 may be a low-penetrant risk factor for HNC, and G allele may confer nasopharyngeal cancer susceptibility. PMID:26945408

  12. Aberrant splicing of the DMP1-ARF-MDM2-p53 pathway in cancer.

    PubMed

    Inoue, Kazushi; Fry, Elizabeth A

    2016-07-01

    Alternative splicing (AS) of mRNA precursors is a ubiquitous mechanism for generating numerous transcripts with different activities from one genomic locus in mammalian cells. The gene products from a single locus can thus have similar, dominant-negative or even opposing functions. Aberrant AS has been found in cancer to express proteins that promote cell growth, local invasion and metastasis. This review will focus on the aberrant splicing of tumor suppressor/oncogenes that belong to the DMP1-ARF-MDM2-p53 pathway. Our recent study shows that the DMP1 locus generates both tumor-suppressive DMP1α (p53-dependent) and oncogenic DMP1β (p53-independent) splice variants, and the DMP1β/α ratio increases with neoplastic transformation of breast epithelial cells. This process is associated with high DMP1β protein expression and shorter survival of breast cancer (BC) patients. Accumulating pieces of evidence show that ARF is frequently inactivated by aberrant splicing in human cancers, demonstrating its involvement in human malignancies. Splice variants from the MDM2 locus promote cell growth in culture and accelerate tumorigenesis in vivo. Human cancers expressing these splice variants are associated with advanced stage/metastasis, and thus have negative clinical impacts. Although they lack most of the p53-binding domain, their activities are mostly dependent on p53 since they bind to wild-type MDM2. The p53 locus produces splice isoforms that have either favorable (β/γ at the C-terminus) or negative impact (Δ40, Δ133 at the N-terminus) on patients' survival. As the oncogenic AS products from these loci are expressed only in cancer cells, they may eventually become targets for molecular therapies.

  13. Homozygous mdm2 SNP309 cancer cells with compromised transcriptional elongation at p53 target genes are sensitive to induction of p53-independent cell death.

    PubMed

    Rosso, Melissa; Polotskaia, Alla; Bargonetti, Jill

    2015-10-27

    A single nucleotide polymorphism (T to G) in the mdm2 P2 promoter, mdm2 SNP309, leads to MDM2 overexpression promoting chemotherapy resistant cancers. Two mdm2 G/G SNP309 cancer cell lines, MANCA and A875, have compromised wild-type p53 that co-localizes with MDM2 on chromatin. We hypothesized that MDM2 in these cells inhibited transcription initiation at the p53 target genes p21 and puma. Surprisingly, following etoposide treatment transcription initiation occurred at the compromised target genes in MANCA and A875 cells similar to the T/T ML-1 cell line. In all cell lines tested there was equally robust recruitment of total and initiated RNA polymerase II (Pol II). We found that knockdown of MDM2 in G/G cells moderately increased expression of subsets of p53 target genes without increasing p53 stability. Importantly, etoposide and actinomycin D treatments increased histone H3K36 trimethylation in T/T, but not G/G cells, suggesting a G/G correlated inhibition of transcription elongation. We therefore tested a chemotherapeutic agent (8-amino-adenosine) that induces p53-independent cell death for higher clinically relevant cytotoxicity. We demonstrated that T/T and G/G mdm2 SNP309 cells were equally sensitive to 8-amino-adenosine induced cell death. In conclusion for cancer cells overexpressing MDM2, targeting MDM2 may be less effective than inducing p53-independent cell death.

  14. Antisense anti-MDM2 oligonucleotides as a novel therapeutic approach to human breast cancer: in vitro and in vivo activities and mechanisms.

    PubMed

    Wang, H; Nan, L; Yu, D; Agrawal, S; Zhang, R

    2001-11-01

    The mouse double minute 2 (MDM2) oncogene has been suggested as a target for cancer therapy. It is amplified or overexpressed in many human cancers, including breast cancer, and MDM2 levels are associated with poor prognosis of several human cancers, including breast cancer, ovarian cancer, osteosarcoma, and lymphoma. In the present study, we investigated the functions of MDM2 oncogene in the growth of breast cancer and the potential value of MDM2 as a drug target for cancer therapy by inhibiting MDM2 expression with a specific antisense antihuman-MDM2 oligonucleotide (oligo). The selected antisense mixed-backbone oligo was evaluated for its in vitro and in vivo antitumor activity in human breast cancer models: MCF-7 cell line containing wild-type p53 and MDA-MB-468 cell line containing mutant p53. In MCF-7 cells, p53 and p21 levels were elevated, resulting from specific inhibition of MDM2 expression by the antisense oligo (AS). In MDA-MB-468 cells, after inhibition of MDM2 expression, p21 levels were elevated, although p53 levels remained unchanged. After i.p. administration of the antisense anti-MDM2 oligo, in vivo antitumor activity occurred in a dose-dependent manner in nude mice bearing MCF-7 or MDA-MB-468 xenografts. In both models, in vivo synergistically or additive therapeutic effects of MDM2 inhibition and the clinically used cancer chemotherapeutic agents irinotecan, 5-fluorouracil, and paclitaxel (Taxol) were observed. These results suggest that MDM2 have a role in tumor growth through both p53-dependent and p53-independent mechanisms. We speculate that MDM2 inhibitors, such as ASs, have a broad spectrum of antitumor activities in human breast cancers, regardless of p53 status. This study should provide a basis for future development of anti-MDM2 ASs as cancer therapeutic agents used alone or in combination with conventional chemotherapeutics.

  15. ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage.

    PubMed

    Maya, R; Balass, M; Kim, S T; Shkedy, D; Leal, J F; Shifman, O; Moas, M; Buschmann, T; Ronai, Z; Shiloh, Y; Kastan, M B; Katzir, E; Oren, M

    2001-05-01

    The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258-260 and 393-395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.

  16. Regulation of the Abundance of Kaposi’s Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2

    PubMed Central

    Chang, Tzu-Hsuan; Chen, Lee-Wen; Shih, Ying-Ju; Chang, Li-Kwan; Liu, Shih-Tung; Chang, Pey-Jium

    2016-01-01

    The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50. PMID:27698494

  17. A systematic review and meta-analysis of MDM2 polymorphisms in osteosarcoma susceptibility.

    PubMed

    Bilbao-Aldaiturriaga, Nerea; Askaiturrieta, Ziortza; Granado-Tajada, Itsasne; Goričar, Katja; Dolžan, Vita; For The Slovenian Osteosarcoma Study Group; Garcia-Miguel, Purificación; Garcia de Andoin, Nagore; Martin-Guerrero, Idoia; Garcia-Orad, Africa

    2016-10-01

    Two polymorphisms in the murine double minute 2 (MDM2) gene (rs1690916 and rs2279744) have been associated with the risk of osteosarcoma (OS). When we analyzed these two polymorphisms in two new independents cohorts (Spanish and Slovenian), we found no association. In order to clarify this, we conducted a meta-analysis including six populations, with a total of 246 OS patients and 1,760 controls for rs1690916; and 433 OS patients and 1,959 controls for rs2279744. Pooled odds ratio risks and corresponding 95% CI were estimated to assess the possible associations. Our results showed that these two polymorphisms were not associated with the susceptibility of OS under any genetic model studied. In conclusion, the present meta-analysis indicates that MDM2 rs1690916 and rs2279744 cannot be considered as genetic risk factors for OS susceptibility in the different populations. Therefore, the influence of these two polymorphisms on the risk of OS may be less important than previously suggested. Future studies are needed to confirm these results. PMID:27438225

  18. MUC1 oncoprotein suppresses activation of the ARF-MDM2-p53 pathway

    PubMed Central

    Raina, Deepak; Ahmad, Rehan; Chen, Dongshu; Kumar, Shailendra; Kharbanda, Surender; Kufe, Donald

    2011-01-01

    The MUC1 oncoprotein interacts with the c-Abl tyrosine kinase and blocks nuclear targeting of c-Abl in the apoptotic response to DNA damage. Mutation of the MUC1 cytoplasmic domain at Tyr-60 disrupts the MUC1-c-Abl interaction. The present results demonstrate that the MUC1(Y60F) mutant is a potent inducer of the ARF tumor suppressor. MUC1(Y60F) induces transcription of the ARF locus by a c-Abl-dependent mechanism that promotes CUL-4A-mediated nuclear export of the replication protein Cdc6. The functional significance of these findings is that MUC1(Y60F)-induced ARF expression and thereby inhibition of MDM2 results in the upregulation of p53 and the homeodomain interacting protein kinase 2 (HIPK2) serine/threonine kinase. HIPK2-mediated phosphorylation of p53 on Ser-46 was further associated with a shift from expression of the cell cycle arrest-related p21 gene to the apoptosis-related PUMA gene. We also show that the MUC1(Y60F) mutant functions as dominant negative inhibitor of tumorigenicity. These findings indicate that the oncogenic function of MUC1 is conferred by suppressing activation of the ARF-MDM2-p53 pathway. PMID:18981727

  19. PTCH-1 and MDM2 expression in ameloblastoma from a West African sub-population: implication for chemotherapeutics

    PubMed Central

    Udeabor, Samuel Ebele; Adisa, Akinyele Olumuyiwa; Lawal, Ahmed Oluwatoyin; Barbeck, Mike; Booms, Patrick; Sader, Robert Alexander; Ghanaati, Shahram

    2015-01-01

    Introduction Ameloblastoma is a slow growing, painless odontogenic swelling which can attain sizes that result in severe deformities of the craniofacial complex. It is the most commonly encountered odontogenic tumor in Nigeria. Surgical intervention is currently the method of treatment; however identification of altered molecular pathways may inform chemotherapeutic potential. The Protein Patched homolog 1 (PTCH-1) is overexpressed in ameloblastoma. Also, mutation in the MDM2 gene can reduce the tumor suppressor function of p53 and promote ameloblastoma growth. No study however has characterized the molecular profile of African cases of ameloblastoma with a view to developing chemotherapeutic alternatives. The objective was to characterize the PTCH-1 genetic profile of Ameloblastoma in Nigerian patients as a first step in investigating its potential for chemotherapeutic intervention. Methods Twenty-eight FFPE blocks of ameloblastoma cases from Nigerian patients were prepared for antibody processing to PTCH-1 (Polyclonal Anti-PTCH antibody ab39266) and MDM2 (Monoclonal Anti-MDM2 antibody (2A10) ab16895). Cytoplasmic brown staining was considered as positive for PTCH while nuclear staining was positive for MDM2. Results Moderate and strong expressions for PTCH in ameloblast and stellate reticulum were 78.6% and 60.7% respectively. Only 3 (10.7%) cases expressed MDM2. Conclusion The importance of our study is that it supports, in theory, anti-PTCH/SHH chemotherapeutics for Nigerian ameloblastoma cases and also infers the possible additional use of anti-p53 agents. PMID:27386018

  20. Discovery of Highly Potent p53-MDM2 Antagonists and Structural Basis for Anti-Acute Myeloid Leukemia Activities

    PubMed Central

    2015-01-01

    The inhibition of p53-MDM2 interaction is a promising new approach to non-genotoxic cancer treatment. A potential application for drugs blocking the p53-MDM2 interaction is acute myeloid leukemia (AML) due to the occurrence of wild type p53 (wt p53) in the majority of patients. Although there are very promising preclinical results of several p53-MDM2 antagonists in early development, none of the compounds have yet proven the utility as a next generation anticancer agent. Herein we report the design, synthesis and optimization of YH239-EE (ethyl ester of the free carboxylic acid compound YH239), a potent p53-MDM2 antagonizing and apoptosis-inducing agent characterized by a number of leukemia cell lines as well as patient-derived AML blast samples. The structural basis of the interaction between MDM2 (the p53 receptor) and YH239 is elucidated by a co-crystal structure. YH239-EE acts as a prodrug and is the most potent compound that induces apoptosis in AML cells and patient samples. The observed superior activity compared to reference compounds provides the preclinical basis for further investigation and progression of YH239-EE. PMID:24405416

  1. How To Design a Successful p53-MDM2/X Interaction Inhibitor: A Thorough Overview Based on Crystal Structures.

    PubMed

    Estrada-Ortiz, Natalia; Neochoritis, Constantinos G; Dömling, Alexander

    2016-04-19

    A recent therapeutic strategy in oncology is based on blocking the protein-protein interaction between the murine double minute (MDM) homologues MDM2/X and the tumor-suppressor protein p53. Inhibiting the binding between wild-type (WT) p53 and its negative regulators MDM2 and/or MDMX has become an important target in oncology to restore the antitumor activity of p53, the so-called guardian of our genome. Interestingly, based on the multiple disclosed compound classes and structural analysis of small-molecule-MDM2 adducts, the p53-MDM2 complex is perhaps the best studied and most targeted protein-protein interaction. Several classes of small molecules have been identified as potent, selective, and efficient inhibitors of the p53-MDM2/X interaction, and many co-crystal structures with the protein are available. Herein we review the properties as well as preclinical and clinical studies of these small molecules and peptides, categorized by scaffold type. A particular emphasis is made on crystallographic structures and the observed binding modes of these compounds, including conserved water molecules present.

  2. Relationship Between Murine Double Minute 2 (MDM2) T309G Polymorphism and Endometrial Cancer Risk: A Meta-Analysis

    PubMed Central

    Xue, Zhuowei; Zhu, Xiaolu; Teng, Yincheng

    2016-01-01

    Background Endometrial cancer is one of the most common cancers in female patients. Many studies have investigated the association between the MDM2 T309G genotype and endometrial cancer incidence, but the results have been inconclusive. Material/Methods We performed a systematic search in PubMed and Web of Science databases (update until October 21, 2015) for all English-language publications. The associations are indicated as pooled odds ratio (OR) and 95% confidence intervals (CI). Results We identified 8 relevant publications (9 case-control studies), including 2188 cases and 4654 controls, that assessed the relationship between MDM2 T309G polymorphism and endometrial cancer risk. There was a significant association between MDM2 T309G polymorphism and endometrial cancer risk in the overall population in the recessive model (OR=1.61; 95% CI: 1.19–2.19; P=0.002). In the subgroup of different ethnic populations, the subgroup analysis showed MDM2 T309G polymorphism was significantly associated with increased endometrial cancer risk in Caucasians (OR=1.75; 95% CI: 1.16–2.63; P=0.007). No similar result was found in Asians. Conclusions Our meta-analysis provides evidence that MDM2 T309G polymorphism is associated with endometrial cancer, especially in Caucasians. PMID:27604213

  3. Relationship Between Murine Double Minute 2 (MDM2) T309G Polymorphism and Endometrial Cancer Risk: A Meta-Analysis.

    PubMed

    Xue, Zhuowei; Zhu, Xiaolu; Teng, Yincheng

    2016-01-01

    BACKGROUND Endometrial cancer is one of the most common cancers in female patients. Many studies have investigated the association between the MDM2 T309G genotype and endometrial cancer incidence, but the results have been inconclusive. MATERIAL AND METHODS We performed a systematic search in PubMed and Web of Science databases (update until October 21, 2015) for all English-language publications. The associations are indicated as pooled odds ratio (OR) and 95% confidence intervals (CI). RESULTS We identified 8 relevant publications (9 case-control studies), including 2188 cases and 4654 controls, that assessed the relationship between MDM2 T309G polymorphism and endometrial cancer risk. There was a significant association between MDM2 T309G polymorphism and endometrial cancer risk in the overall population in the recessive model (OR=1.61; 95% CI: 1.19-2.19; P=0.002). In the subgroup of different ethnic populations, the subgroup analysis showed MDM2 T309G polymorphism was significantly associated with increased endometrial cancer risk in Caucasians (OR=1.75; 95% CI: 1.16-2.63; P=0.007). No similar result was found in Asians. CONCLUSIONS Our meta-analysis provides evidence that MDM2 T309G polymorphism is associated with endometrial cancer, especially in Caucasians. PMID:27604213

  4. In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas.

    PubMed

    Bozzi, Fabio; Conca, Elena; Laurini, Erik; Posocco, Paola; Lo Sardo, Alessandra; Jocollè, Genny; Sanfilippo, Roberta; Gronchi, Alessandro; Perrone, Federica; Tamborini, Elena; Pelosi, Giuseppe; Pierotti, Marco A; Maestro, Roberta; Pricl, Sabrina; Pilotti, Silvana

    2013-11-01

    The molecular marker of well-differentiated/de-differentiated liposarcomas is MDM2 gene amplification coupled with protein overexpression and wild-type TP53. MDMX is a recently identified MDM2 homolog and its presence in this tumor is unexplored. Our aim was to investigate the role of full-length MDM2 and MDMX proteins and their isoforms in surgical specimens of well-differentiated/de-differentiated liposarcomas in view of Nutlin-3A (a MDM2 inhibitor) treatment. Frozen and matched formalin-fixed, paraffin-embedded material from surgical specimens was examined by means of: (1) fluorescence in situ hybridization to determine MDM2 and MDMX gene copy numbers; (2) RT-PCR and densitometry to analyze alternative splicing forms of mdm2 and mdmx; (3) immunoblotting and immunohistochemistry to assess the corresponding translated proteins; and (4) in vitro and in silico assays to determine their affinity for Nutlin-3A. All these cases showed MDM2 gene amplification with an MDMX disomic pattern. In all cases, the full-length mdm2 transcript was associated with the mdm2-b transcript, with ratios ranging from 0.07 to 5.6, and both were translated into protein; mdmx and mdmx-s were co-transcripted, with ratios ranging from 0.1 to 5.6. MDMX-S was frequently more upregulated than MDMX at both transcriptional and protein level. Each case showed different amounts of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and the corresponding proteins. In vitro assays showed that Nutlin-3A was ineffective against MDM2-B and was unable to disrupt the MDMX/TP53 and MSMX-S/TP53 complexes. Molecular simulations confirmed these in vitro findings by showing that MDM2 has high Nutlin-3A affinity, followed by MDMX-S, MDMX, and MDM2-B. Nutlin-3A is predicted to be a good therapeutic option for well-differentiated/de-differentiated liposarcomas. However, our findings predict heterogeneous responses depending on the relative expression of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and proteins. PMID

  5. The central acidic domain of MDM2 is critical in inhibition of retinoblastoma-mediated suppression of E2F and cell growth.

    PubMed

    Sdek, Patima; Ying, Haoqiang; Zheng, Hongwu; Margulis, Alexander; Tang, Xiaoren; Tian, Kui; Xiao, Zhi-Xiong Jim

    2004-12-17

    Retinoblastoma (Rb) protein is a paradigm of tumor suppressors. Inactivation of Rb plays a critical role in the development of human malignancies. MDM2, an oncogene frequently found amplified and overexpressed in a variety of human tumors and cancers, directly interacts and inhibits the p53 tumor suppressor protein. In addition, MDM2 has been shown to stimulate E2F transactivation activity and promote S-phase entry independent of p53, yet the mechanism of which is still not fully understood. In this study, we demonstrate that MDM2 specifically binds to Rb C-pocket and that the central acidic domain of MDM2 is essential for Rb interaction. In addition, we show that overexpression of MDM2 reduces Rb-E2F complexes in vivo. Moreover, the ectopic expression of the wild type MDM2, but not mutant MDM2 defective in Rb interaction, stimulates E2F transactivation activity and inhibits Rb growth suppression function. Taken together, these results suggest that MDM2-mediated inhibition of Rb likely contributes to MDM2 oncogenic activity. PMID:15485814

  6. Single-nucleotide polymorphism (c.309T>G) in the MDM2 gene and lung cancer risk.

    PubMed

    Enokida, Yasuaki; Shimizu, Kimihiro; Kakegawa, Seiichi; Atsumi, Jun; Takase, Yoshiaki; Miyamae, Yohei; Nagashima, Toshiteru; Ohtaki, Yoichi; Kaira, Kyoichi; Sunaga, Noriaki; Yanagitani, Noriko; Yoshino, Reiko; Tsunekawa, Katsuhiko; Igai, Hitoshi; Kamiyoshihara, Mitsuhiro; Usui, Kengo; Lezhava, Alexander; Tomizawa, Yoshio; Ishikawa, Toshihisa; Murakami, Masami; Hayashizaki, Yoshihide; Takeyoshi, Izumi

    2014-09-01

    Murine double minute 2 (MDM2) is a negative regulator of p53. A single-nucleotide polymorphism (SNP) (rs2279744: c.309T>G) in the promoter region of the MDM2 gene has been shown to result in higher levels of MDM2 RNA and protein. Regarding the contribution of c.309T>G in the MDM2 gene to the lung cancer risk, previous studies are conflicting. In order to evaluate the association between c.309T>G and the lung cancer risk, a case-control study was performed. The MDM2 genotypes were determined in 762 lung cancer patients and in 700 cancer-free control subjects using the Smart Amplification Process. Statistical adjustment was performed for gender, age and pack-years of smoking. The distributions of c.309T>G (T/T, T/G, G/G) were 20.1, 49.7, 30.2% in the case group and 21.7, 47.9, 30.4% in the healthy-control group. There were no overall associations between the MDM2 genotypes and the risk of lung cancer [T/G genotype: Adjusted odds ratio (AOR), 1.30; 95% confidence interval (CI), 0.88-1.93; and G/G genotype: AOR, 1.18; 95% CI, 0.78-1.80]. The subgroup analysis of gender, histology, smoking status and epidermal growth factor receptor mutation status also indicated that there was no association with lung cancer. Additionally, the genotypes did not have an effect on the age at the time of diagnosis of lung cancer (P=0.25). In conclusion, the G allele frequency in the lung cancer cases was 0.551, which was similar to other studies. The results of the present study suggest that the c.309T>G is not significantly associated with lung cancer.

  7. Identification of lineariifolianoid A as a novel dual NFAT1 and MDM2 inhibitor for human cancer therapy

    PubMed Central

    Qin, Jiang-Jiang; Sarkar, Sushanta; Voruganti, Sukesh; Agarwal, Rajesh; Wang, Wei; Zhang, Ruiwen

    2016-01-01

    Abstract There is an increasing interest in development of novel anticancer agents that target oncogenes. We have recently discovered that nuclear factor of activated T cells 1 (NFAT1) is a novel regulator of the Mouse Double Minute 2 (MDM2) oncogene and the NFAT1-MDM2 pathway has been implicated in human cancer development and progression, justifying that targeting the NFAT1-MDM2 pathway could be a novel strategy for discovery and development of novel cancer therapeutics. The present study was designed to examine the anticancer activity and underlying mechanisms of action of lineariifolianoid A (LinA), a novel natural product inhibitor of the NFAT1-MDM2 pathway. The cytotoxicity of LinA was first tested in various human cancer cell lines in comparison with normal cell lines. The results showed that the breast cancer cells were highly sensitive to LinA treatment. We next demonstrated the effects of LinA on cell proliferation, colony formation, cell cycle progression, and apoptosis in breast cancer MCF7 and MDA-MB-231 cells, in dose-dependent and p53-independent manners. LinA also inhibited the migration and invasion of these cancer cells. Our mechanistic studies further indicated that its anticancer activities were attributed to its inhibitory effects on the NFAT1-MDM2 pathway and modulatory effects on the expression of key proteins involved in cell cycle progression, apoptosis, and DNA damage. In summary, LinA is a novel NFAT1-MDM2 inhibitor and may be developed as a preventive and therapeutic agent against human cancer. PMID:27533941

  8. A re-examination of the MDM2/p53 interaction leads to revised design criteria for novel inhibitors.

    PubMed

    Vasilevich, Natalya I; Afanasyev, Ilya I; Kovalskiy, Dmitry A; Genis, Dmitry V; Kochubey, Valery S

    2014-11-01

    The general model of epitope-type MDM2 inhibitor was developed based on the structural information on the complexes between MDM2 and various low molecular weight ligands found in the PDB database. Application of this model to our in-house library has led us to a new scaffold capable of interrupting protein-protein interactions. A synthetic library based on this and related scaffolds resulted in new classes of compounds that possess biochemical and cellular activity and good pharmacokinetic properties. We assume that such general approach to PPI inhibitors design may be useful for the development of inhibitors of various PPI types, including Bcl/XL.

  9. Design, synthesis and biological evaluation of sulfamide and triazole benzodiazepines as novel p53-MDM2 inhibitors.

    PubMed

    Yu, Zhiliang; Zhuang, Chunlin; Wu, Yuelin; Guo, Zizhao; Li, Jin; Dong, Guoqiang; Yao, Jianzhong; Sheng, Chunquan; Miao, Zhenyuan; Zhang, Wannian

    2014-01-01

    A series of sulfamide and triazole benzodiazepines were obtained with the principle of bioisosterism. The p53-murine double minute 2 (MDM2) inhibitory activity and in vitro antitumor activity were evaluated. Most of the novel benzodiazepines exhibited moderate protein binding inhibitory activity. Particularly, triazole benzodiazepines showed good inhibitory activity and antitumor potency. Compound 16 had promising antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 4.17 μM, which was much better than that of nutlin-3. The molecular docking model also successfully predicted that this class of compounds mimicked the three critical residues of p53 binding to MDM2.

  10. The Role of p53 and MDM2 in Head and Neck Cancer

    PubMed Central

    Denaro, N.; Lo Nigro, C.; Natoli, G.; Russi, E. G.; Adamo, V.; Merlano, M. C.

    2011-01-01

    Head and neck cancer is a complex disorder that includes mostly squamous cell carcinomas that can develop in the throat, larynx, nose, sinuses, and mouth. Etiopathogenesis is due to tobacco and alcohol consumption and to infection by human papillomavirus (HPV) type 16/18. Tumors often develop within preneoplastic fields of genetically altered cells. Most head and neck cancers result from multistep accumulation of genetic alterationsm resulting in clonal outgrowth of transformed cells. These DNA changes are caused by a variety of mechanisms like endogenous mutations and exogenous mutations. Dysregulated molecular pathway includes alterations of critical inhibitor of cyclin CDK complexes, inactivating mutations of p53 gene, and activation of oncogenes and growth factors. This paper attempts to review the role of p53 and MDM2 genetic aberrations and pathways in head and neck cancer. PMID:23724261

  11. The nucleolar SUMO-specific protease SMT3IP1/SENP3 attenuates Mdm2-mediated p53 ubiquitination and degradation

    SciTech Connect

    Nishida, Tamotsu; Yamada, Yoshiji

    2011-03-11

    Research highlights: {yields} SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. {yields} SMT3IP1 competes with p53 for binding to the central acidic domain of Mdm2. {yields} SMT3IP1 binding to Mdm2 inhibits Mdm2-mediated p53 ubiquitination and degradation. {yields} We postulate that SMT3IP1 acts as a new regulator of the p53-Mdm2 pathway. -- Abstract: SUMO (small ubiquitin-like modifier) modification plays multiple roles in several cellular processes. Sumoylation is reversibly regulated by SUMO-specific proteases. SUMO-specific proteases have recently been implicated in cell proliferation and early embryogenesis, but the underlying mechanisms remain unknown. Here, we show that a nucleolar SUMO-specific protease, SMT3IP1/SENP3, controls the p53-Mdm2 pathway. We found that SMT3IP1 interacts with p53 and Mdm2, and desumoylates both proteins. Overexpression of SMT3IP1 in cells resulted in the accumulation of Mdm2 in the nucleolus and increased stability of the p53 protein. In addition, SMT3IP1 bound to the acidic domain of Mdm2, which also mediates the p53 interaction, and competed with p53 for binding. Increasing expression of SMT3IP1 suppressed Mdm2-mediated p53 ubiquitination and subsequent proteasomal degradation. Interestingly, the desumoylation activity of SMT3IP1 was not necessary for p53 stabilization. These results suggest that SMT3IP1 is a new regulator of the p53-Mdm2 pathway.

  12. Impact of MDM2 309T>G polymorphism on sarcomagenesis

    PubMed Central

    Liu, Hua-Wei; Ni, Ming; Li, Xiang; Chen, Hui; Zhang, Guo-Qiang; Chai, Wei; Xu, Meng; Zhou, Yong-Gang; Chen, Ji-Ying; Wang, Yan

    2015-01-01

    Background: A series of epidemiological studies have attempted to evaluate the impact of 309T>G polymorphism in MDM2 gene frequently identified as a susceptibility loci for various cancers on malignant sarcomas, however the reported conclusions remain inconsistent and elusive. We pooled all usable data sets in order to systematically assess the association between 309T>G polymorphism and sarcoma risk. Methods: To identify as many informative studies with complete data as possible, we searched a number of databases (PubMed, EBSCO, BIOSIS, the Cochrane Library, ISI Web of Science, Wiley Online Library and Embase). Inclusion criteria were defined to select the eligible studies. The fixed effects meta-analysis was properly used to calculate the pooled ORs and 95% CIs. Major findings: We eventually identified six studies evaluating the association of sarcoma risk with 309T>G polymorphism. People with 309-GG were found to have 43% greater risk of sarcoma relative to people with 309-TT (OR, 1.43; 95% CI, 1.01~2.03; Pheterogeneity, 0.45). In the G vs. T genetic model, the risk reduced to 19% (OR, 1.19; 95% CI, 1.01~1.40; Pheterogeneity, 0.50). Statistical data showed no significant heterogeneity or publication bias in the meta-analysis. Conclusion: These data demonstrate that 309T>G polymorphism located within the MDM2 gene may act as modifier factor for sarcomas. A weakness of this analysis is that the findings cannot be explainable when the subtypes are separated and additional larger investigations are needed to identify the role of 309T>G polymorphism in each form of sarcoma. PMID:26550195

  13. Differential regulation of the p21/WAF-1 and mdm2 genes after high-dose UV irradiation: p53-dependent and p53-independent regulation of the mdm2 gene.

    PubMed Central

    Wu, L.; Levine, A. J.

    1997-01-01

    BACKGROUND: DNA damage in mammalian cells stabilizes the p53 protein which then functions as a cell cycle checkpoint by leading to growth arrest or apoptosis. p53 is a transcription factor and positively regulates the expression of the p21/WAF-1 gene and the mdm2 gene. After high-dose UV irradiation, p53 increases the expression of the p21/WAF-1 gene immediately (2 to 5 hours after irradiation) while the induction of the mdm2 gene is delayed (8 to 12 hours after irradiation). Experiments presented here explore this differential expression of two different p53-regulated genes. MATERIALS AND METHODS: IP-Western (protein) and Northern (mRNA) blot experiments are used to follow mdm2 and p21/WAF-1 expression in primary rat or mouse cells after a low-dose (4 J/m2) or a high-dose (20 J/M2) of UV irradiation. Northern blot and nuclear run-on experiments are employed to study mRNA stability as well as transcription rates of selected genes. RESULTS: After high-dose UV irradiation, p53 is rapidly stabilized and the expression of p21/WAF1 is immediately increased. By contrast, both protein and mRNA levels of mdm2 first decrease in a p53-independent manner, and later increase in a p53-dependent manner. The initial decline of mdm2 expression following high-dose UV irradiation is UV-dosage dependent and regulated at the level of transcription. CONCLUSION: p53 regulates two genes, p21/WAF1 (blocks cell cycle progression) and mdm2 (reverses p53 activity), that mediate opposite actions. This process is regulated in a temporal fashion after high-dose UV irradiation, so that cell cycle progression can be halted while DNA repair continues prior to reversal of p53-mediated arrest by mdm2. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 PMID:9260156

  14. A Novel Interaction between TFII-I and Mdm2 with a Negative Effect on TFII-I Transcriptional Activity

    PubMed Central

    Cetkovská, Kateřina; Šustová, Hana; Kosztyu, Pavlína; Uldrijan, Stjepan

    2015-01-01

    Williams-Beuren syndrome-associated transcription factor TFII-I plays a critical regulatory role in bone and neural tissue development and in immunity, in part by regulating cell proliferation in response to mitogens. Mdm2, a cellular oncogene responsible for the loss of p53 tumor suppressor activity in a significant proportion of human cancers, was identified in this study as a new binding partner for TFII-I and a negative regulator of TFII-I-mediated transcription. These findings suggest a new p53-independent mechanism by which increased Mdm2 levels found in human tumors could influence cancer cells. In addition to that, we present data indicating that TFII-I is an important cellular regulator of transcription from the immediate-early promoter of human cytomegalovirus, a promoter sequence frequently used in mammalian expression vectors, including vectors for gene therapy. Our observation that Mdm2 over-expression can decrease the ability of TFII-I to activate the CMV promoter might have implications for the efficiency of experimental gene therapy based on CMV promoter–derived vectors in cancers with Mdm2 gene amplification. PMID:26656605

  15. Influence of zinc deficiency on AKT-MDM2-P53 signaling axes in normal and malignant human prostate cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    With prostate being the highest zinc-accumulating tissue before the onset of cancer, the effects of physiologic levels of zinc on Akt-Mdm2-p53 and Akt-p21 signaling axes in human normal prostate epithelial cells (PrEC) and malignant prostate LNCaP cells were examined. Cells were cultured for 6 d in...

  16. Probing the origin of structural stability of single and double stapled p53 peptide analogs bound to MDM2.

    PubMed

    Guo, Zuojun; Streu, Kristina; Krilov, Goran; Mohanty, Udayan

    2014-06-01

    The stabilization of secondary structure is believed to play an important role in the peptide-protein binding interaction. In this study, the α-helical conformation and structural stability of single and double stapled all-hydrocarbon cross-linked p53 peptides when bound and unbound to MDM2 are investigated. We determined the effects of the peptide sequence, the stereochemistry of the cross-linker, the conformation of the double bond in the alkene bridge, and the length of the bridge, to the relative stability of the α-helix structure. The binding affinity calculations by WaterMap provided over one hundred hydration sites in the MDM2 binding pocket where water density is greater than twice that of the bulk, and the relative value of free energy released by displacing these hydration sites. In agreement with the experimental data, potentials of mean force obtained by weighted histogram analysis methods indicated the order of peptides from lowest to highest binding affinity. Our study provides a comprehensive rationalization of the relationship between peptide stapling strategy, the secondary structural stability, and the binding affinity of p53/MDM2 complex. We hope our efforts can help to further the development of a new generation p53/MDM2 inhibitors that can reactivate the function of p53 as tumor suppressor gene.

  17. NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2.

    PubMed

    Liu, Xiaofeng; Tan, Yuqin; Zhang, Chunfeng; Zhang, Ying; Zhang, Liangliang; Ren, Pengwei; Deng, Hongkui; Luo, Jianyuan; Ke, Yang; Du, Xiaojuan

    2016-03-01

    As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2-p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53-mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor. PMID:26882543

  18. A Novel Interaction between TFII-I and Mdm2 with a Negative Effect on TFII-I Transcriptional Activity.

    PubMed

    Cetkovská, Kateřina; Šustová, Hana; Kosztyu, Pavlína; Uldrijan, Stjepan

    2015-01-01

    Williams-Beuren syndrome-associated transcription factor TFII-I plays a critical regulatory role in bone and neural tissue development and in immunity, in part by regulating cell proliferation in response to mitogens. Mdm2, a cellular oncogene responsible for the loss of p53 tumor suppressor activity in a significant proportion of human cancers, was identified in this study as a new binding partner for TFII-I and a negative regulator of TFII-I-mediated transcription. These findings suggest a new p53-independent mechanism by which increased Mdm2 levels found in human tumors could influence cancer cells. In addition to that, we present data indicating that TFII-I is an important cellular regulator of transcription from the immediate-early promoter of human cytomegalovirus, a promoter sequence frequently used in mammalian expression vectors, including vectors for gene therapy. Our observation that Mdm2 over-expression can decrease the ability of TFII-I to activate the CMV promoter might have implications for the efficiency of experimental gene therapy based on CMV promoter-derived vectors in cancers with Mdm2 gene amplification. PMID:26656605

  19. Structural Exploration and Conformational Transitions in MDM2 upon DHFR Interaction from Homo sapiens: A Computational Outlook for Malignancy via Epigenetic Disruption.

    PubMed

    Banerjee, Arundhati; Ray, Sujay

    2016-01-01

    Structural basis for exploration into MDM2 and MDM2-DHFR interaction plays a vital role in analyzing the obstruction in folate metabolism, nonsynthesis of purines, and further epigenetic regulation in Homo sapiens. Therefore, it leads to suppression of normal cellular behavior and malignancy. This has been earlier documented via yeast two-hybrid assays. So, with a novel outlook, this study explores the molecular level demonstration of the best satisfactory MDM2 model selection after performing manifold modeling techniques. Z-scores and other stereochemical features were estimated for comparison. Further, protein-protein docking was executed with MDM2 and the experimentally validated X-ray crystallographic DHFR. Residual disclosure from the best suited simulated protein complex disclosed 18 side chain and 3 ionic interactions to strongly accommodate MDM2 protein into the pocket-like zone in DHFR due to the positive environment by charged residues. Lysine residues from MDM2 played a predominant role. Moreover, evaluation from varied energy calculations, folding rate, and net area for solvent accessibility implied the active participation of MDM2 with DHFR. Fascinatingly, conformational transitions from coils to helices and β-sheets after interaction with DHFR affirm the conformational strength and firmer interaction of human MDM2-DHFR. Therefore, this probe instigates near-future clinical research and interactive computational investigations with mutations. PMID:27213086

  20. Structural Exploration and Conformational Transitions in MDM2 upon DHFR Interaction from Homo sapiens: A Computational Outlook for Malignancy via Epigenetic Disruption

    PubMed Central

    Banerjee, Arundhati; Ray, Sujay

    2016-01-01

    Structural basis for exploration into MDM2 and MDM2-DHFR interaction plays a vital role in analyzing the obstruction in folate metabolism, nonsynthesis of purines, and further epigenetic regulation in Homo sapiens. Therefore, it leads to suppression of normal cellular behavior and malignancy. This has been earlier documented via yeast two-hybrid assays. So, with a novel outlook, this study explores the molecular level demonstration of the best satisfactory MDM2 model selection after performing manifold modeling techniques. Z-scores and other stereochemical features were estimated for comparison. Further, protein-protein docking was executed with MDM2 and the experimentally validated X-ray crystallographic DHFR. Residual disclosure from the best suited simulated protein complex disclosed 18 side chain and 3 ionic interactions to strongly accommodate MDM2 protein into the pocket-like zone in DHFR due to the positive environment by charged residues. Lysine residues from MDM2 played a predominant role. Moreover, evaluation from varied energy calculations, folding rate, and net area for solvent accessibility implied the active participation of MDM2 with DHFR. Fascinatingly, conformational transitions from coils to helices and β-sheets after interaction with DHFR affirm the conformational strength and firmer interaction of human MDM2-DHFR. Therefore, this probe instigates near-future clinical research and interactive computational investigations with mutations. PMID:27213086

  1. The role of NPM, p14arf and MDM2 in precursors of bronchial squamous cell carcinoma.

    PubMed

    Mascaux, C; Bex, F; Martin, B; Burny, A; Haller, A; Paesmans, M; Willard-Gallo, K; Ninane, V; Sculier, J-P

    2008-09-01

    Murine double minute clone 2 (MDM2), p14 alternate reading frame (p14arf), and nucleophosmin (NPM) regulate p53 activity. A total of 200 biopsies, including normal bronchial, pre-invasive and invasive tissues, were examined for changes in NPM, p14arf, MDM2 and p53 expression patterns by immunohistochemistry and immunofluorescence with confocal microscopy. NPM and p14arf displayed a diffuse nuclear staining in most normal bronchial tissue. The fraction of biopsies displaying an increased MDM2 staining or a nucleolar relocalisation of NPM increased at mild and moderate dysplasia, respectively. Two different modifications occurred in p14arf expression, i.e. its loss or its nucleolar relocalisation, both increasing at severe dysplasia and both being associated with high MDM2 expression. In addition, the nucleolar relocalisation of p14arf was associated with that of NPM. Immunofluorescence staining indicated that NPM and p14arf either co-localised in the nucleoplasm or in the nucleoli, before and as a result of severe dysplasia, respectively. MDM2 was not detected in the nucleoli. Thus, changes occur in murine double minute clone 2, p14 alternate reading frame and nucleophosmin level of expression and/or cellular distribution during early steps of lung carcinogenesis. Their relative localisation as determined by immunofluorescence, supports the hypothesis that p14 alternate reading frame nucleolar relocalisation impairs p14 alternate reading frame-murine double minute clone 2 complex formation and that nucleophosmin might sequester p14 alternate reading frame. The demonstration of this hypothesis requires further functional studies.

  2. p53-independent roles of MDM2 in NF-κB signaling: implications for cancer therapy, wound healing, and autoimmune diseases.

    PubMed

    Thomasova, Dana; Mulay, Shrikant R; Bruns, Hauke; Anders, Hans-Joachim

    2012-12-01

    Murine double minute-2 (MDM2) is an intracellular molecule with multiple biologic functions. It serves as a negative regulator of p53 and thereby limits cell cycle arrest and apoptosis. Because MDM2 blockade suppresses tumor cell growth in vitro and in vivo, respective MDM2 inhibition is currently evaluated as anti-cancer therapy in clinical trials. However, the anti-proliferative effects of MDM2 inhibition also impair regenerative cell growth upon tissue injury. This was so far documented for tubular repair upon postischemic acute kidney injury and might apply to wound healing responses in general. Furthermore, MDM2 has numerous p53-independent effects. As a new entry, MDM2 was identified to act as a co-transcription factor for nuclear factor-kappa-light-enhancer of activated B cells (NF-κB) at cytokine promoters. This explains the potent anti-inflammatory effects of MDM2 inhibitors in vitro and in vivo. For example, the NF-κB-antagonistic and p53-agonistic activities of MDM2 inhibitors elicit potent therapeutic effects on experimental lymphoproliferative autoimmune disorders such as systemic lupus erythematosus. In this review, we discuss the classic p53-dependent, the recently discovered p53-independent, and the NF-κB-agonistic biologic functions of MDM2. We describe its complex regulatory role on p53 and NF-κB signaling and name areas of research that may help to foresee previously unexpected effects or potential alternative indications of therapeutic MDM2 blockade. PMID:23308042

  3. Triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer

    PubMed Central

    Xiong, Jing; Su, Tiefen; Qu, Zhiling; Yang, Qin; Wang, Yu; Li, Jiansha; Zhou, Sheng

    2016-01-01

    Triptolide has been shown to exhibit anticancer activity. However, its mechanism of action is not clearly defined. Herein we report a novel signaling pathway, MDM2/Akt, is involved in the anticancer mechanism of triptolide. We observed that triptolide inhibits MDM2 expression in human breast cancer cells with either wild-type or mutant p53. This MDM2 inhibition resulted in decreased Akt activation. More specifically, triptolide interfered with the interaction between MDM2 and the transcription factor REST to increase expression of the regulatory subunit of PI3-kinase p85 and consequently inhibit Akt activation. We further showed that, regardless of p53 status, triptolide inhibited proliferation, induced apoptosis, and caused G1 phase cell cycle arrest. Triptolide also enhanced the cytotoxic effect of doxorubicin. MDM2 inhibition plays a causative role in these effects. The inhibitory effect of triptolide on MDM2-mediated Akt activation was eliminated with MDM2 overexpression. MDM2-overexpressing tumor cells, in turn, were less susceptible to the anticancer and chemosensitization effects of triptolide than control cells. Triptolide also exhibited anticancer and chemosensitization effects in nude mouse xenograft model. When it was administered to tumor-bearing nude mice, triptolide inhibited tumor growth and enhanced the antitumor effects of doxorubicin. In summary, triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer. Our study helps to elucidate the p53-independent regulatory function of MDM2 in Akt signaling, offering a novel view of the mechanism by which triptolide functions as an anticancer agent. PMID:27004407

  4. Ewing Sarcoma: influence of TP53 Arg72Pro and MDM2 T309G SNPs.

    PubMed

    Thurow, Helena S; Hartwig, Fernando P; Alho, Clarice S; Silva, Deborah S B S; Roesler, Rafael; Abujamra, Ana Lucia; de Farias, Caroline Brunetto; Brunetto, Algemir Lunardi; Horta, Bernardo L; Dellagostin, Odir A; Collares, Tiago; Seixas, Fabiana K

    2013-08-01

    The Ewing Sarcoma is an important tumor of bone and soft tissue. The SNPs Arg72Pro of TP53 and T309G of MDM2 have been associated with many cancer types and have been differently distributed among populations worldwide. Based on a case-control design, this study aimed to assess the role of these SNPs in 24 Ewing Sarcoma patients, compared to 91 control individuals. DNA samples were extracted from blood and genotyped for both SNPs by PCR-RFLP and confirmed by DNA sequencing. The results showed an association between the G allele of the T309G and Ewing Sarcoma (P=0.02). Comparing to the TT carriers, the risk of G allele carriers was 3.35 (95% CI=1.22-9.21) with P=0.02. At the genotypic level, an association of the TT genotype with the control group (P=0.03) was found. Comparing to the TT genotype, the risk of TG and GG was 2.97 (95% CI=1.03-8.58) with P=0.04 and 5.00 (95% CI=1.23-20.34) with P=0.02, respectively. No associations regarding the Arg72Pro SNP were found. Considering that the T309G has been associated with several types of cancer, including sarcomas, our results indicate that this SNP may also be important to Ewing Sarcoma predisposition.

  5. Targeting the p53-MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients

    PubMed Central

    Trino, Stefania; Iacobucci, Ilaria; Erriquez, Daniela; Laurenzana, Ilaria; De Luca, Luciana; Ferrari, Anna; Luserna Di Rorà, Andrea Ghelli; Papayannidis, Cristina; Derenzini, Enrico; Simonetti, Giorgia; Lonetti, Annalisa; Venturi, Claudia; Cattina, Federica; Ottaviani, Emanuela; Abbenante, Maria Chiara; Russo, Domenico; Perini, Giovanni; Musto, Pellegrino; Martinelli, Giovanni

    2016-01-01

    MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph−) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph− ALL. PMID:26887044

  6. Targeting the p53-MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients.

    PubMed

    Trino, Stefania; Iacobucci, Ilaria; Erriquez, Daniela; Laurenzana, Ilaria; De Luca, Luciana; Ferrari, Anna; Di Rorà, Andrea Ghelli Luserna; Papayannidis, Cristina; Derenzini, Enrico; Simonetti, Giorgia; Lonetti, Annalisa; Venturi, Claudia; Cattina, Federica; Ottaviani, Emanuela; Abbenante, Maria Chiara; Russo, Domenico; Perini, Giovanni; Musto, Pellegrino; Martinelli, Giovanni

    2016-03-15

    MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph- ALL. PMID:26887044

  7. Loss of oocytes due to conditional ablation of Murine double minute 2 (Mdm2) gene is p53-dependent and results in female sterility.

    PubMed

    Livera, Gabriel; Uzbekov, Rustem; Jarrier, Peggy; Fouchécourt, Sophie; Duquenne, Clotilde; Parent, Anne-Simone; Marine, Jean-Christophe; Monget, Philippe

    2016-08-01

    Murine double minute 2 and 4 (Mdm2, Mdm4) are major p53-negative regulators, preventing thus uncontrolled apoptosis induction in numerous cell types, although their function in the female germ line has received little attention. In the present work, we have generated mice with specific invalidation of Mdm2 and Mdm4 genes in the mouse oocyte (Mdm2(Ocko) and Mdm4(Ocko) mice), to test their implication in survival of these germ cells. Most of the Mdm2(Ocko) but not Mdm4(Ocko) mice were sterile, with a dramatic reduction of the weight of ovaries and genital tract, a strong increase in follicle-stimulating hormone and luteinizing hormone serum levels, and a reduction of anti-mullerian hormone serum levels. Histological analyses revealed an obvious decrease of the number of growing follicles beyond the primary stage in Mdm2(Ocko) ovaries in comparison to controls, with a pronounced increase in the apparition of primary atretic follicles, most being devoid of oocyte. Similar phenotypes were observed with Mdm2(Ocko) Mdm4(Ocko) ovaries, with no worsening of the phenotype. However, we failed to detect any increase in p53 level in mutant oocytes, nor any other apoptotic marker, introgression of this targeted invalidation in p53-/- mice restored the fertility of females. This study is the first to show that Mdm2, but not Mdm4, has a critical role in oocyte survival and would be involved in premature ovarian insufficiency phenotype. PMID:27364741

  8. Disruption of the 5S RNP-Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia.

    PubMed

    Jaako, P; Debnath, S; Olsson, K; Zhang, Y; Flygare, J; Lindström, M S; Bryder, D; Karlsson, S

    2015-11-01

    Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

  9. Ribosomal protein S7 regulates arsenite-induced GADD45α expression by attenuating MDM2-mediated GADD45α ubiquitination and degradation.

    PubMed

    Gao, Ming; Li, Xiaoguang; Dong, Wen; Jin, Rui; Ma, Hanghang; Yang, Pingxun; Hu, Meiru; Li, Yi; Hao, Yi; Yuan, Shengtao; Huang, Junjian; Song, Lun

    2013-05-01

    The stress-responding protein, GADD45α, plays important roles in cell cycle checkpoint, DNA repair and apoptosis. In our recent study, we demonstrate that GADD45α undergoes a dynamic ubiquitination and degradation in vivo, which process can be blocked by the cytotoxic reagent, arsenite, resulting in GADD45α accumulation to activate JNKs cell death pathway, thereby revealing a novel mechanism for the cellular GADD45α functional regulation. But the factors involved in GADD45α stability modulations are unidentified. Here, we demonstrated that MDM2 was an E3 ubiquitin ligase for GADD45α. One of MDM2-binding partner, ribosomal protein S7, interacted with and stabilized GADD45α through preventing the ubiquitination and degradation of GADD45α mediated by MDM2. This novel function of S7 is unrelated to p53 but seems to depend on S7/MDM2 interaction, for the S7 mutant lacking MDM2-binding ability lost its function to stabilize GADD45α. Further investigations indicated that arsenite treatment enhanced S7-MDM2 interaction, resulting in attenuation of MDM2-dependent GADD45α ubiquitination and degradation, thereby leading to GADD45α-dependent cell death pathway activation. Silencing S7 expression suppressed GADD45α-dependent cytotoxicity induced by arsenite. Our findings thus identify a novel function of S7 in control of GADD45α stabilization under both basal and stress conditions and its significance in mediating arsenite-induced cellular stress.

  10. MDM2 polymorphism associated with the development of cervical lesions in women infected with Human papillomavirus and using of oral contraceptives

    PubMed Central

    2014-01-01

    Background The MDM2 gene is the major negative regulator of p53, a tumor suppressor protein. Single nucleotide polymorphism in promoter region of MDM2 gene leads to increased expression resulting in higher levels of MDM2 protein. This event increases the attenuation of the p53 pathway. Polymorphisms in this gene can interfere in the regulation of cellular proliferation. We evaluated whether MDM2 SNP309 (rs2278744) associated or not with the use of oral contraceptive can heighten susceptibility to development of cervical lesions in women HPV infected. Methods MDM2 SNP309 (rs2278744) was genotyped in a total of 287 patients using the PCR-RFLP technique. The results were analyzed by UNPHASED v.3.121 and SNPStats programs. Results The three groups (SIL, LSIL and HSIL) showed no significant differences in either genotype or allelic frequencies for MDM2 polymorphisms, except when HSIL was compared with LSIL (p = 0.037; OR = 1.81). Furthermore, in the analysis of contraceptives, a significant association was found between the use of contraceptives and the MDM2 variant in the development of high-grade cervical lesions for the TG genotype (p = 0.019; OR = 2.21) when HSIL was compared with control. When HSIL was compared with LSIL (p = 0.006; OR = 2.27). Conclusion The results of this study suggest that MDM2 SNP309 might be a good marker for assessing the progression of LSIL to HSIL. In addition, they also show that oral contraceptives alone, did not have any effect on the progression or development of cervical lesions. However, they may act synergistically with MDM2 SNP309 (rs2278744) and HPV infection in the development of cervical lesions. PMID:25075210

  11. Targeting RING domains of Mdm2-MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells.

    PubMed

    Wu, W; Xu, C; Ling, X; Fan, C; Buckley, B P; Chernov, M V; Ellis, L; Li, F; Muñoz, I G; Wang, X

    2015-12-31

    Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2-p53 interface or MdmX ((MDM4), mouse double minute 4)-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2-MdmX really interesting new gene (RING)-RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2-MdmX RING-RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2-MdmX RING domain inhibitors)) that specifically inhibit Mdm2-MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2-MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2-MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.

  12. On the interaction mechanisms of a p53 peptide and nutlin with the MDM2 and MDMX proteins: a Brownian dynamics study.

    PubMed

    ElSawy, Karim M; Verma, Chandra S; Joseph, Thomas L; Lane, David P; Twarock, Reidun; Caves, Leo S D

    2013-02-01

    The interaction of p53 with its regulators MDM2 and MDMX plays a major role in regulating the cell cycle. Inhibition of this interaction has become an important therapeutic strategy in oncology. Although MDM2 and MDMX share a very high degree of sequence/structural similarity, the small-molecule inhibitor nutlin appears to be an efficient inhibitor only of the p53-MDM2 interaction. Here, we investigate the mechanism of interaction of nutlin with these two proteins and contrast it with that of p53 using Brownian dynamics simulations. In contrast to earlier attempts to examine the bound states of the partners, here we locate initial reaction events in these interactions by identifying the regions of space around MDM2/MDMX, where p53/nutlin experience associative encounters with prolonged residence times relative to that in bulk solution. We find that the initial interaction of p53 with MDM2 is long-lived relative to nutlin, but, unlike nutlin, it takes place at the N- and C termini of the MDM2 protein, away from the binding site, suggestive of an allosteric mechanism of action. In contrast, nutlin initially interacts with MDM2 directly at the clefts of the binding site. The interaction of nutlin with MDMX, however, is very short-lived compared with MDM2 and does not show such direct initial interactions with the binding site. Comparison of the topology of the electrostatic potentials of MDM2 and MDMX and the locations of the initial encounters with p53/nutlin in tandem with structure-based sequence alignment revealed that the origin of the diminished activity of nutlin toward MDMX relative to MDM2 may stem partly from the differing topologies of the electrostatic potentials of the two proteins. Glu25 and Lys51 residues underpin these topological differences and appear to collectively play a key role in channelling nutlin directly toward the binding site on the MDM2 surface and are absent in MDMX. The results, therefore, provide new insight into the mechanism of p53

  13. Regulation of the p14ARF-Mdm2-p53 pathway: an overview in breast cancer.

    PubMed

    Agrawal, Anshu; Yang, Jianhui; Murphy, Richard F; Agrawal, Devendra K

    2006-10-01

    Knowledge of the roles of proteins that are abnormally suppressed or activated due to mutation in the DNA sequences of the common tumor suppressor genes, p14ARF and p53, is critical to the understanding the pathogenesis of breast cancer. Mdm2 is a mediator for the function of both p14ARF and p53. In this review article factors including Pokemon, Geminin, Twist, and Apigenin, which control the action of individual proteins in the p14ARF-Mdm2-p53 pathway in breast cancer as well the consequences of mutation 7 of p53 are discussed. The complexity of interaction of components of the pathway and the underlying development of cancer is emphasized. Opportunities for future therapeutic innovations are indicated. PMID:16919268

  14. Design, Synthesis and Biological Evaluation of Sulfamide and Triazole Benzodiazepines as Novel p53-MDM2 Inhibitors

    PubMed Central

    Yu, Zhiliang; Zhuang, Chunlin; Wu, Yuelin; Guo, Zizhao; Li, Jin; Dong, Guoqiang; Yao, Jianzhong; Sheng, Chunquan; Miao, Zhenyuan; Zhang, Wannian

    2014-01-01

    A series of sulfamide and triazole benzodiazepines were obtained with the principle of bioisosterism. The p53-murine double minute 2 (MDM2) inhibitory activity and in vitro antitumor activity were evaluated. Most of the novel benzodiazepines exhibited moderate protein binding inhibitory activity. Particularly, triazole benzodiazepines showed good inhibitory activity and antitumor potency. Compound 16 had promising antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 4.17 μM, which was much better than that of nutlin-3. The molecular docking model also successfully predicted that this class of compounds mimicked the three critical residues of p53 binding to MDM2. PMID:25198897

  15. Regulation of the p14ARF-Mdm2-p53 pathway: an overview in breast cancer.

    PubMed

    Agrawal, Anshu; Yang, Jianhui; Murphy, Richard F; Agrawal, Devendra K

    2006-10-01

    Knowledge of the roles of proteins that are abnormally suppressed or activated due to mutation in the DNA sequences of the common tumor suppressor genes, p14ARF and p53, is critical to the understanding the pathogenesis of breast cancer. Mdm2 is a mediator for the function of both p14ARF and p53. In this review article factors including Pokemon, Geminin, Twist, and Apigenin, which control the action of individual proteins in the p14ARF-Mdm2-p53 pathway in breast cancer as well the consequences of mutation 7 of p53 are discussed. The complexity of interaction of components of the pathway and the underlying development of cancer is emphasized. Opportunities for future therapeutic innovations are indicated.

  16. Association of MDM2 SNP309 and TP53 Arg72Pro polymorphisms with risk of endometrial cancer

    PubMed Central

    YONEDA, TOMOKO; KUBOYAMA, AYUMI; KATO, KIYOKO; OHGAMI, TATSUHIRO; OKAMOTO, KANAKO; SAITO, TOSHIAKI; WAKE, NORIO

    2013-01-01

    The incidence of endometrial cancer, a common gynecological malignancy, is increasing in Japan. We have previously shown that the ER/MDM2/p53/p21 pathway plays an important role in endometrial carcinogenesis. In the present study, we investigated the effects of germline single nucleotide polymorphisms in murine double minute 2 (MDM2) SNP309, TP53 Arg72Pro, ESR1 PvuII and XbaI, and p21 codon 31 on endometrial cancer risk. We evaluated these polymorphisms in DNA samples from 125 endometrial cancer cases and 200 controls using polymerase chain reaction-based restriction fragment length polymorphism. The association of each genetic polymorphism with endometrial cancer was examined by the odds ratio and 95% confidence interval, which were obtained using logistic regression analysis. The SNP309 GG genotype non-significantly increased the risk of endometrial cancer. The 95% confidence interval for the GG genotype vs. the TT genotype of MDM2 SNP309 was 1.76 (0.93–3.30). Endometrial cancer was not associated with tested SNP genotypes for TP53, ESR1 and p21. The combination of SNP309 GG + TG and TP53 codon 72 Arg/Arg significantly increased endometrial cancer risk. The adjusted OR was 2.53 (95% confidence interval, 1.03–6.21) and P for the interaction was 0.04. This result was supported by in vitro data showing that endometrial cancer cell lines with the SNP309 G allele failed to show growth inhibition by treatment with RITA, which reduces p53-MDM2 binding. The presence of the SNP309 G allele and TP53 codon 72 Arg/Arg genotype is associated with an increased risk of endometrial cancer in Japanese women. PMID:23624782

  17. NOLC1, an Enhancer of Nasopharyngeal Carcinoma Progression, Is Essential for TP53 to Regulate MDM2 Expression

    PubMed Central

    Hwang, Yu-Chyi; Lu, Tung-Ying; Huang, Dah-Yeou; Kuo, Yuan-Sung; Kao, Cheng-Fu; Yeh, Ning-Hsing; Wu, Han-Chung; Lin, Chin-Tarng

    2009-01-01

    Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. At present, its etiological factors are not well defined. To identify which genetic alterations might be involved in NPC pathogenesis, we identified genes that were differentially expressed in NPC cell lines and normal nasomucosal cells using subtractive hybridization and microarray analysis. Most NPC cell lines and biopsy specimens were found to have higher expression levels of the gene encoding nucleolar and coiled-body phosphoprotein 1 (NOLC1) as compared with normal cells. Severe combined immunodeficiency mice bearing NPC xenografts derived from NOLC1-short hairpin-RNA-transfected animals were found to have 82% lower levels of tumor growth than control mice as well as marked tumor cell apoptosis. Measuring the expression levels of genes related to cell growth, apoptosis, and angiogenesis, we found that the MDM2 gene was down-regulated in the transfectants. Both co-transfection and chromatin immunoprecipitation experiments showed that tumor protein 53-regulated expression of the MDM2 gene requires co-activation of NOLC1. These findings suggest that NOLC1 plays a role in the regulation of tumorigenesis of NPC and demonstrate that both NOLC1 and tumor protein 53 work together synergistically to activate the MDM2 promoter in NPC cells. PMID:19541936

  18. TP53 and MDM2 single nucleotide polymorphisms influence survival in non-del(5q) myelodysplastic syndromes

    PubMed Central

    Sallman, David A.; Basiorka, Ashley A.; Irvine, Brittany A.; Zhang, Ling; Epling-Burnette, P.K.; Rollison, Dana E.; Mallo, Mar; Sokol, Lubomir; Solé, Francesc; Maciejewski, Jaroslaw; List, Alan F.

    2015-01-01

    P53 is a key regulator of many cellular processes and is negatively regulated by the human homolog of murine double minute-2 (MDM2) E3 ubiquitin ligase. Single nucleotide polymorphisms (SNPs) of either gene alone, and in combination, are linked to cancer susceptibility, disease progression, and therapy response. We analyzed the interaction of TP53 R72P and MDM2 SNP309 SNPs in relationship to outcome in patients with myelodysplastic syndromes (MDS). Sanger sequencing was performed on DNA isolated from 208 MDS cases. Utilizing a novel functional SNP scoring system ranging from +2 to −2 based on predicted p53 activity, we found statistically significant differences in overall survival (OS) (p = 0.02) and progression-free survival (PFS) (p = 0.02) in non-del(5q) MDS patients with low functional scores. In univariate analysis, only IPSS and the functional SNP score predicted OS and PFS in non-del(5q) patients. In multivariate analysis, the functional SNP score was independent of IPSS for OS and PFS. These data underscore the importance of TP53 R72P and MDM2 SNP309 SNPs in MDS, and provide a novel scoring system independent of IPSS that is predictive for disease outcome. PMID:26416416

  19. Mdm2-p53 signaling regulates epidermal stem cell senescence and premature aging phenotypes in mouse skin.

    PubMed

    Gannon, Hugh S; Donehower, Lawrence A; Lyle, Stephen; Jones, Stephen N

    2011-05-01

    The p53 transcription factor is activated by various types of cell stress or DNA damage and induces the expression of genes that control cell growth and inhibit tumor formation. Analysis of mice that express mutant forms of p53 suggest that inappropriate p53 activation can alter tissue homeostasis and life span, connecting p53 tumor suppressor functions with accelerated aging. However, other mouse models that display increased levels of wildtype p53 in various tissues fail to corroborate a link between p53 and aging phenotypes, possibly due to the retention of signaling pathways that negatively regulate p53 activity in these models. In this present study, we have generated mice lacking Mdm2 in the epidermis. Deletion of Mdm2, the chief negative regulator of p53, induced an aging phenotype in the skin of mice, including thinning of the epidermis, reduced wound healing, and a progressive loss of fur. These phenotypes arise due to an induction of p53-mediated senescence in epidermal stem cells and a gradual loss of epidermal stem cell function. These results reveal that activation of endogenous p53 by ablation of Mdm2 can induce accelerated aging phenotypes in mice.

  20. TP53 and MDM2 single nucleotide polymorphisms influence survival in non-del(5q) myelodysplastic syndromes.

    PubMed

    McGraw, Kathy L; Cluzeau, Thomas; Sallman, David A; Basiorka, Ashley A; Irvine, Brittany A; Zhang, Ling; Epling-Burnette, P K; Rollison, Dana E; Mallo, Mar; Sokol, Lubomir; Solé, Francesc; Maciejewski, Jaroslaw; List, Alan F

    2015-10-27

    P53 is a key regulator of many cellular processes and is negatively regulated by the human homolog of murine double minute-2 (MDM2) E3 ubiquitin ligase. Single nucleotide polymorphisms (SNPs) of either gene alone, and in combination, are linked to cancer susceptibility, disease progression, and therapy response. We analyzed the interaction of TP53 R72P and MDM2 SNP309 SNPs in relationship to outcome in patients with myelodysplastic syndromes (MDS). Sanger sequencing was performed on DNA isolated from 208 MDS cases. Utilizing a novel functional SNP scoring system ranging from +2 to -2 based on predicted p53 activity, we found statistically significant differences in overall survival (OS) (p = 0.02) and progression-free survival (PFS) (p = 0.02) in non-del(5q) MDS patients with low functional scores. In univariate analysis, only IPSS and the functional SNP score predicted OS and PFS in non-del(5q) patients. In multivariate analysis, the functional SNP score was independent of IPSS for OS and PFS. These data underscore the importance of TP53 R72P and MDM2 SNP309 SNPs in MDS, and provide a novel scoring system independent of IPSS that is predictive for disease outcome. PMID:26416416

  1. The Role of miR-18b in MDM2-p53 Pathway Signaling and Melanoma Progression

    PubMed Central

    2013-01-01

    Background Although p53 is inactivated by point mutations in many tumors, melanomas infrequently harbor mutations in the p53 gene. Here we investigate the biological role of microRNA-18b (miR-18b) in melanoma by targeting the MDM2-p53 pathway. Methods Expression of miR-18b was examined in nevi (n = 48) and melanoma (n = 92) samples and in melanoma cell lines and normal melanocytes. Immunoblotting was performed to determine the expression of various proteins regulated by miR-18b. The effects of miR-18b overexpression in melanoma cell lines were investigated using assays of colony formation, cell viability, migration, invasion, and cell cycle and in a xenograft model (n = 10 mice per group). Chromatin immunoprecipitation and methylation assays were performed to determine the mechanism of microRNA silencing. Results Expression of miR-18b was substantially reduced in melanoma specimens and cell lines by virtue of hypermethylation and was reinduced (by 1.5- to 5.3-fold) in melanoma cell lines after 5-AZA-deoxycytidine treatment. MDM2 was identified as a target of miR-18b action, and overexpression of miR-18b in melanoma cells was accompanied by 75% reduced MDM2 expression and 2.5-fold upregulation of p53, resulting in 70% suppression of melanoma cell colony formation. The effects of miR-18b overexpression on the p53 pathway and on melanoma cell growth were reversed by MDM2 overexpression. Stable overexpression of miR-18b produced potent tumor suppressor activity, as evidenced by suppressed melanoma cell viability, induction of apoptosis, and reduced tumor growth in vivo. miR-18b overexpression suppressed melanoma cell migration and invasiveness and reversed epithelial-to-mesenchymal transition. Conclusions Our results demonstrate a novel role for miR-18b as a tumor suppressor in melanoma, identify the MDM2-p53 pathway as a target of miR-18b action, and suggest miR-18b overexpression as a novel strategy to reactivate the p53 pathway in human tumors. PMID:23365201

  2. Discovery of a novel class of highly potent inhibitors of the p53-MDM2 interaction by structure-based design starting from a conformational argument.

    PubMed

    Furet, Pascal; Masuya, Keiichi; Kallen, Joerg; Stachyra-Valat, Thérèse; Ruetz, Stephan; Guagnano, Vito; Holzer, Philipp; Mah, Robert; Stutz, Stefan; Vaupel, Andrea; Chène, Patrick; Jeay, Sébastien; Schlapbach, Achim

    2016-10-01

    The p53-MDM2 interaction is an anticancer drug target under investigation in the clinic. Our compound NVP-CGM097 is one of the small molecule inhibitors of this protein-protein interaction currently evaluated in cancer patients. As part of our effort to identify new classes of p53-MDM2 inhibitors that could lead to additional clinical candidates, we report here the design of highly potent inhibitors having a pyrazolopyrrolidinone core structure. The conception of these new inhibitors originated in a consideration on the MDM2 bound conformation of the dihydroisoquinolinone class of inhibitors to which NVP-CGM097 belongs. This work forms the foundation of the discovery of HDM201, a second generation p53-MDM2 inhibitor that recently entered phase I clinical trial.

  3. Exploiting the MDM2-CK1α protein-protein interface to develop novel biologics that induce UBL-kinase-modification and inhibit cell growth.

    PubMed

    Huart, Anne-Sophie; MacLaine, Nicola J; Narayan, Vikram; Hupp, Ted R

    2012-01-01

    Protein-protein interactions forming dominant signalling events are providing ever-growing platforms for the development of novel Biologic tools for controlling cell growth. Casein Kinase 1 α (CK1α) forms a genetic and physical interaction with the murine double minute chromosome 2 (MDM2) oncoprotein resulting in degradation of the p53 tumour suppressor. Pharmacological inhibition of CK1 increases p53 protein level and induces cell death, whilst small interfering RNA-mediated depletion of CK1α stabilizes p53 and induces growth arrest. We mapped the dominant protein-protein interface that stabilizes the MDM2 and CK1α complex in order to determine whether a peptide derived from the core CK1α-MDM2 interface form novel Biologics that can be used to probe the contribution of the CK1-MDM2 protein-protein interaction to p53 activation and cell viability. Overlapping peptides derived from CK1α were screened for dominant MDM2 binding sites using (i) ELISA with recombinant MDM2; (ii) cell lysate pull-down towards endogenous MDM2; (iii) MDM2-CK1α complex-based competition ELISA; and (iv) MDM2-mediated ubiquitination. One dominant peptide, peptide 35 was bioactive in all four assays and its transfection induced cell death/growth arrest in a p53-independent manner. Ectopic expression of flag-tagged peptide 35 induced a novel ubiquitin and NEDD8 modification of CK1α, providing one of the first examples whereby NEDDylation of a protein kinase can be induced. These data identify an MDM2 binding motif in CK1α which when isolated as a small peptide can (i) function as a dominant negative inhibitor of the CK1α-MDM2 interface, (ii) be used as a tool to study NEDDylation of CK1α, and (iii) reduce cell growth. Further, this approach provides a technological blueprint, complementing siRNA and chemical biology approaches, by exploiting protein-protein interactions in order to develop Biologics to manipulate novel types of signalling pathways such as cross-talk between

  4. The polymorphisms of P53 codon 72 and MDM2 SNP309 and renal cell carcinoma risk in a low arsenic exposure area

    SciTech Connect

    Huang, Chao-Yuan; Su, Chien-Tien; Chu, Jan-Show; Huang, Shu-Pin; Pu, Yeong-Shiau; Yang, Hsiu-Yuan; Chung, Chi-Jung; Wu, Chia-Chang; Hsueh, Yu-Mei

    2011-12-15

    Our recent study demonstrated the increased risk of renal cell carcinoma (RCC) associated with high urinary total arsenic levels among people living in a low arsenic exposure area. Genomic instability is important in arsenic carcinogenesis. This study evaluated the relationship between the polymorphisms of p53, p21, and MDM2, which plays a role in gene stability, and the arsenic-related RCC risk. Here, we found that p53 Pro/Pro genotype and MDM2 SNP309 GG genotype significantly increased RCC risk compared to the p53 Arg/Arg genotype and MDM2 SNP309 TT genotype. RCC patients with the p53Arg/Arg genotype had a signicantly low percentage of inorganic arsenic, a low percentage of monomethylarsonic acid (MMA), and a high percentage of dimethylarsinic acid (DMA), which indicates efcient arsenic methylation capacity. Subjects with the p53 Arg/Pro + Pro/Pro genotype or MDM2 SNP309 TG + GG genotype, in conjunction with high urinary total arsenic ({>=} 14.02 {mu}g/L), had a signicantly higher RCC risk than those with the p53 Arg/Arg or MDM2 SNP309 TT genotypes and low urinary total arsenic. Taken together, this is the first study to show that a variant genotype of p53 Arg{sup 72}Pro or MDM2 SNP309 may modify the arsenic-related RCC risk even in a non-obvious arsenic exposure area. -- Highlights: Black-Right-Pointing-Pointer Subjects with p53 Pro/Pro or MDM2 GG genotype significantly increased RCC risk. Black-Right-Pointing-Pointer A significant multiplicative joint effect of p53 and p21 on RCC risk. Black-Right-Pointing-Pointer RCC patients with p53 Arg/Arg genotype had efficient arsenic methylation capacity. Black-Right-Pointing-Pointer Joint effect of p53 or MDM2 genotype and high urinary total arsenic on RCC risk.

  5. P15, MDM2, NF-κB, and Bcl-2 expression in primary bone tumor and correlation with tumor formation and metastasis

    PubMed Central

    Qian, Guibin; Hao, Songnan; Yang, Dawei; Meng, Qinggang

    2015-01-01

    Primary bone tumor is one of the most common malignant tumors in skeletal system. It seriously affected bone movement and development with unclear pathogenesis. In this paper, rabbit VX-2 malignant bone tumor model was applied to explore apoptotic genes P15, MDM2, NF-κB and Bcl-2 correlation with primary bone tumor occurrence and metastasis. 0.3 ml rabbit VX-2 tumor cell suspension (1×106/ml) was injected to the marrow cavity of the right tibia condyle to establish the rabbit malignant bone tumor model, while equal amount of the saline was injected to the left tibia as control. Real-time PCR was applied to determine P15, MDM2, NF-κB and Bcl-2 expression level. Immunohistochemistry was performed to detect the abovementioned genes expression in lung, stomach, kidney and bladder. Compared with control, P15 expression level in the inoculation site surrounding tissues decreased obviously following the inoculate time elongation (P<0.05), while Bcl-2, MDM2 and NF-κB expression significantly increased (P<0.05). Bcl-2 showed significant correlation with MDM2 and NF-κB (P<0.05). At the 2, 4, 6 weeks, Bcl-2, MDM2 and NF-κB in lung, Bcl-2 in kidney, and Bcl-2 and MDM2 in bladder positively expressed (P<0.05), whereas P15 gene exhibited no significant positive expression in these tissues (P>0.05). P15, MDM2, NF-κB, and Bcl-2 genes expression levels can effectively reflect malignant bone tumor growth of rabbit tibia. MDM2, NF-κB and Bcl-2 genes involved in primary bone tumors metastasis directly. It has important clinical significance for early diagnosis and treatment of primary bone tumor. PMID:26823818

  6. Apoptosis of Sertoli cells after conditional ablation of murine double minute 2 (Mdm2) gene is p53-dependent and results in male sterility

    PubMed Central

    Fouchécourt, S; Livera, G; Messiaen, S; Fumel, B; Parent, A-S; Marine, J-C; Monget, P

    2016-01-01

    Beside its well-documented role in carcinogenesis, the function of p53 family has been more recently revealed in development and female reproduction, but it is still poorly documented in male reproduction. We specifically tested this possibility by ablating Mdm2, an E3 ligase that regulates p53 protein stability and transactivation function, specifically in Sertoli cells (SCs) using the AMH-Cre line and created the new SC-Mdm2−/− line. Heterozygous SC-Mdm2−/+ adult males were fertile, but SC-Mdm2−/− males were infertile and exhibited: a shorter ano-genital distance, an extra duct along the vas deferens that presents a uterus-like morphology, degenerated testes with no organized seminiferous tubules and a complete loss of differentiated germ cells. In adults, testosterone levels as well as StAR, P450c17 (Cyp17a1) and P450scc (Cyp11a1) mRNA levels decreased significantly, and both plasma LH and FSH levels increased. A detailed investigation of testicular development indicated that the phenotype arose during fetal life, with SC-Mdm2−/− testes being much smaller at birth. Interestingly, Leydig cells remained present until adulthood and fetal germ cells abnormally initiated meiosis. Inactivation of Mdm2 in SCs triggered p53 activation and apoptosis as early as 15.5 days post conception with significant increase in apoptotic SCs. Importantly, testis development occurred normally in SC-Mdm2−/− lacking p53 mice (SC-Mdm2−/−p53−/−) and accordingly, these mice were fertile indicating that the aforementioned phenotypes are entirely p53-dependent. These data not only highlight the importance of keeping p53 in check for proper testicular development and male fertility but also certify the critical role of SCs in the maintenance of meiotic repression. PMID:26470726

  7. Estrogen receptor alpha (ERα/ESR1) mediates the p53-independent overexpression of MDM4/MDMX and MDM2 in human breast cancer

    PubMed Central

    Swetzig, Wendy M.; Wang, Jianmin; Das, Gokul M.

    2016-01-01

    MDM2 and MDM4 are heterodimeric, non-redundant oncoproteins that potently inhibit the p53 tumor suppressor protein. MDM2 and MDM4 also enhance the tumorigenicity of breast cancer cells in in vitro and in vivo models and are overexpressed in primary human breast cancers. Prior studies have characterized Estrogen Receptor Alpha (ERα/ESR1) as a regulator of MDM2 expression and an MDM2- and p53-interacting protein. However, similar crosstalk between ERα and MDM4 has not been investigated. Moreover, signaling pathways that mediate the overexpression of MDM4 in human breast cancer remain to be elucidated. Using the Cancer Genome Atlas (TCGA) breast invasive carcinoma patient cohort, we have analyzed correlations between ERα status and MDM4 and MDM2 expression in primary, treatment-naïve, invasive breast carcinoma samples. We report that the expression of MDM4 and MDM2 is elevated in primary human breast cancers of luminal A/B subtypes and associates with ERα-positive disease, independently of p53 mutation status. Furthermore, in cell culture models, ERα positively regulates MDM4 and MDM2 expression via p53-independent mechanisms, and these effects can be blocked by the clinically-relevant endocrine therapies fulvestrant and tamoxifen. Additionally, ERα also positively regulates p53 expression. Lastly, we report that endogenous MDM4 negatively regulates ERα expression and forms a protein complex with ERα in breast cancer cell lines and primary human breast tumor tissue. This suggests direct signaling crosstalk and negative feedback loops between ERα and MDM4 expression in breast cancer cells. Collectively, these novel findings implicate ERα as a central component of the p53-MDM2-MDM4 signaling axis in human breast cancer. PMID:26909605

  8. Induction of apoptosis in non-small cell lung cancer by downregulation of MDM2 using pH-responsive PMPC-b-PDPA/siRNA complex nanoparticles.

    PubMed

    Yu, Haijun; Zou, Yonglong; Jiang, Lei; Yin, Qi; He, Xinyu; Chen, Lingli; Zhang, Zhiwen; Gu, Wangwen; Li, Yaping

    2013-04-01

    Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancer caused human death. In this work, we selected oncogene mouse double minute 2 (MDM2) as a therapeutic target for NSCLC treatment and proposed that sufficient MDM2 knockdown could inhibit tumor growth via induction of cell cycle arrest and cancer cell apoptosis. On this regard, a new pH-responsive diblock copolymer of poly(methacryloyloxy ethyl phosphorylcholine)-block-poly(diisopropanolamine ethyl methacrylate) (PMPC-b-PDPA)/siRNA-MDM2 complex nanoparticle with minimized surface charge and suitable particle size was designed and developed for siRNA-MDM2 delivery in vitro and in vivo. The experimental results showed that the nanoparticles were spherical with particle size around 50 nm. MDM2 knockdown in p53 mutant NSCLC H2009 cells induced significant cell cycle arrest, apoptosis and growth inhibition through upregulation of p21 and activation of caspase-3. Furthermore, the growth of H2009 xenograft tumor in nude mice was inhibited via repeated injection of PMPC-b-PDPA/siRNA-MDM2 complex nanoparticles. These results suggested that PMPC-b-PDPA/siRNA complex nanoparticles targeting a unique set of oncogenes could be developed into a new therapeutic approach for NSCLC treatment. PMID:23352573

  9. Glycogen synthase kinase 3β inhibitors protect hippocampal neurons from radiation-induced apoptosis by regulating MDM2-p53 pathway

    PubMed Central

    Thotala, D K; Hallahan, D E; Yazlovitskaya, E M

    2012-01-01

    Exposure of the brain to ionizing radiation can cause neurocognitive deficiencies. The pathophysiology of these neurological changes is complex and includes radiation-induced apoptosis in the subgranular zone of the hippocampus. We have recently found that inhibition of glycogen synthase kinase 3β (GSK-3β) resulted in significant protection from radiation-induced apoptosis in hippocampal neurons. The molecular mechanisms of this cytoprotection include abrogation of radiation-induced accumulation of p53. Here we show that pretreatment of irradiated HT-22 hippocampal-derived neurons with small molecule inhibitors of GSK-3β SB216763 or SB415286, or with GSK-3β-specific shRNA resulted in accumulation of the p53-specific E3 ubiquitin ligase MDM2. Knockdown of MDM2 using specific shRNA or chemical inhibition of MDM2-p53 interaction prevented the protective changes triggered by GSK-3β inhibition in irradiated HT-22 neurons and restored radiation cytotoxicity. We found that this could be due to regulation of apoptosis by subcellular localization and interaction of GSK-3β, p53 and MDM2. These data suggest that the mechanisms of radioprotection by GSK-3β inhibitors in hippocampal neurons involve regulation of MDM2-dependent p53 accumulation and interactions between GSK-3β, MDM2 and p53. PMID:21738215

  10. Stabilization of p53 in Influenza A Virus-infected Cells Is Associated with Compromised MDM2-mediated Ubiquitination of p53*

    PubMed Central

    Wang, Xiaodu; Deng, Xufang; Yan, Wenjun; Zhu, Zixiang; Shen, Yang; Qiu, Yafeng; Shi, Zixue; Shao, Donghua; Wei, Jianchao; Xia, Xianzhu; Ma, Zhiyong

    2012-01-01

    Influenza A virus (IAV) induces apoptosis of infected cells. In response to IAV infection, p53, a tumor suppressor involved in regulating apoptosis and host antiviral defense, accumulates and becomes activated. This study was undertaken to examine the mechanism of p53 accumulation in IAV-infected cells. Here we show that p53 accumulation in IAV-infected cells results from protein stabilization, which was associated with compromised Mdm2-mediated ubiquitination of p53. In IAV-infected cells, p53 was stabilized and its half-life was remarkably extended. The ladders of polyubiquitinated p53 were not detectable in the presence of the proteasome inhibitor MG132 and were less sensitive to proteasome-mediated degradation. IAV infection did not affect the abundance of Mdm2, a major ubiquitin E3 ligase responsible for regulating p53 ubiquitination and degradation, but weakened the interaction between p53 and Mdm2. Viral nucleoprotein (NP) was able to increase the transcriptional activity and stability of p53. Furthermore, NP was found to associate with p53 and to impair the p53-Mdm2 interaction and Mdm2-mediated p53 ubiquitination, demonstrating its role in inhibiting Mdm2-mediated p53 ubiquitination and degradation. PMID:22474335

  11. Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors.

    PubMed

    Li, Yizhu; Saini, Priyanka; Sriraman, Anusha; Dobbelstein, Matthias

    2015-10-20

    Pharmacological inhibition of the cell cycle regulatory kinase Wee1 represents a promising strategy to eliminate cancer cells. Wee1 inhibitors cooperate with chemotherapeutics, e. g. nucleoside analogues, pushing malignant cells from S phase towards premature mitosis and death. However, considerable toxicities are observed in preclinical and clinical trials. A high proportion of tumor cells can be distinguished from all other cells of a patient's body by inactivating mutations in the tumor suppressor p53. Here we set out to develop an approach for the selective protection of p53-proficient cells against the cytotoxic effects of Wee1 inhibitors. We pretreated such cells with Nutlin-3a, a prototype inhibitor of the p53-antagonist Mdm2. The resulting transient cell cycle arrest effectively increased the survival of cells that were subsequently treated with combinations of the Wee1 inhibitor MK-1775 and/or the nucleoside analogue gemcitabine. In this constellation, Nutlin-3a reduced caspase activation and diminished the phosphorylation of Histone 2AX, an indicator of the DNA damage response. Both effects were strictly dependent on the presence of p53. Moreover, Nutlin pre-treatment reduced the fraction of cells that were undergoing premature mitosis in response to Wee1 inhibition. We conclude that the pre-activation of p53 through Mdm2 antagonists serves as a viable option to selectively protect p53-proficient cells against the cytotoxic effects of Wee1 inhibitors, especially when combined with a nucleoside analogue. Thus, Mdm2 antagonists might prove useful to avoid unwanted side effects of Wee1 inhibitors. On the other hand, when a tumor contains wild type p53, care should be taken not to induce its activity before applying Wee1 inhibitors. PMID:26431163

  12. Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors

    PubMed Central

    Dobbelstein, Matthias

    2015-01-01

    Pharmacological inhibition of the cell cycle regulatory kinase Wee1 represents a promising strategy to eliminate cancer cells. Wee1 inhibitors cooperate with chemotherapeutics, e. g. nucleoside analogues, pushing malignant cells from S phase towards premature mitosis and death. However, considerable toxicities are observed in preclinical and clinical trials. A high proportion of tumor cells can be distinguished from all other cells of a patient's body by inactivating mutations in the tumor suppressor p53. Here we set out to develop an approach for the selective protection of p53-proficient cells against the cytotoxic effects of Wee1 inhibitors. We pretreated such cells with Nutlin-3a, a prototype inhibitor of the p53-antagonist Mdm2. The resulting transient cell cycle arrest effectively increased the survival of cells that were subsequently treated with combinations of the Wee1 inhibitor MK-1775 and/or the nucleoside analogue gemcitabine. In this constellation, Nutlin-3a reduced caspase activation and diminished the phosphorylation of Histone 2AX, an indicator of the DNA damage response. Both effects were strictly dependent on the presence of p53. Moreover, Nutlin pre-treatment reduced the fraction of cells that were undergoing premature mitosis in response to Wee1 inhibition. We conclude that the pre-activation of p53 through Mdm2 antagonists serves as a viable option to selectively protect p53-proficient cells against the cytotoxic effects of Wee1 inhibitors, especially when combined with a nucleoside analogue. Thus, Mdm2 antagonists might prove useful to avoid unwanted side effects of Wee1 inhibitors. On the other hand, when a tumor contains wild type p53, care should be taken not to induce its activity before applying Wee1 inhibitors. PMID:26431163

  13. Np9, a cellular protein of retroviral ancestry restricted to human, chimpanzee and gorilla, binds and regulates ubiquitin ligase MDM2.

    PubMed

    Heyne, Kristina; Kölsch, Kathrin; Bruand, Marine; Kremmer, Elisabeth; Grässer, Friedrich A; Mayer, Jens; Roemer, Klaus

    2015-01-01

    Humans and primates are long-lived animals with long reproductive phases. One factor that appears to contribute to longevity and fertility in humans, as well as to cancer-free survival, is the transcription factor and tumor suppressor p53, controlled by its main negative regulator MDM2. However, p53 and MDM2 homologs are found throughout the metazoan kingdom from Trichoplacidae to Hominidae. Therefore the question arises, if p53/MDM2 contributes to the shaping of primate features, then through which mechanisms. Previous findings have indicated that the appearances of novel p53-regulated genes and wild-type p53 variants during primate evolution are important in this context. Here, we report on another mechanism of potential relevance. Human endogenous retrovirus K subgroup HML-2 (HERV-K(HML-2)) type 1 proviral sequences were formed in the genomes of the predecessors of contemporary Hominoidea and can be identified in the genomes of Nomascus leucogenys (gibbon) up to Homo sapiens. We previously reported on an alternative splicing event in HERV-K(HML-2) type 1 proviruses that can give rise to nuclear protein of 9 kDa (Np9). We document here the evolution of Np9-coding capacity in human, chimpanzee and gorilla, and show that the C-terminal half of Np9 binds directly to MDM2, through a domain of MDM2 that is known to be contacted by various cellular proteins in response to stress. Np9 can inhibit the MDM2 ubiquitin ligase activity toward p53 in the cell nucleus, and can support the transactivation of genes by p53. Our findings point to the possibility that endogenous retrovirus protein Np9 contributes to the regulation of the p53-MDM2 pathway specifically in humans, chimpanzees and gorillas.

  14. Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53-MDM2 interaction.

    PubMed

    Soares, Joana; Pereira, Nuno A L; Monteiro, Ângelo; Leão, Mariana; Bessa, Cláudia; Dos Santos, Daniel J V A; Raimundo, Liliana; Queiroz, Glória; Bisio, Alessandra; Inga, Alberto; Pereira, Clara; Santos, Maria M M; Saraiva, Lucília

    2015-01-23

    One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53-MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53-MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53(+/+)) and in its isogenic derivative without p53 (HCT116 p53(-/-)). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53(+/+) but not in p53(-/-) HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53-MDM2 interaction in HCT116 p53(+/+) cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of

  15. Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53-MDM2 interaction.

    PubMed

    Soares, Joana; Pereira, Nuno A L; Monteiro, Ângelo; Leão, Mariana; Bessa, Cláudia; Dos Santos, Daniel J V A; Raimundo, Liliana; Queiroz, Glória; Bisio, Alessandra; Inga, Alberto; Pereira, Clara; Santos, Maria M M; Saraiva, Lucília

    2015-01-23

    One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53-MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53-MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53(+/+)) and in its isogenic derivative without p53 (HCT116 p53(-/-)). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53(+/+) but not in p53(-/-) HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53-MDM2 interaction in HCT116 p53(+/+) cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of

  16. Lead Optimization of 2-Phenylindolylglyoxylyldipeptide Murine Double Minute (MDM)2/Translocator Protein (TSPO) Dual Inhibitors for the Treatment of Gliomas.

    PubMed

    Daniele, Simona; La Pietra, Valeria; Barresi, Elisabetta; Di Maro, Salvatore; Da Pozzo, Eleonora; Robello, Marco; La Motta, Concettina; Cosconati, Sandro; Taliani, Sabrina; Marinelli, Luciana; Novellino, Ettore; Martini, Claudia; Da Settimo, Federico

    2016-05-26

    In glioblastoma multiforme (GBM), translocator protein (TSPO) and murine double minute (MDM)2/p53 complex represent two druggable targets. We recently reported the first dual binder 3 possessing a higher anticancer effect in GBM cells than the standards PK11195 1 or Nutlin-3 2 singularly applied. Herein, through a structure-activity relationship study, we developed derivatives 4-10 with improved potencies toward both TSPO and MDM2. As a result, compound 9: (i) reactivated the p53 functionality; (ii) inhibited the viability of two human GBM cells; (iii) impaired the proliferation of glioma cancer stem cells (CSCs), more resistant to chemotherapeutics and responsible of GBM recurrence; (iv) sensitized GBM cells and CSCs to the activity of temozolomide; (v) directed its effects preferentially toward tumor cells with respect to healthy ones. Thus, 9 may represent a promising cytotoxic agent, which is worthy of being further developed for a therapeutic approach against GBM, where the downstream p53 signaling is intact and TSPO is overexpressed.

  17. A stapled peptide antagonist of MDM2 carried by polymeric micelles sensitizes glioblastoma to temozolomide treatment through p53 activation.

    PubMed

    Chen, Xishan; Tai, Lingyu; Gao, Jie; Qian, Jianchang; Zhang, Mingfei; Li, Beibei; Xie, Cao; Lu, Linwei; Lu, Wuyuan; Lu, Weiyue

    2015-11-28

    Antagonizing MDM2 and MDMX to activate the tumor suppressor protein p53 is an attractive therapeutic paradigm for the treatment of glioblastoma multiforme (GBM). However, challenges remain with respect to the poor ability of p53 activators to efficiently cross the blood-brain barrier and/or blood-brain tumor barrier and to specifically target tumor cells. To circumvent these problems, we developed a cyclic RGD peptide-conjugated poly(ethylene glycol)-co-poly(lactic acid) polymeric micelle (RGD-M) that carried a stapled peptide antagonist of both MDM2 and MDMX (sPMI). The peptide-carrying micelle RGD-M/sPMI was prepared via film-hydration method with high encapsulation efficiency and loading capacity as well as ideal size distribution. Micelle encapsulation dramatically increased the solubility of sPMI, thus alleviating its serum sequestration. In vitro studies showed that RGD-M/sPMI efficiently inhibited the proliferation of glioma cells in the presence of serum by activating the p53 signaling pathway. Further, RGD-M/sPMI exerted potent tumor growth inhibitory activity against human glioblastoma in nude mouse xenograft models. Importantly, the combination of RGD-M/sPMI and temozolomide--a standard chemotherapy drug for GBM increased antitumor efficacy against glioblastoma in experimental animals. Our results validate a combination therapy using p53 activators with temozolomide as a more effective treatment for GBM. PMID:26428461

  18. Long lasting MDM2/Translocator protein modulator: a new strategy for irreversible apoptosis of human glioblastoma cells

    PubMed Central

    Zappelli, Elisa; Marinelli, Luciana; Novellino, Ettore; Da Settimo, Federico; Taliani, Sabrina; Trincavelli, Maria L.; Martini, Claudia

    2016-01-01

    The development of multi-target drugs and irreversible modulators of deregulated signalling proteins is the major challenge for improving glioblastoma multiforme (GBM) treatment. Reversible single-target drugs are not sufficient to sustain a therapeutic effect over time and may favour the activation of alternative signalling pathways and the onset of resistance phenomena. Thus, a multi-target compound that has a long-lasting mechanism of action might have a greater and longer life span of anti-proliferative activity. Recently, a dual-target indol-3ylglyoxyldipeptide derivative, designed to bind to the Translocator Protein (TSPO) and reactivate p53 function via dissociation from its physiological inhibitor, murine double minute 2 (MDM2), has been developed as a potent GBM pro-apoptotic agent. In this study, this derivative was chemically modified to irreversibly bind MDM2 and TSPO. The new compound elicited a TSPO-mediated mitochondrial membrane dissipation and restored p53 activity, triggering a long-lasting apoptosis of GBM cells. These effects were sustained over time, involved a stable activation of extracellular signal regulated kinases and were specifically observed in cancer cells, in which these protein kinases are deregulated. Dual-targeting and irreversible binding properties combined in the same molecule may represent a useful strategy to overcome the time-limited effects elicited by classical chemotherapies. PMID:26761214

  19. Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction

    PubMed Central

    Pettersson, Mariell; Quant, Maria; Min, Jaeki; Iconaru, Luigi; Kriwacki, Richard W.; Waddell, M. Brett; Guy, R. Kiplin; Luthman, Kristina; Grøtli, Morten

    2015-01-01

    The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2) via a direct protein—protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay. PMID:26427060

  20. Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

    PubMed

    Pettersson, Mariell; Quant, Maria; Min, Jaeki; Iconaru, Luigi; Kriwacki, Richard W; Waddell, M Brett; Guy, R Kiplin; Luthman, Kristina; Grøtli, Morten

    2015-01-01

    The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2) via a direct protein-protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay.

  1. Probing Origin of Binding Difference of inhibitors to MDM2 and MDMX by Polarizable Molecular Dynamics Simulation and QM/MM-GBSA Calculation

    NASA Astrophysics Data System (ADS)

    Chen, Jianzhong; Wang, Jinan; Zhang, Qinggang; Chen, Kaixian; Zhu, Weiliang

    2015-11-01

    Binding abilities of current inhibitors to MDMX are weaker than to MDM2. Polarizable molecular dynamics simulations (MD) followed by Quantum mechanics/molecular mechanics generalized Born surface area (QM//MM-GBSA) calculations were performed to investigate the binding difference of inhibitors to MDM2 and MDMX. The predicted binding free energies not only agree well with the experimental results, but also show that the decrease in van der Walls interactions of inhibitors with MDMX relative to MDM2 is a main factor of weaker bindings of inhibitors to MDMX. The analyses of dihedral angles based on MD trajectories suggest that the closed conformation formed by the residues M53 and Y99 in MDMX leads to a potential steric clash with inhibitors and prevents inhibitors from arriving in the deep of MDMX binding cleft, which reduces the van der Waals contacts of inhibitors with M53, V92, P95 and L98. The calculated results using the residue-based free energy decomposition method further prove that the interaction strength of inhibitors with M53, V92, P95 and L98 from MDMX are obviously reduced compared to MDM2. We expect that this study can provide significant theoretical guidance for designs of potent dual inhibitors to block the p53-MDM2/MDMX interactions.

  2. Proneness to UV-induced apoptosis in human fibroblasts defective in transcription coupled repair is associated with the lack of Mdm2 transactivation.

    PubMed

    Conforti, G; Nardo, T; D'Incalci, M; Stefanini, M

    2000-05-18

    The apoptotic response and the level of expression of p53 and of three genes transcriptionally activated by p53 (Mdm2, p21 and bax) were investigated in UV-sensitive cells from patients with xeroderma pigmentosum (XP) or Cockayne syndrome (CS). These disorders are due to different genetic defects affecting transcription-coupled repair (TCR) and/or global genome repair (GGR), the nucleotide excision repair subpathways which remove UV-induced lesions from the transcribed strand of active genes or from the rest of the genome, respectively. After 20 J/m2 UV light, normal and GGR-defective XP-C fibroblasts showed rapid increase in p53, late induction of Mdm2 and no evidence of apoptosis even 96 h after irradiation. In contrast, in XP-A (defective in GGR and TCR), CS-A and CS-B (defective only in TCR) fibroblasts, the p53 increase was not followed by Mdm2 induction and the persistence of high levels of p53, due to the lack of its degradation by Mdm2, was associated with the appearance of apoptosis. Besides indicating that the persistence of DNA damage in the transcribed strand of active genes leads to apoptosis, these findings provide the first evidence that the lack of activation of Mdm2 plays a key role in the cascade of events leading to apoptosis. Oncogene (2000). PMID:10851071

  3. Histone deacetylase inhibitor sodium butyrate suppresses proliferation and promotes apoptosis in osteosarcoma cells by regulation of the MDM2–p53 signaling

    PubMed Central

    Xie, Chuhai; Wu, Boyi; Chen, Binwei; Shi, Qunwei; Guo, Jianhong; Fan, Ziwen; Huang, Yan

    2016-01-01

    Histone deacetylase inhibitors have been reported to induce tumor cell growth arrest, differentiation, and apoptosis. This study aimed to investigate the effects of one histone deacetylase inhibitor – sodium butyrate (SB) – on osteosarcoma (OS) cell proliferation and apoptosis and also the molecular mechanisms by which SB exerts regulatory effects on OS cells. U2OS and MG63 cells were treated with SB at various concentrations. Then, cell proliferation and apoptosis were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and flow cytometry assays, respectively; the expression of Ki67, Bax, Bcl-2, MDM2, and p53 proteins was determined by using Western blot assay. The results showed that SB suppressed proliferation in a concentration-dependent manner and promoted apoptosis of OS cells. In addition, SB enhanced p53 expression and decreased MDM2 expression, indicating that SB can regulate MDM2–p53 feedback loop. p53 inhibited proliferation and promoted apoptosis, whereas MDM2 promoted proliferation and suppressed apoptosis, which indicated that functional effect of SB on OS cell lines at least in part depended on the MDM2–p53 signaling. We also explored the effect of SB on OS cells in vivo and found that SB suppressed the growth of OS cells with no noticeable effect on activity and body weight of mice in vivo. These findings will offer new clues for OS development and progression and offer SB as a potent targeted agent for OS treatment. PMID:27445491

  4. Probing Origin of Binding Difference of inhibitors to MDM2 and MDMX by Polarizable Molecular Dynamics Simulation and QM/MM-GBSA Calculation

    PubMed Central

    Chen, Jianzhong; Wang, Jinan; Zhang, Qinggang; Chen, Kaixian; Zhu, Weiliang

    2015-01-01

    Binding abilities of current inhibitors to MDMX are weaker than to MDM2. Polarizable molecular dynamics simulations (MD) followed by Quantum mechanics/molecular mechanics generalized Born surface area (QM//MM-GBSA) calculations were performed to investigate the binding difference of inhibitors to MDM2 and MDMX. The predicted binding free energies not only agree well with the experimental results, but also show that the decrease in van der Walls interactions of inhibitors with MDMX relative to MDM2 is a main factor of weaker bindings of inhibitors to MDMX. The analyses of dihedral angles based on MD trajectories suggest that the closed conformation formed by the residues M53 and Y99 in MDMX leads to a potential steric clash with inhibitors and prevents inhibitors from arriving in the deep of MDMX binding cleft, which reduces the van der Waals contacts of inhibitors with M53, V92, P95 and L98. The calculated results using the residue-based free energy decomposition method further prove that the interaction strength of inhibitors with M53, V92, P95 and L98 from MDMX are obviously reduced compared to MDM2. We expect that this study can provide significant theoretical guidance for designs of potent dual inhibitors to block the p53-MDM2/MDMX interactions. PMID:26616018

  5. Histone deacetylase inhibitor sodium butyrate suppresses proliferation and promotes apoptosis in osteosarcoma cells by regulation of the MDM2-p53 signaling.

    PubMed

    Xie, Chuhai; Wu, Boyi; Chen, Binwei; Shi, Qunwei; Guo, Jianhong; Fan, Ziwen; Huang, Yan

    2016-01-01

    Histone deacetylase inhibitors have been reported to induce tumor cell growth arrest, differentiation, and apoptosis. This study aimed to investigate the effects of one histone deacetylase inhibitor - sodium butyrate (SB) - on osteosarcoma (OS) cell proliferation and apoptosis and also the molecular mechanisms by which SB exerts regulatory effects on OS cells. U2OS and MG63 cells were treated with SB at various concentrations. Then, cell proliferation and apoptosis were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and flow cytometry assays, respectively; the expression of Ki67, Bax, Bcl-2, MDM2, and p53 proteins was determined by using Western blot assay. The results showed that SB suppressed proliferation in a concentration-dependent manner and promoted apoptosis of OS cells. In addition, SB enhanced p53 expression and decreased MDM2 expression, indicating that SB can regulate MDM2-p53 feedback loop. p53 inhibited proliferation and promoted apoptosis, whereas MDM2 promoted proliferation and suppressed apoptosis, which indicated that functional effect of SB on OS cell lines at least in part depended on the MDM2-p53 signaling. We also explored the effect of SB on OS cells in vivo and found that SB suppressed the growth of OS cells with no noticeable effect on activity and body weight of mice in vivo. These findings will offer new clues for OS development and progression and offer SB as a potent targeted agent for OS treatment. PMID:27445491

  6. Hydrophobic Interactions Are a Key to MDM2 Inhibition by Polyphenols as Revealed by Molecular Dynamics Simulations and MM/PBSA Free Energy Calculations.

    PubMed

    Verma, Sharad; Grover, Sonam; Tyagi, Chetna; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-01-01

    p53, a tumor suppressor protein, has been proven to regulate the cell cycle, apoptosis, and DNA repair to prevent malignant transformation. MDM2 regulates activity of p53 and inhibits its binding to DNA. In the present study, we elucidated the MDM2 inhibition potential of polyphenols (Apigenin, Fisetin, Galangin and Luteolin) by MD simulation and MM/PBSA free energy calculations. All polyphenols bind to hydrophobic groove of MDM2 and the binding was found to be stable throughout MD simulation. Luteolin showed the highest negative binding free energy value of -173.80 kJ/mol followed by Fisetin with value of -172.25 kJ/mol. It was found by free energy calculations, that hydrophobic interactions (vdW energy) have major contribution in binding free energy.

  7. Hydrophobic Interactions Are a Key to MDM2 Inhibition by Polyphenols as Revealed by Molecular Dynamics Simulations and MM/PBSA Free Energy Calculations.

    PubMed

    Verma, Sharad; Grover, Sonam; Tyagi, Chetna; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-01-01

    p53, a tumor suppressor protein, has been proven to regulate the cell cycle, apoptosis, and DNA repair to prevent malignant transformation. MDM2 regulates activity of p53 and inhibits its binding to DNA. In the present study, we elucidated the MDM2 inhibition potential of polyphenols (Apigenin, Fisetin, Galangin and Luteolin) by MD simulation and MM/PBSA free energy calculations. All polyphenols bind to hydrophobic groove of MDM2 and the binding was found to be stable throughout MD simulation. Luteolin showed the highest negative binding free energy value of -173.80 kJ/mol followed by Fisetin with value of -172.25 kJ/mol. It was found by free energy calculations, that hydrophobic interactions (vdW energy) have major contribution in binding free energy. PMID:26863418

  8. Hydrophobic Interactions Are a Key to MDM2 Inhibition by Polyphenols as Revealed by Molecular Dynamics Simulations and MM/PBSA Free Energy Calculations

    PubMed Central

    Verma, Sharad; Grover, Sonam; Tyagi, Chetna; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-01-01

    p53, a tumor suppressor protein, has been proven to regulate the cell cycle, apoptosis, and DNA repair to prevent malignant transformation. MDM2 regulates activity of p53 and inhibits its binding to DNA. In the present study, we elucidated the MDM2 inhibition potential of polyphenols (Apigenin, Fisetin, Galangin and Luteolin) by MD simulation and MM/PBSA free energy calculations. All polyphenols bind to hydrophobic groove of MDM2 and the binding was found to be stable throughout MD simulation. Luteolin showed the highest negative binding free energy value of -173.80 kJ/mol followed by Fisetin with value of -172.25 kJ/mol. It was found by free energy calculations, that hydrophobic interactions (vdW energy) have major contribution in binding free energy. PMID:26863418

  9. Stress-Induced Isoforms of MDM2 and MDM4 Correlate with High-Grade Disease and an Altered Splicing Network in Pediatric Rhabdomyosarcoma12

    PubMed Central

    Jacob, Aishwarya G; O'Brien, Dennis; Singh, Ravi K; Comiskey, Daniel F; Littleton, Robert M; Mohammad, Fuad; Gladman, Jordan T; Widmann, Maria C; Jeyaraj, Selvi C; Bolinger, Cheryl; Anderson, James R; Barkauskas, Donald A; Boris-Lawrie, Kathleen; Chandler, Dawn S

    2013-01-01

    Pediatric rhabdomyosarcoma (RMS) is a morphologically and genetically heterogeneous malignancy commonly classified into three histologic subtypes, namely, alveolar, embryonal, and anaplastic. An issue that continues to challenge effective RMS patient prognosis is the dearth of molecular markers predictive of disease stage irrespective of tumor subtype. Our study involving a panel of 70 RMS tumors has identified specific alternative splice variants of the oncogenes Murine Double Minute 2 (MDM2) and MDM4 as potential biomarkers for RMS. Our results have demonstrated the strong association of genotoxic-stress inducible splice forms MDM2-ALT1 (91.6% Intergroup Rhabdomyosarcoma Study Group stage 4 tumors) and MDM4-ALT2 (90.9% MDM4-ALT2-positive T2 stage tumors) with high-risk metastatic RMS. Moreover, MDM2-ALT1-positive metastatic tumors belonged to both the alveolar (50%) and embryonal (41.6%) subtypes, making this the first known molecular marker for high-grade metastatic disease across the most common RMS subtypes. Furthermore, our results show that MDM2-ALT1 expression can function by directly contribute to metastatic behavior and promote the invasion of RMS cells through a matrigel-coated membrane. Additionally, expression of both MDM2-ALT1 and MDM4-ALT2 increased anchorage-independent cell-growth in soft agar assays. Intriguingly, we observed a unique coordination in the splicing of MDM2-ALT1 and MDM4-ALT2 in approximately 24% of tumor samples in a manner similar to genotoxic stress response in cell lines. To further explore splicing network alterations with possible relevance to RMS disease, we used an exon microarray approach to examine stress-inducible splicing in an RMS cell line (Rh30) and observed striking parallels between stress-responsive alternative splicing and constitutive splicing in RMS tumors. PMID:24027430

  10. MDM2 promoter SNP55 (rs2870820) affects risk of colon cancer but not breast-, lung-, or prostate cancer.

    PubMed

    Helwa, Reham; Gansmo, Liv B; Romundstad, Pål; Hveem, Kristian; Vatten, Lars; Ryan, Bríd M; Harris, Curtis C; Lønning, Per E; Knappskog, Stian

    2016-01-01

    Two functional SNPs (SNP285G > C; rs117039649 and SNP309T > G; rs2279744) have previously been reported to modulate Sp1 transcription factor binding to the promoter of the proto-oncogene MDM2, and to influence cancer risk. Recently, a third SNP (SNP55C > T; rs2870820) was also reported to affect Sp1 binding and MDM2 transcription. In this large population based case-control study, we genotyped MDM2 SNP55 in 10,779 Caucasian individuals, previously genotyped for SNP309 and SNP285, including cases of colon (n = 1,524), lung (n = 1,323), breast (n = 1,709) and prostate cancer (n = 2,488) and 3,735 non-cancer controls, as well as 299 healthy African-Americans. Applying the dominant model, we found an elevated risk of colon cancer among individuals harbouring SNP55TT/CT genotypes compared to the SNP55CC genotype (OR = 1.15; 95% CI = 1.01-1.30). The risk was found to be highest for left-sided colon cancer (OR = 1.21; 95% CI = 1.00-1.45) and among females (OR = 1.32; 95% CI = 1.01-1.74). Assessing combined genotypes, we found the highest risk of colon cancer among individuals harbouring the SNP55TT or CT together with the SNP309TG genotype (OR = 1.21; 95% CI = 1.00-1.46). Supporting the conclusions from the risk estimates, we found colon cancer cases carrying the SNP55TT/CT genotypes to be diagnosed at younger age as compared to SNP55CC (p = 0.053), in particular among patients carrying the SNP309TG/TT genotypes (p = 0.009). PMID:27624283

  11. MDM2 promoter SNP55 (rs2870820) affects risk of colon cancer but not breast-, lung-, or prostate cancer

    PubMed Central

    Helwa, Reham; Gansmo, Liv B.; Romundstad, Pål; Hveem, Kristian; Vatten, Lars; Ryan, Bríd M.; Harris, Curtis C.; Lønning, Per E.; Knappskog, Stian

    2016-01-01

    Two functional SNPs (SNP285G > C; rs117039649 and SNP309T > G; rs2279744) have previously been reported to modulate Sp1 transcription factor binding to the promoter of the proto-oncogene MDM2, and to influence cancer risk. Recently, a third SNP (SNP55C > T; rs2870820) was also reported to affect Sp1 binding and MDM2 transcription. In this large population based case-control study, we genotyped MDM2 SNP55 in 10,779 Caucasian individuals, previously genotyped for SNP309 and SNP285, including cases of colon (n = 1,524), lung (n = 1,323), breast (n = 1,709) and prostate cancer (n = 2,488) and 3,735 non-cancer controls, as well as 299 healthy African-Americans. Applying the dominant model, we found an elevated risk of colon cancer among individuals harbouring SNP55TT/CT genotypes compared to the SNP55CC genotype (OR = 1.15; 95% CI = 1.01–1.30). The risk was found to be highest for left-sided colon cancer (OR = 1.21; 95% CI = 1.00–1.45) and among females (OR = 1.32; 95% CI = 1.01–1.74). Assessing combined genotypes, we found the highest risk of colon cancer among individuals harbouring the SNP55TT or CT together with the SNP309TG genotype (OR = 1.21; 95% CI = 1.00–1.46). Supporting the conclusions from the risk estimates, we found colon cancer cases carrying the SNP55TT/CT genotypes to be diagnosed at younger age as compared to SNP55CC (p = 0.053), in particular among patients carrying the SNP309TG/TT genotypes (p = 0.009). PMID:27624283

  12. The preparation of kits for labelling macromolecules of biological interest with technetium-99m.

    PubMed

    Zanelli, G D; Ellison, D; Barrowcliffe, M P

    1987-04-01

    A method has been developed for producing 'kits' for labelling porphyrins, dextrans, myoglobin and cytochromes with technetium-99m. The kits have a shelf life of at least 3 months and can be reconstituted by the simple addition of 99TcmO4 from a commercial generator diluted in a buffer. The most suitable buffer was found to be isotonic citrate/saline at pH 9.0 to 9.5. When the kits are prepared as described and reconstituted with the above buffer, labelling yields of more than 95% are routinely achieved and polymerization or stacking of the labelled compounds is prevented.

  13. AMPK Reverses the Mesenchymal Phenotype of Cancer Cells by Targeting the Akt-MDM2-Foxo3a Signaling Axis

    PubMed Central

    Chou, Chih-Chien; Lee, Kuen-Haur; Lai, I-Lu; Wang, Dasheng; Mo, Xiaokui; Kulp, Samuel K.; Shapiro, Charles L.; Chen, Ching-Shih

    2014-01-01

    In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacological activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis. PMID:24994714

  14. AMPK reverses the mesenchymal phenotype of cancer cells by targeting the Akt-MDM2-Foxo3a signaling axis.

    PubMed

    Chou, Chih-Chien; Lee, Kuen-Haur; Lai, I-Lu; Wang, Dasheng; Mo, Xiaokui; Kulp, Samuel K; Shapiro, Charles L; Chen, Ching-Shih

    2014-09-01

    In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacologic activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis.

  15. AMPK reverses the mesenchymal phenotype of cancer cells by targeting the Akt-MDM2-Foxo3a signaling axis.

    PubMed

    Chou, Chih-Chien; Lee, Kuen-Haur; Lai, I-Lu; Wang, Dasheng; Mo, Xiaokui; Kulp, Samuel K; Shapiro, Charles L; Chen, Ching-Shih

    2014-09-01

    In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacologic activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis. PMID:24994714

  16. Celecoxib Treatment Alters p53 and MDM2 Expression via COX-2 Crosstalk in A549 Cells.

    PubMed

    Gharghabi, Mehdi; Rezaei, Farhang; Mir Mohammadrezaei, Fereshteh; Ghahremani, Mohammad Hossein

    2016-01-01

    Cyclooxygenase-2 (COX-2) has a pivotal role in the pathogenesis of the lung cancer. It is known that COX-2 negatively regulates the activity of a number of tumor suppressors, including p53. Consequently, inhibition of COX-2 signaling is anticipated to be a promising approach to stabilize p53 functionality. In this regard, we investigated the effect of COX-2 signaling blockade on p53 and COX-2expression in A549 cells. Cell viability was assessed using MTT and protein expression was measured using Western Blot assay. Results revealed that Celecoxib dose-dependently induced growth inhibition within 24 h. However, prolonged exposure to the drug up to 48 h led to increase cell viability compared to the corresponding control. Western blot analysis demonstrated that Celecoxib could augment p53 expression within 24 h, independently of COX-2 inhibition. In contrast, Celecoxib treatment not only returned p53 to the control level, but also strikingly induced COX-2 expression within 48 h. Of further relevance, Celecoxib exposure could significantly result in MDM2 elevation at 48 h. These findings represent p53 as a molecular target being interconnected with COX-2 signaling axis upon Celecoxib treatment. Moreover, our data point toward the possibility that Celecoxib treatment may not be a proper therapeutic strategy in lung cancer cells owing to its potential role in the activation of oncogenes, including COX-2 and MDM2 which seemingly confers a chemoresistance circumstance to the cell. Consequently, these results underscore intensive preclinical assessment prior to applying COX-2 inhibitors in the treatment of lung tumors. PMID:27642319

  17. Associations between the MDM2 promoter P1 polymorphism del1518 (rs3730485) and incidence of cancer of the breast, lung, colon and prostate

    PubMed Central

    Gansmo, Liv B.; Vatten, Lars; Romundstad, Pål; Hveem, Kristian; Ryan, Bríd M.; Harris, Curtis C.; Knappskog, Stian; Lønning, Per E.

    2016-01-01

    The MDM2 promoter region contains several polymorphisms, some of which have been associated with MDM2 expression, cancer risk and age at cancer onset. del1518 (rs3730485) is an indel polymorphism residing in the MDM2 promoter P1 and is in almost complete linkage disequilibrium with the MDM2 promoter P2 polymorphism SNP309T>G (rs2279744). Cancer risk assessments of del1518 have previously been conducted in relatively small Chinese populations only. In this study we assessed the genotype distribution of del1518 among healthy Caucasians, African Americans and Chinese, and we estimated the Odds Ratios (OR) for incident cancer of the breast, colon, lung and prostate (n=7,081) as compared to controls (n=3,749) in a large Caucasian (Norwegian) cohort. We found the genotypes of the del1518 to vary significantly between healthy Caucasians, African-Americans and Chinese (p< 1×10−5). Further, we found a positive association of the del1518 del-allele with risk of colon cancer (dominant model: OR = 1.15; 95 % CI = 1.01 – 1.31). Stratifying according to SNP309 status, this association remained among carriers of the SNP309TG genotype (OR = 1.21; 95 % CI = 1.01 – 1.46), but with no clear association among carriers of the SNP309TT genotype. In conclusion, our findings suggest del1518 to be associated with increased risk of colon cancer. PMID:27081698

  18. The MDM2–p53–pyruvate carboxylase signalling axis couples mitochondrial metabolism to glucose-stimulated insulin secretion in pancreatic β-cells

    PubMed Central

    Li, Xiaomu; Cheng, Kenneth K. Y.; Liu, Zhuohao; Yang, Jin-Kui; Wang, Baile; Jiang, Xue; Zhou, Yawen; Hallenborg, Philip; Hoo, Ruby L. C.; Lam, Karen S. L.; Ikeda, Yasuhiro; Gao, Xin; Xu, Aimin

    2016-01-01

    Mitochondrial metabolism is pivotal for glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. However, little is known about the molecular machinery that controls the homeostasis of intermediary metabolites in mitochondria. Here we show that the activation of p53 in β-cells, by genetic deletion or pharmacological inhibition of its negative regulator MDM2, impairs GSIS, leading to glucose intolerance in mice. Mechanistically, p53 activation represses the expression of the mitochondrial enzyme pyruvate carboxylase (PC), resulting in diminished production of the TCA cycle intermediates oxaloacetate and NADPH, and impaired oxygen consumption. The defective GSIS and mitochondrial metabolism in MDM2-null islets can be rescued by restoring PC expression. Under diabetogenic conditions, MDM2 and p53 are upregulated, whereas PC is reduced in mouse β-cells. Pharmacological inhibition of p53 alleviates defective GSIS in diabetic islets by restoring PC expression. Thus, the MDM2–p53–PC signalling axis links mitochondrial metabolism to insulin secretion and glucose homeostasis, and could represent a therapeutic target in diabetes. PMID:27265727

  19. The expression of MDM2/CDK4 gene product in the differential diagnosis of well differentiated liposarcoma and large deep-seated lipoma

    PubMed Central

    Pilotti, S; Torre, G Della; Mezzelani, A; Tamborini, E; Azzarelli, A; Sozzi, G; Pierotti, M A

    2000-01-01

    Ordinary lipomas are cytogenetically characterized by a variety of balanced rearrangements involving chromosome segment 12q13–15, whereas well differentiated liposarcomas (WDL) show supernumerary ring and giant marker chromosomes, known to contain amplified 12q sequences. The tight correlation between the presence of ring chromosomes and both amplification and overexpression of MDM2 and CDK4 genes suggests the exploration of the possibility that immunocytochemistry (ICC) might assist in the differential diagnosis of lipoma-like well differentiated liposarcomas (LL-WDL) and large deep-seated lipomas (LDSL). For this purpose, 21 cases of the former and 19 cases of the latter tumours were analysed by ICC and, according to the availability of material, by molecular and cytogenetic approaches. All lipomas displayed a null MDM2/CDK4 phenotype, whereas all LL-WDL showed MDM2/CDK4 or CDK4 phenotypes. Southern blot analysis performed on 16 suitable cases, complemented by fluorescence in situ hybridization and classical cytogenetic analysis in 11 cases, was consistent with, and further supported the immunophenotyping data. In conclusion, MDM2/CDK4 product-based immunophenotyping appears to represent a valuable method for the categorization of arguable LDSL. © 2000 Cancer Research Campaign PMID:10755400

  20. Non-linear feedback control of the p53 protein-mdm2 inhibitor system using the derivative-free non-linear Kalman filter.

    PubMed

    Rigatos, Gerasimos G

    2016-06-01

    It is proven that the model of the p53-mdm2 protein synthesis loop is a differentially flat one and using a diffeomorphism (change of state variables) that is proposed by differential flatness theory it is shown that the protein synthesis model can be transformed into the canonical (Brunovsky) form. This enables the design of a feedback control law that maintains the concentration of the p53 protein at the desirable levels. To estimate the non-measurable elements of the state vector describing the p53-mdm2 system dynamics, the derivative-free non-linear Kalman filter is used. Moreover, to compensate for modelling uncertainties and external disturbances that affect the p53-mdm2 system, the derivative-free non-linear Kalman filter is re-designed as a disturbance observer. The derivative-free non-linear Kalman filter consists of the Kalman filter recursion applied on the linearised equivalent of the protein synthesis model together with an inverse transformation based on differential flatness theory that enables to retrieve estimates for the state variables of the initial non-linear model. The proposed non-linear feedback control and perturbations compensation method for the p53-mdm2 system can result in more efficient chemotherapy schemes where the infusion of medication will be better administered. PMID:27187988

  1. Non-linear feedback control of the p53 protein-mdm2 inhibitor system using the derivative-free non-linear Kalman filter.

    PubMed

    Rigatos, Gerasimos G

    2016-06-01

    It is proven that the model of the p53-mdm2 protein synthesis loop is a differentially flat one and using a diffeomorphism (change of state variables) that is proposed by differential flatness theory it is shown that the protein synthesis model can be transformed into the canonical (Brunovsky) form. This enables the design of a feedback control law that maintains the concentration of the p53 protein at the desirable levels. To estimate the non-measurable elements of the state vector describing the p53-mdm2 system dynamics, the derivative-free non-linear Kalman filter is used. Moreover, to compensate for modelling uncertainties and external disturbances that affect the p53-mdm2 system, the derivative-free non-linear Kalman filter is re-designed as a disturbance observer. The derivative-free non-linear Kalman filter consists of the Kalman filter recursion applied on the linearised equivalent of the protein synthesis model together with an inverse transformation based on differential flatness theory that enables to retrieve estimates for the state variables of the initial non-linear model. The proposed non-linear feedback control and perturbations compensation method for the p53-mdm2 system can result in more efficient chemotherapy schemes where the infusion of medication will be better administered.

  2. Functional inactivation of endogenous MDM2 and CHIP by HSP90 causes aberrant stabilization of mutant p53 in human cancer cells.

    PubMed

    Li, Dun; Marchenko, Natalia D; Schulz, Ramona; Fischer, Victoria; Velasco-Hernandez, Talia; Talos, Flaminia; Moll, Ute M

    2011-05-01

    The tight control of wild-type p53 by mainly MDM2 in normal cells is permanently lost in tumors harboring mutant p53, which exhibit dramatic constitutive p53 hyperstabilization that far exceeds that of wild-type p53 tumors. Importantly, mutant p53 hyperstabilization is critical for oncogenic gain of function of mutant p53 in vivo. Current insight into the mechanism of this dysregulation is fragmentary and largely derived from ectopically constructed cell systems. Importantly, mutant p53 knock-in mice established that normal mutant p53 tissues have sufficient enzymatic reserves in MDM2 and other E3 ligases to maintain full control of mutant p53. We find that in human cancer cells, endogenous mutant p53, despite its ability to interact with MDM2, suffers from a profound lack of ubiquitination as the root of its degradation defect. In contrast to wild-type p53, the many mutant p53 proteins which are conformationally aberrant are engaged in complexes with the HSP90 chaperone machinery to prevent its aggregation. In contrast to wild-type p53 cancer cells, we show that in mutant p53 cancer cells, this HSP90 interaction blocks the endogenous MDM2 and CHIP (carboxy-terminus of Hsp70-interacting protein) E3 ligase activity. Interference with HSP90 either by RNA interference against HSF1, the transcriptional regulator of the HSP90 pathway, or by direct knockdown of Hsp90 protein or by pharmacologic inhibition of Hsp90 activity with 17AAG (17-allylamino-17-demethoxygeldanamycin) destroys the complex, liberates mutant p53, and reactivates endogenous MDM2 and CHIP to degrade mutant p53. Of note, 17AAG induces a stronger viability loss in mutant p53 than in wild-type p53 cancer cells. Our data support the rationale that suppression of mutant p53 levels in vivo in established cancers might achieve clinically significant effects.

  3. Differential response between the p53 ubiquitin-protein ligases Pirh2 and MdM2 following DNA damage in human cancer cells

    SciTech Connect

    Duan Wenrui; Gao, Li; Wu Xin; Zhang Yang; Otterson, Gregory A.; Villalona-Calero, Miguel A. . E-mail: Miguel.villalona@osumc.edu

    2006-10-15

    Pirh2, a recently identified ubiquitin-protein ligase, has been reported to promote p53 degradation. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of MDM2. Like MDM2, Pirh2 is thought to participate in an autoregulatory feedback loop that controls p53 function. We have previously reported that Pirh2 was overexpressed in human and murine lung cancers as compared to uninvolved lung tissue. Pirh2 increase could potentially cause degradation of wildtype p53 and reduce its tumor suppression function in the lung tumor cells. Since Pirh2 has been reported to be transactivated by p53, however, the mechanisms by which a high level of Pirh2 expression is maintained in tumor cells despite low level of wildtype p53 protein are unclear. In order to evaluate p53 involvement in the transactivation of Pirh2, we evaluated Pirh2, MDM2, p53 and p21 expression with Western blot analysis and real time PCR after {gamma} irradiation or cisplatin DNA damage treatment using human cancer cell lines containing wildtype (A549, MCF-7), mutant (H719) and null (H1299) p53. Surprisingly, Pirh2 expression was not affected by the presence of wildtype p53 in the cancer cells. In contrast, MDM2 was upregulated by wildtype p53 in A549 and MCF-7 cells and was absent from the H1299 and the H719 cells. We conclude that Pirh2 operates in a distinct manner from MDM2 in response to DNA damage in cancer cells. Pirh2 elevation in p53 null cells indicates the existence of additional molecular mechanisms for Pirh2 upregulation and suggests that p53 is not the sole target of Pirh2 ubiquitin ligase activity.

  4. A Functional Polymorphism (rs937283) in the MDM2 Promoter Region is Associated with Poor Prognosis of Retinoblastoma in Chinese Han Population.

    PubMed

    Jiao, Yongfa; Jiang, Zhongming; Wu, Yuxia; Chen, Xiaochong; Xiao, Xing; Yu, Haiying

    2016-01-01

    The effect of single nucleotide polymorphisms (SNPs) at MDM2 has been investigated in several cancer types. Three MDM2 SNPs(rs937283, rs2270744 and rs769412) have previously been suggested to be positively correlated with cancer. In this study, we aimed to explore the association of rs937283, rs2270744 and rs769412 polymorphisms with retinoblastoma (RB) risk, clinicopathological characteristics, and prognosis. Compared with wild-type genotype AA at rs937283, individuals carrying AG and GG genotype had a significantly increased risk for developing RB (OR = 1.86, 95% CI 1.13-3.08; OR = 2.48, 95% CI 1.10-5.62, respectively). RB patients with allele G at rs937283 were more susceptible to invasion and high tumor aggression (OR = 2.42, 95% CI 1.43-4.11; OR = 2.15, 95% CI 1.27-3.64, respectively). Kaplan-Meier curves and log-rank results revealed that RB patients harboring genotype GG and G allele at rs937283 had worse survival (P < 0.02 and P < 0.01, respectively). In addition, the A to G substitution at rs937283 significantly enhanced the transcription activity of the MDM2 gene in vitro. In vivo, we found that MDM2 mRNA and protein were overexpressed in individuals who carried the G allele at rs937283. This study suggested that the MDM2 rs937283 polymorphism is a novel functional SNP both in vitro and in vivo as well as a biomarker for poor prognosis in RB. PMID:27506496

  5. A Functional Polymorphism (rs937283) in the MDM2 Promoter Region is Associated with Poor Prognosis of Retinoblastoma in Chinese Han Population

    PubMed Central

    Jiao, Yongfa; Jiang, Zhongming; Wu, Yuxia; Chen, Xiaochong; Xiao, Xing; Yu, Haiying

    2016-01-01

    The effect of single nucleotide polymorphisms (SNPs) at MDM2 has been investigated in several cancer types. Three MDM2 SNPs(rs937283, rs2270744 and rs769412) have previously been suggested to be positively correlated with cancer. In this study, we aimed to explore the association of rs937283, rs2270744 and rs769412 polymorphisms with retinoblastoma (RB) risk, clinicopathological characteristics, and prognosis. Compared with wild-type genotype AA at rs937283, individuals carrying AG and GG genotype had a significantly increased risk for developing RB (OR = 1.86, 95% CI 1.13–3.08; OR = 2.48, 95% CI 1.10–5.62, respectively). RB patients with allele G at rs937283 were more susceptible to invasion and high tumor aggression (OR = 2.42, 95% CI 1.43–4.11; OR = 2.15, 95% CI 1.27–3.64, respectively). Kaplan-Meier curves and log-rank results revealed that RB patients harboring genotype GG and G allele at rs937283 had worse survival (P < 0.02 and P < 0.01, respectively). In addition, the A to G substitution at rs937283 significantly enhanced the transcription activity of the MDM2 gene in vitro. In vivo, we found that MDM2 mRNA and protein were overexpressed in individuals who carried the G allele at rs937283. This study suggested that the MDM2 rs937283 polymorphism is a novel functional SNP both in vitro and in vivo as well as a biomarker for poor prognosis in RB. PMID:27506496

  6. Numerical and Experimental Analysis of the p53-mdm2 Regulatory Pathway

    NASA Astrophysics Data System (ADS)

    van Leeuwen, Ingeborg M. M.; Sanders, Ian; Staples, Oliver; Lain, Sonia; Munro, Alastair J.

    The p53 tumour suppressor plays key regulatory roles in various fundamental biological processes, including development, ageing and cell differentiation. It is therefore known as "the guardian of the genome" and is currently the most extensively studied protein worldwide. Besides members of the biomedical community, who view p53 as a promising target for novel anti-cancer therapies, the complex network of protein interactions modulating p53's activity has captivated the attention of theoreticians and modellers due to the possible occurrence of oscillations in protein levels in response to stress. This paper presents new insights into the behaviour of the p53 network, which we acquired by combining mathematical and experimental techniques. Notably, our data raises the question of whether the discrete p53 pulses in single cells, observed using fluorescent labelling, could in fact be an artefact. Furthermore, we propose a new model for the p53 pathway that is amenable to analysis by computational methods developed within the OPAALS project.

  7. The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

    PubMed

    Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J

    2016-08-15

    Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. PMID:27528756

  8. The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

    PubMed

    Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J

    2016-08-15

    Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease.

  9. Investigating the effect of autoinduction in cynomolgus monkeys of a novel anticancer MDM2 antagonist, idasanutlin, and relevance to humans.

    PubMed

    Glenn, Kelli J; Yu, Li J; Reddy, Micaela B; Fretland, Adrian J; Parrott, Neil; Hussain, Sazzad; Palacios, Mary; Vazvaei, Faye; Zhi, Jianguo; Tuerck, Dietrich

    2016-08-01

    1. Idasanutlin (RG7388) is a potent p53-MDM2 antagonist currently in clinical development for treatment of cancer. The purpose of the present studies was to investigate the cause of marked decrease in plasma exposure after repeated oral administration of RG7388 in monkeys and whether the autoinduction observed in monkeys is relevant to humans. 2. In monkey liver and intestinal microsomes collected after repeated oral administration of RG7388 to monkeys, significantly increased activities of homologue CYP3A8 were observed (ex vivo). Investigation using a physiologically based pharmacokinetic (PBPK) model suggested that the loss of exposure was primarily due to induction of metabolism in the gut of monkeys. 3. Studies in monkey and human primary hepatocytes showed that CYP3A induction by RG7388 only occurred in monkey hepatocytes but not in human hepatocytes, which suggests the observed CYP3A induction is monkey specific. 4. The human PK data obtained from the first cohorts confirmed the lack of relevant induction as predicted by the human hepatocytes and the PBPK modelling based on no induction in humans. PMID:26586447

  10. The importance of ribosome production, and the 5S RNP–MDM2 pathway, in health and disease

    PubMed Central

    Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J.

    2016-01-01

    Ribosomes are abundant, large RNA–protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. PMID:27528756

  11. Whole genome sequence analysis links chromothripsis to EGFR, MDM2, MDM4, and CDK4 amplification in glioblastoma

    PubMed Central

    Furgason, John M.; Koncar, Robert F.; Michelhaugh, Sharon K.; Sarkar, Fazlul H.; Mittal, Sandeep; Sloan, Andrew E.; Barnholtz-Sloan, Jill S.; Bahassi, El Mustapha

    2015-01-01

    Background Findings based on recent advances in next-generation sequence analysis suggest that, in some tumors, a single catastrophic event, termed chromothripsis, results in several simultaneous tumorigenic alterations. Previous studies have suggested that glioblastoma (GBM) may exhibit chromothripsis at a higher rate (39%) than other tumors (9%). Primary glioblastoma is an aggressive form of brain cancer that typically appears suddenly in older adults. With aggressive treatment, the median survival time is only 15 months. Their acute onset and widespread genomic instability indicates that chromothripsis may play a key role in their initiation and progression. GBMs are often characterized by EGFR amplification, CDKN2A and PTEN deletion, although approximately 20% of GBMs harbor additional amplifications in MDM2 or MDM4 with CDK4. Methods We used the chromothripsis prediction tool, Shatterproof, in conjunction with a custom whole genome sequence analysis pipeline in order to generate putative regions of chromothripsis. The data derived from this study was further expanded on using fluorescence in situ hybridization (FISH) analysis and susceptibility studies with colony formation assays. Results We show that primary GBMs are associated with higher chromothripsis scores and establish a link between chromothripsis and gene amplification of receptor tyrosine kinases (RTKs), as well as modulators of the TP53 and RB1 pathways. Conclusions Utilizing a newly introduced bioinformatic tool, we provide evidence that chromothripsis is associated with the formation of amplicons containing several oncogenes involved in key pathways that are likely essential for post-chromothriptic cell survival. PMID:26328271

  12. Synergistic roles of p53 and HIF1α in human renal cell carcinoma-cell apoptosis responding to the inhibition of mTOR and MDM2 signaling pathways

    PubMed Central

    Liu, Qing-jun; Shen, Hong-liang; Lin, Jun; Xu, Xiu-hong; Ji, Zheng-guo; Han, Xiao; Shang, Dong-hao; Yang, Pei-qian

    2016-01-01

    Introduction mTOR and MDM2 signaling pathways are frequently deregulated in cancer development, and inhibition of mTOR or MDM2 independently enhances carcinoma-cell apoptosis. However, responses to mTOR and MDM2 antagonists in renal cell carcinoma (RCC) remain unknown. Materials and methods A498 cells treated with MDM2 antagonist MI-319 and/or mTOR inhibitor rapamycin were employed in the present study. Cell apoptosis and Western blot analysis were performed. Results and conclusion We found that the MDM2 inhibitor MI-319 induced RCC cell apoptosis mainly dependent on p53 overexpression, while the mTOR antagonist rapamycin promoted RCC cell apoptosis primarily through upregulation of HIF1α expression. Importantly, strong synergistic effects of MI-319 and rapamycin combinations at relatively low concentrations on RCC cell apoptosis were observed. Depletion of p53 or HIF1α impaired both antagonist-elicited apoptoses to differential extents, corresponding to their expression changes responding to chemical treatments, and double knockdown of p53 and HIF1α remarkably hindered MI-319- or rapamycin-induced apoptosis, suggesting that both p53 and HIF1α are involved in MDM2 or mTOR antagonist-induced apoptosis. Collectively, we propose that concurrent activation of p53 and HIF1α may effectively result in cancer-cell apoptosis, and that combined MDM2 antagonists and mTOR inhibitors may be useful in RCC therapy. PMID:26937175

  13. In vitro cytotoxicity and in vivo efficacy, pharmacokinetics and metabolism of pyrazole-based small-molecule inhibitors of Mdm2/4-p53 interaction

    PubMed Central

    Christner, Susan M.; Clausen, Dana M.; Beumer, Jan H.; Parise, Robert A.; Guo, Jianxia; Huang, Yijun; Dömling, Alexander S; Eiseman, Julie L.

    2016-01-01

    Purpose The interaction of p53, with its negative regulators Mdm2/4 has been widely studied [1]. In p53+/+ cells, expression of Mdm2/4 leads to p53 turnover, inhibition of downstream transcription, decreasing cell cycle arrest or apoptosis. We report in vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of YH264, YH263 and WW751, three proposed small molecule inhibitors of the Mdm2/4-p53 interaction. Methods MTT cytotoxicity assays were performed and alterations in proteins were examined using Western blots. Mice were dosed 150 mg/kg YH264 or YH263 iv or po QDx5. Mice were iv dosed 88 mg/kg, 57 mg/kg, or 39 mg/kg WW751 for three, five, or five days. YH264, YH263 and WW751 and metabolites were quantitated by LC-MS/MS. Results IC50 values for YH264, YH263 and WW751 against p53 wild type HCT 116 cells after 72 h of incubation were 18.3 ± 2.3 μM, 8.9 ± 0.6 μM, and 3.1 ± 0.2 μM respectively. Only YH264 appeared to affect p53 expression in vitro. None of the compounds affected the growth of HCT 116 xenografts in C.B-17 SCID mice. YH264 plasma half-life was 147 min; YH263 plasma half-life was 263 min; and WW751 plasma half-life was less than 120 min. Conclusions Despite dosing the mice at the maximum soluble doses, we could not achieve tumor concentrations equivalent to the intracellular concentrations required to inhibit cell growth in vitro. YH263 and WW751 do not appear to affect p53/Mdm2 and none of the three were active in a subcutaneous HCT116 p53+/+ xenograft model. PMID:26050209

  14. The use of ion mobility mass spectrometry to probe modulation of the structure of p53 and of MDM2 by small molecule inhibitors

    PubMed Central

    Dickinson, Eleanor R.; Jurneczko, Ewa; Nicholson, Judith; Hupp, Ted R.; Zawacka-Pankau, Joanna; Selivanova, Galina; Barran, Perdita E.

    2015-01-01

    Developing drug-like molecules to inhibit the interactions formed by disordered proteins is desirable due to the high correlation of disorder with protein implicated in disease, but is challenging due in part to the lack of atomistically resolved and resolvable structures from conformationally dynamic systems. Ion mobility mass spectrometry (IM-MS) is well-positioned to assess protein ligand interactions along with the effect of a given inhibitor on conformation. Here we demonstrate the use of IM-MS to characterize the effect of two inhibitors RITA and Nutlin-3 on their respective binding partners: p53 and MDM2. RITA binds N-terminal transactivation domain of p53 (Np53) weakly, preventing direct observation of the complex in the gas phase. Nonetheless, upon incubation with RITA, we observe an alteration in the charge state distribution and in the conformational distributions adopted by Np53 in the gas phase. This finding supports the hypothesis that RITAs mode of action proceeds via a conformational change in p53. Circular dichroism corroborates our gas phase findings, showing a slight increase in secondary structure content on ligand incubation, and HDX-MS experiments also highlight the dynamic properties of this protein. Using the same approach we present data to show the effect of Nutlin-3 binding to the N-terminal domain of MDM2 (N-MDM2), N-MDM2 presents as at least two conformational families in the absence of Nutlin-3. Upon Nutlin-3 binding, the protein undergoes a compaction event similar to that exhibited by RITA on Np53. This multi-technique approach highlights the inherent disorder in these systems; and in particular exemplifies the power of IM-MS as a technique to study transient interactions between small molecule inhibitors and intrinsically disordered proteins. PMID:26217671

  15. Markov models of the apo-MDM2 lid region reveal diffuse yet two-state binding dynamics and receptor poses for computational docking

    NASA Astrophysics Data System (ADS)

    Mukherjee, Sudipto; Pantelopulos, George A.; Voelz, Vincent A.

    2016-08-01

    MDM2 is a negative regulator of p53 activity and an important target for cancer therapeutics. The N-terminal lid region of MDM2 modulates interactions with p53 via competition for its binding cleft, exchanging slowly between docked and undocked conformations in the absence of p53. To better understand these dynamics, we constructed Markov State Models (MSMs) from large collections of unbiased simulation trajectories of apo-MDM2, and find strong evidence for diffuse, yet two-state folding and binding of the N-terminal region to the p53 receptor site. The MSM also identifies holo-like receptor conformations highly suitable for computational docking, despite initiating trajectories from closed-cleft receptor structures unsuitable for docking. Fixed-anchor docking studies using a test set of high-affinity small molecules and peptides show simulated receptor ensembles achieve docking successes comparable to cross-docking studies using crystal structures of receptors bound by alternative ligands. For p53, the best-scoring receptor structures have the N-terminal region lid region bound in a helical conformation mimicking the bound structure of p53, suggesting lid region association induces receptor conformations suitable for binding. These results suggest that MD + MSM approaches can sample binding-competent receptor conformations suitable for computational peptidomimetic design, and that inclusion of disordered regions may be essential to capturing the correct receptor dynamics.

  16. Influence of the nuclear membrane, active transport, and cell shape on the Hes1 and p53-Mdm2 pathways: insights from spatio-temporal modelling.

    PubMed

    Sturrock, Marc; Terry, Alan J; Xirodimas, Dimitris P; Thompson, Alastair M; Chaplain, Mark A J

    2012-07-01

    There are many intracellular signalling pathways where the spatial distribution of the molecular species cannot be neglected. These pathways often contain negative feedback loops and can exhibit oscillatory dynamics in space and time. Two such pathways are those involving Hes1 and p53-Mdm2, both of which are implicated in cancer. In this paper we further develop the partial differential equation (PDE) models of Sturrock et al. (J. Theor. Biol., 273:15-31, 2011) which were used to study these dynamics. We extend these PDE models by including a nuclear membrane and active transport, assuming that proteins are convected in the cytoplasm towards the nucleus in order to model transport along microtubules. We also account for Mdm2 inhibition of p53 transcriptional activity. Through numerical simulations we find ranges of values for the model parameters such that sustained oscillatory dynamics occur, consistent with available experimental measurements. We also find that our model extensions act to broaden the parameter ranges that yield oscillations. Hence oscillatory behaviour is made more robust by the inclusion of both the nuclear membrane and active transport. In order to bridge the gap between in vivo and in silico experiments, we investigate more realistic cell geometries by using an imported image of a real cell as our computational domain. For the extended p53-Mdm2 model, we consider the effect of microtubule-disrupting drugs and proteasome inhibitor drugs, obtaining results that are in agreement with experimental studies.

  17. p21-Activated kinase 6 (PAK6) inhibits prostate cancer growth via phosphorylation of androgen receptor and tumorigenic E3 ligase murine double minute-2 (Mdm2).

    PubMed

    Liu, Tong; Li, Yang; Gu, Hui; Zhu, Ge; Li, Jiabin; Cao, Liu; Li, Feng

    2013-02-01

    The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.

  18. Markov models of the apo-MDM2 lid region reveal diffuse yet two-state binding dynamics and receptor poses for computational docking

    PubMed Central

    Mukherjee, Sudipto; Pantelopulos, George A.; Voelz, Vincent A.

    2016-01-01

    MDM2 is a negative regulator of p53 activity and an important target for cancer therapeutics. The N-terminal lid region of MDM2 modulates interactions with p53 via competition for its binding cleft, exchanging slowly between docked and undocked conformations in the absence of p53. To better understand these dynamics, we constructed Markov State Models (MSMs) from large collections of unbiased simulation trajectories of apo-MDM2, and find strong evidence for diffuse, yet two-state folding and binding of the N-terminal region to the p53 receptor site. The MSM also identifies holo-like receptor conformations highly suitable for computational docking, despite initiating trajectories from closed-cleft receptor structures unsuitable for docking. Fixed-anchor docking studies using a test set of high-affinity small molecules and peptides show simulated receptor ensembles achieve docking successes comparable to cross-docking studies using crystal structures of receptors bound by alternative ligands. For p53, the best-scoring receptor structures have the N-terminal region lid region bound in a helical conformation mimicking the bound structure of p53, suggesting lid region association induces receptor conformations suitable for binding. These results suggest that MD + MSM approaches can sample binding-competent receptor conformations suitable for computational peptidomimetic design, and that inclusion of disordered regions may be essential to capturing the correct receptor dynamics. PMID:27538695

  19. Markov models of the apo-MDM2 lid region reveal diffuse yet two-state binding dynamics and receptor poses for computational docking.

    PubMed

    Mukherjee, Sudipto; Pantelopulos, George A; Voelz, Vincent A

    2016-01-01

    MDM2 is a negative regulator of p53 activity and an important target for cancer therapeutics. The N-terminal lid region of MDM2 modulates interactions with p53 via competition for its binding cleft, exchanging slowly between docked and undocked conformations in the absence of p53. To better understand these dynamics, we constructed Markov State Models (MSMs) from large collections of unbiased simulation trajectories of apo-MDM2, and find strong evidence for diffuse, yet two-state folding and binding of the N-terminal region to the p53 receptor site. The MSM also identifies holo-like receptor conformations highly suitable for computational docking, despite initiating trajectories from closed-cleft receptor structures unsuitable for docking. Fixed-anchor docking studies using a test set of high-affinity small molecules and peptides show simulated receptor ensembles achieve docking successes comparable to cross-docking studies using crystal structures of receptors bound by alternative ligands. For p53, the best-scoring receptor structures have the N-terminal region lid region bound in a helical conformation mimicking the bound structure of p53, suggesting lid region association induces receptor conformations suitable for binding. These results suggest that MD + MSM approaches can sample binding-competent receptor conformations suitable for computational peptidomimetic design, and that inclusion of disordered regions may be essential to capturing the correct receptor dynamics. PMID:27538695

  20. mTOR inhibitors blunt the p53 response to nucleolar stress by regulating RPL11 and MDM2 levels.

    PubMed

    Goudarzi, Kaveh M; Nistér, Monica; Lindström, Mikael S

    2014-01-01

    Mechanistic target of rapamycin (mTOR) is a master regulator of cell growth through its ability to stimulate ribosome biogenesis and mRNA translation. In contrast, the p53 tumor suppressor negatively controls cell growth and is activated by a wide range of insults to the cell. The mTOR and p53 signaling pathways are connected by a number of different mechanisms. Chemotherapeutics that inhibit ribosome biogenesis often induce nucleolar stress and activation of p53. Here we have investigated how the p53 response to nucleolar stress is affected by simultaneous mTOR inhibition in osteosarcoma and glioma cell lines. We found that inhibitors of the mTOR pathway including rapamycin, wortmannin, and caffeine blunted the p53 response to nucleolar stress induced by actinomycin D. Synthetic inhibitors of mTOR (temsirolimus, LY294.002 and PP242) also impaired actinomycin D triggered p53 stabilization and induction of p21. Ribosomal protein (RPL11) is known to be required for p53 protein stabilization following nucleolar stress. Treatment of cells with mTOR inhibitors may lead to reduced synthesis of RPL11 and thereby destabilize p53. We found that rapamycin mimicked the effect of RPL11 depletion in terms of blunting the p53 response to nucleolar stress. However, the extent to which the levels of p53 and RPL11 were reduced by rapamycin varied between cell lines. Additional mechanisms whereby rapamycin blunts the p53 response to nucleolar stress are likely to be involved. Indeed, rapamycin increased the levels of endogenous MDM2 despite inhibition of its phosphorylation at Ser-166. Our findings may have implications for the design of combinatorial cancer treatments with mTOR pathway inhibitors.

  1. Pokemon enhances proliferation, cell cycle progression and anti-apoptosis activity of colorectal cancer independently of p14ARF-MDM2-p53 pathway.

    PubMed

    Zhao, Yi; Yao, Yun-hong; Li, Li; An, Wei-fang; Chen, Hong-zen; Sun, Li-ping; Kang, Hai-xian; Wang, Sen; Hu, Xin-rong

    2014-12-01

    Pokemon has been showed to directly suppress p14(ARF) expression and also to overexpress in multiple cancers. However, p14(ARF)-MDM2-p53 pathway is usually aberrant in colorectal cancer (CRC). The aim is to confirm whether Pokemon plays a role in CRC and explore whether Pokemon works through p14(ARF)-MDM2-p53 pathway in CRC. Immunohistochemistry for Pokemon, p14(ARF) and Mtp53 protein was applied to 45 colorectal epitheliums (CREs), 42 colorectal adenomas (CRAs) and 66 CRCs. Pokemon was knocked down with RNAi technique in CRC cell line Lovo to detect mRNA expression of p14(ARF) with qRT-PCR, cell proliferation with CCK8 assay, and cell cycle and apoptosis with flowcytometry analysis. The protein expression rates were significantly higher in CRC (75.8%) than in CRE (22.2 %) or CRA (38.1%) for Pokemon and higher in CRC (53.0%) than in CRE (0) or CRA (4.8%) for Mtp53, but not significantly different in CRC (86.4 %) versus CRE (93.3%) or CRA (90.5 %) for p14(ARF). Higher expression rate of Pokemon was associated with lymph node metastasis and higher Duke's stage. After knockdown of Pokemon in Lovo cells, the mRNA level of p14(ARF) was not significantly changed, the cell proliferation ability was decreased by 20.6%, cell cycle was arrested by 55.7% in G0/G1 phase, and apoptosis rate was increased by 19.0%. Pokemon enhanced the oncogenesis of CRC by promoting proliferation, cell cycle progression and anti-apoptosis activity of CRC cells independently of p14(ARF)-MDM2-p53 pathway. This finding provided a novel idea for understanding and further studying the molecular mechanism of Pokemon on carcinogenesis of CRC. PMID:25367850

  2. Pokemon enhances proliferation, cell cycle progression and anti-apoptosis activity of colorectal cancer independently of p14ARF-MDM2-p53 pathway.

    PubMed

    Zhao, Yi; Yao, Yun-hong; Li, Li; An, Wei-fang; Chen, Hong-zen; Sun, Li-ping; Kang, Hai-xian; Wang, Sen; Hu, Xin-rong

    2014-12-01

    Pokemon has been showed to directly suppress p14(ARF) expression and also to overexpress in multiple cancers. However, p14(ARF)-MDM2-p53 pathway is usually aberrant in colorectal cancer (CRC). The aim is to confirm whether Pokemon plays a role in CRC and explore whether Pokemon works through p14(ARF)-MDM2-p53 pathway in CRC. Immunohistochemistry for Pokemon, p14(ARF) and Mtp53 protein was applied to 45 colorectal epitheliums (CREs), 42 colorectal adenomas (CRAs) and 66 CRCs. Pokemon was knocked down with RNAi technique in CRC cell line Lovo to detect mRNA expression of p14(ARF) with qRT-PCR, cell proliferation with CCK8 assay, and cell cycle and apoptosis with flowcytometry analysis. The protein expression rates were significantly higher in CRC (75.8%) than in CRE (22.2 %) or CRA (38.1%) for Pokemon and higher in CRC (53.0%) than in CRE (0) or CRA (4.8%) for Mtp53, but not significantly different in CRC (86.4 %) versus CRE (93.3%) or CRA (90.5 %) for p14(ARF). Higher expression rate of Pokemon was associated with lymph node metastasis and higher Duke's stage. After knockdown of Pokemon in Lovo cells, the mRNA level of p14(ARF) was not significantly changed, the cell proliferation ability was decreased by 20.6%, cell cycle was arrested by 55.7% in G0/G1 phase, and apoptosis rate was increased by 19.0%. Pokemon enhanced the oncogenesis of CRC by promoting proliferation, cell cycle progression and anti-apoptosis activity of CRC cells independently of p14(ARF)-MDM2-p53 pathway. This finding provided a novel idea for understanding and further studying the molecular mechanism of Pokemon on carcinogenesis of CRC.

  3. Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway.

    PubMed

    Wu, Chien-Ting; Lin, Tung-Yi; Hsu, Hsien-Yeh; Sheu, Fuu; Ho, Chau-Mei; Chen, Edmund I-T

    2011-12-01

    Ling Zhi-8 (LZ-8), an immunomodulatory protein, is derived from and has been cloned from the medicinal mushroom Ganoderma lucidum (Reishi or Ling Zhi); this protein exhibits immunomodulating and antitumor properties. We investigated the effects of recombinant LZ-8 protein (rLZ-8) on the proliferation of A549 human lung cancer cells. Here, we showed that rLZ-8 inhibits cell growth and that this is correlated with increased G(1) arrest. The treatment of A549 cells with rLZ-8 activated p53 and p21 expression, and both the G(1) arrest and the antigrowth effect were found to be p53 dependent. It was further demonstrated that rLZ-8 inhibited tumor growth in mice transplanted with Lewis lung carcinoma cells. Interestingly, rLZ-8 treatment was found to lead to nucleolar stress (or ribosomal stress) as evidenced by inhibition of precursor ribosomal RNA synthesis and reduced polysome formation in A549 cells. These changes resulted in an increasing binding of ribosomal protein S7 to MDM2 and a decreased interaction between MDM2 and p53. Taking these results together, we have identified a novel rLZ-8 antitumor function that positively modulates p53 via ribosomal stress and inhibits lung cancer cell growth in vitro and in vivo. Our current results suggest that rLZ-8 may have potential as a therapeutic intervention for the treatment of cancers that contain wild-type p53 and high expression of MDM2.

  4. Dual inhibition of protein kinase C and p53-MDM2 or PKC and mTORC1 are novel efficient therapeutic approaches for uveal melanoma

    PubMed Central

    Dahmani, Ahmed; Raymondie, Chloé; Cassoux, Nathalie; Piperno-Neumann, Sophie; Némati, Fariba; Laurent, Cécile; De Koning, Leanne; Halilovic, Ensar; Jeay, Sebastien; Wylie, Andrew; Emery, Caroline; Roman-Roman, Sergio

    2016-01-01

    Uveal melanoma (UM) is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified. Novel therapeutic approaches are therefore urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway, leading to the use of PKC inhibitors as a rational strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models (PDXs), several PKC inhibitor-based combinations were tested in vivo using the PKC inhibitor AEB071. The most promising approaches were further investigated in vitro using our unique panel of UM cell lines. When combined with AEB071, the two agents CGM097 (p53-MDM2 inhibitor) and RAD001 (mTORC1 inhibitor) demonstrated greater activity than single agents, with tumor regression observed in several UM PDXs. Follow-up studies in UM cell lines on these two drug associations confirmed their combination activity and ability to induce cell death. While no effective treatment currently exists for metastatic uveal melanoma, we have discovered using our unique panel of preclinical models that combinations between PKC/mTOR inhibitors and PKC/p53-MDM2 inhibitors are two novel and very effective therapeutic approaches for this disease. Together, our study reveals that combining PKC and p53-MDM2 or mTORC1 inhibitors may provide significant clinical benefit for UM patients. PMID:27507190

  5. Genetic Variants in the p14ARF/MDM2/TP53 Pathway Are Associated with the Prognosis of Esophageal Squamous Cell Carcinoma Patients Treated with Radical Resection

    PubMed Central

    Li, Jing; Tang, Yang; Huang, Liu; Yu, Qianqian; Hu, Guangyuan; Yuan, Xianglin

    2016-01-01

    The p14ARF/MDM2/ TP53 pathway is known to play an important role in tumor progression by cell cycle control, although the association between this pathway and the prognosis of esophageal squamous cell carcinoma (ESCC) is unclear. In this study, we explored the association between genetic variants in the p14ARF/MDM2/TP53 pathway and prognosis in ESCC patients with radical resection. 124 ESCC patients with radical resection were included in this retrospective study and genotyped using the MassArray method. According to multivariate Cox hazard analysis and multiple testing, the TC/CC genotype of p14ARF rs3814960 was shown to be strongly related to a decreased overall survival (OS) (HR = 2.77, 95% CI: 1.33–5.75, P = 0.006, Pc = 0.030) and disease-free survival (DFS) (HR = 2.45, 95% CI: 1.30–4.61, P = 0.005, Pc = 0.025). Moreover, patients with the DEL/A +AA genotype of MDM2 rs34886328 had a notably increased OS (HR = 0.27, 95% CI: 0.13–0.56, P = 4.7×10−4, Pc = 0.003) and DFS (HR = 0.22, 95% CI: 0.11–0.43, P = 1.1×10−5, Pc = 6.6×10−5). We also found that these two SNPs had a cumulative effect on the prognosis of ESCC, with the OS (P < 0.001) and DFS (P < 0.001) being shortest for patients carrying both of these unfavorable genotypes. In conclusion, genetic variants of the p14ARF/MDM2/TP53 pathway are significantly related to OS and DFS, and may be predictors of the prognosis of ESCC after surgery. We speculate the individuals with the TC/CC genotype of p14ARF rs3814960 and/or the DEL/DEL genotype of MDMD2 rs34886328 should have more aggressive treatment and may greatly benefit from early prediction and prevention of an unfavorable prognosis by genotyping before the initiation of therapy. These findings should be further validated in a larger population. PMID:27414035

  6. Genetic Variants in the p14ARF/MDM2/TP53 Pathway Are Associated with the Prognosis of Esophageal Squamous Cell Carcinoma Patients Treated with Radical Resection.

    PubMed

    Li, Jing; Tang, Yang; Huang, Liu; Yu, Qianqian; Hu, Guangyuan; Yuan, Xianglin

    2016-01-01

    The p14ARF/MDM2/ TP53 pathway is known to play an important role in tumor progression by cell cycle control, although the association between this pathway and the prognosis of esophageal squamous cell carcinoma (ESCC) is unclear. In this study, we explored the association between genetic variants in the p14ARF/MDM2/TP53 pathway and prognosis in ESCC patients with radical resection. 124 ESCC patients with radical resection were included in this retrospective study and genotyped using the MassArray method. According to multivariate Cox hazard analysis and multiple testing, the TC/CC genotype of p14ARF rs3814960 was shown to be strongly related to a decreased overall survival (OS) (HR = 2.77, 95% CI: 1.33-5.75, P = 0.006, Pc = 0.030) and disease-free survival (DFS) (HR = 2.45, 95% CI: 1.30-4.61, P = 0.005, Pc = 0.025). Moreover, patients with the DEL/A +AA genotype of MDM2 rs34886328 had a notably increased OS (HR = 0.27, 95% CI: 0.13-0.56, P = 4.7×10-4, Pc = 0.003) and DFS (HR = 0.22, 95% CI: 0.11-0.43, P = 1.1×10-5, Pc = 6.6×10-5). We also found that these two SNPs had a cumulative effect on the prognosis of ESCC, with the OS (P < 0.001) and DFS (P < 0.001) being shortest for patients carrying both of these unfavorable genotypes. In conclusion, genetic variants of the p14ARF/MDM2/TP53 pathway are significantly related to OS and DFS, and may be predictors of the prognosis of ESCC after surgery. We speculate the individuals with the TC/CC genotype of p14ARF rs3814960 and/or the DEL/DEL genotype of MDMD2 rs34886328 should have more aggressive treatment and may greatly benefit from early prediction and prevention of an unfavorable prognosis by genotyping before the initiation of therapy. These findings should be further validated in a larger population.

  7. Follicular dendritic cell sarcoma: clinicopathologic study of 15 cases with emphasis on novel expression of MDM2, somatostatin receptor 2A, and PD-L1.

    PubMed

    Agaimy, Abbas; Michal, Michael; Hadravsky, Ladislav; Michal, Michal

    2016-08-01

    Follicular dendritic cell sarcoma (FDCS) is a rare low-grade neoplasm with the phenotype of FDC cells. This rare sarcoma has been well known for being mistaken for a variety of neoplasms (mainly meningioma), particularly at extranodal sites. Diagnosis of FDCS mainly relies on characteristic histologic appearance supplemented by immunohistochemistry and electron microscopy. In this study, we reviewed 15 FDCSs retrieved from our consultation files and stained them for newly reported or novel markers (PD-L1, Rb1, MDM2, and somatostatin receptor 2A [SSTR2A]) in addition to conventional FDC markers. Patients were 7 men and 7 women (1 unspecified) with a mean age of 47 years (20-75 years). The tumor site was lymph nodes (6) or spleen (2), both (1) and extranodal sites of head and neck (4) or abdominal cavity (2). Treatment was variable combinations of surgery and aggressive chemotherapy/radiotherapy. Four of 8 patients with follow-up died of disease within 1 to 10 years. All tumors expressed at least 1 FDC marker: CD21 (8/13), CD23 (2/13), CD35 (8/12), CNA.42 (13/14), Clusterin (8/13), Fascin (15/15) and D2-40/podoplanin (7/14). Epstein-Barr virus (EBER-1/2 in situ hybridization) was performed successfully in 10 conventional variants; all were negative. Five of 14 cases (36%) stained strongly for SSTR2A with a distinctive membranous pattern. Residual lymphoid follicles surrounding some of the tumors stained similarly for SSTR2A. Seven (54%) of 13 assessable cases showed moderate to strong membranous staining for PD-L1 in greater than 5% of the neoplastic cells. The Rb1 antigen was lost in 4 (28%) of 14 cases. MDM2 stained less than 5% to 20% of the tumor cells in 5 (36%) of 14 cases; 2 of them showed amplification by fluorescence in situ hybridization (FISH). CDK4 was negative except for weak staining in 1 of 14 cases. This study adds to the existing few clinicopathologic series on FDCS and represents the first study to show MDM2 amplification in this entity. Our results

  8. The isolation, total synthesis and structure elucidation of chlorofusin, a natural product inhibitor of the p53-MDM2 protein-protein interaction

    PubMed Central

    Clark, Ryan C.; Lee, Sang Yeul; Searcey, Mark; Boger, Dale L.

    2009-01-01

    Inhibitors of key protein-protein interactions are emerging as exciting therapeutic targets for the treatment of cancer. One such interaction between MDM2 (HDM2) and p53, that silences the tumour suppression activities of p53, was found to be inhibited by the recently isolated natural product chlorofusin. Synthetic studies on this complex natural product summarized herein have served to reassign its chromophore relative stereochemistry, assign its absolute stereochemistry, and provided access to a series of key analogues and partial structures for biological evaluation. PMID:19642417

  9. Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

    PubMed

    Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

    2015-02-01

    In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

  10. Oral delivery of anti-MDM2 inhibitor SP141-loaded FcRn-targeted nanoparticles to treat breast cancer and metastasis.

    PubMed

    Qin, Jiang-Jiang; Wang, Wei; Sarkar, Sushanta; Zhang, Ruiwen

    2016-09-10

    We have recently discovered a specific Murine Double Minute 2 (MDM2) oncogene inhibitor, called SP141, which exerts potent anticancer activity in various breast cancer models. However, its low oral bioavailability is the major hurdle for moving this drug to clinical trial. The present study was designed to discover and validate a novel nano-oral delivery system for this promising anticancer agent. Herein, we report the preparation, characterization, and evaluation of the efficacy and safety of the SP141-loaded IgG Fc-conjugated maleimidyl-poly(ethylene glycol)-co-poly(ε-caprolactone) (Mal-PEG-PCL) nanoparticles (SP141FcNP) as an orally cancer therapeutic agent. Our results indicated that SP141FcNP showed a biphasic release pattern and increased transepithelial transport in vitro and in vivo with the involvement of FcRn-mediated transcytosis. SP141FcNP also exhibited increased intestinal epithelial permeability, cellular uptake, and oral bioavailability, with extended blood circulation time, increased tumor accumulation, enhanced MDM2 inhibition, and stronger responses in anti-tumor growth and metastasis effects in vitro and in vivo, without apparent host toxicity. Collectively, this newly developed nanoparticle oral delivery system provides a basis for evaluation of SP141 as a potential clinical candidate for cancer therapy. PMID:27394681

  11. Luteolin Prevents H2O2-Induced Apoptosis in H9C2 Cells through Modulating Akt-P53/Mdm2 Signaling Pathway.

    PubMed

    Chang, Hong; Li, Chun; Huo, Kuiyuan; Wang, Qiyan; Lu, Linghui; Zhang, Qian; Wang, Yong; Wang, Wei

    2016-01-01

    Introduction. Luteolin, a falconoid compound in many Chinese herbs and formula, plays important roles in cardiovascular diseases. The underlying mechanism of luteolin remains to be further elaborated. Methods. A model of hydrogen peroxide- (H2O2-) induced H9C2 cells apoptosis was established. Cell viabilities were examined with an MTT assay. 2',7'-Dichlorofluorescin diacetate (DCFH-DA) and flow cytometry were used to detect ROS level and apoptosis rate, respectively. The expressions of signaling proteins related to apoptosis were analyzed by western blot and mRNA levels were detected by real-time polymerase chain reaction (PCR). Quercetin was applied as positive drug. Results. Incubation with various concentrations of H2O2 (0, 50, 100, and 200 μM) for 1 h caused dose-dependent loss of cell viability and 100 μM H2O2 reduced the cell viability to approximately 50%. Treatments with luteolin and quercetin protected cells from H2O2-induced cytotoxicity and reduced cellular ROS level and apoptosis rate. Moreover, luteolin could downregulate the expressions of Bax, caspase-8, cleaved-caspase-3, and p53 in apoptotic signaling pathway. Further study showed that the expressions of Akt, Bcl-2, and Mdm2 were upregulated by luteolin. Conclusion. Luteolin protects H9C2 cells from H2O2-induced apoptosis. The protective and antiapoptotic effects of luteolin could be mediated by regulating the Akt-P53/Mdm2 apoptotic pathway. PMID:27525270

  12. Design, synthesis and in vitro and in vivo antitumour activity of 3-benzylideneindolin-2-one derivatives, a novel class of small-molecule inhibitors of the MDM2-p53 interaction.

    PubMed

    Zheng, Guang-hui; Shen, Jia-jia; Zhan, Yue-chen; Yi, Hong; Xue, Si-tu; Wang, Zhen; Ji, Xing-yue; Li, Zhuo-rong

    2014-06-23

    A novel class of small-molecule inhibitors of MDM2-p53 interaction with a (E)-3-benzylideneindolin-2-one scaffold was identified using an integrated virtual screening strategy that combined both pharmacophore- and structure-based approaches. The hit optimisation identified several compounds with more potent activity than the hit compound and the positive drug nutlin-3a, especially compound 1b, which exhibited both the highest binding affinity to MDM2 (Ki = 0.093 μM) and the most potent antiproliferative activity against HCT116 (wild type p53) cells (GI50 = 13.42 μM). Additionally, 1b dose-dependently inhibited tumour growth in BALB/c mice bearing CT26 colon carcinoma, with no visible sign of toxicity. In summary, compound 1b represents a novel and promising lead structure for the development of anticancer drugs as MDM2-p53 interaction disruptors.

  13. The TP53 Arg72Pro and MDM2 309G>T polymorphisms are not associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers

    PubMed Central

    Sinilnikova, O M; Antoniou, A C; Simard, J; Healey, S; Léoné, M; Sinnett, D; Spurdle, A B; Beesley, J; Chen, X; Greene, M H; Loud, J T; Lejbkowicz, F; Rennert, G; Dishon, S; Andrulis, I L; Domchek, S M; Nathanson, K L; Manoukian, S; Radice, P; Konstantopoulou, I; Blanco, I; Laborde, A L; Durán, M; Osorio, A; Benitez, J; Hamann, U; Hogervorst, F B L; van Os, T A M; Gille, H J P; Peock, S; Cook, M; Luccarini, C; Evans, D G; Lalloo, F; Eeles, R; Pichert, G; Davidson, R; Cole, T; Cook, J; Paterson, J; Brewer, C; Hughes, D J; Coupier, I; Giraud, S; Coulet, F; Colas, C; Soubrier, F; Rouleau, E; Bièche, I; Lidereau, R; Demange, L; Nogues, C; Lynch, H T; Schmutzler, R K; Versmold, B; Engel, C; Meindl, A; Arnold, N; Sutter, C; Deissler, H; Schaefer, D; Froster, U G; Aittomäki, K; Nevanlinna, H; McGuffog, L; Easton, D F; Chenevix-Trench, G; Stoppa-Lyonnet, D

    2009-01-01

    Background: The TP53 pathway, in which TP53 and its negative regulator MDM2 are the central elements, has an important role in carcinogenesis, particularly in BRCA1- and BRCA2-mediated carcinogenesis. A single nucleotide polymorphism (SNP) in the promoter region of MDM2 (309T>G, rs2279744) and a coding SNP of TP53 (Arg72Pro, rs1042522) have been shown to be of functional significance. Methods: To investigate whether these SNPs modify breast cancer risk for BRCA1 and BRCA2 mutation carriers, we pooled genotype data on the TP53 Arg72Pro SNP in 7011 mutation carriers and on the MDM2 309T>G SNP in 2222 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analysed using a Cox proportional hazards model within a retrospective likelihood framework. Results: No association was found between these SNPs and breast cancer risk for BRCA1 (TP53: per-allele hazard ratio (HR)=1.01, 95% confidence interval (CI): 0.93–1.10, Ptrend=0.77; MDM2: HR=0.96, 95%CI: 0.84–1.09, Ptrend=0.54) or for BRCA2 mutation carriers (TP53: HR=0.99, 95%CI: 0.87–1.12, Ptrend=0.83; MDM2: HR=0.98, 95%CI: 0.80–1.21, Ptrend=0.88). We also evaluated the potential combined effects of both SNPs on breast cancer risk, however, none of their combined genotypes showed any evidence of association. Conclusion: There was no evidence that TP53 Arg72Pro or MDM2 309T>G, either singly or in combination, influence breast cancer risk in BRCA1 or BRCA2 mutation carriers. PMID:19707196

  14. Luteolin Prevents H2O2-Induced Apoptosis in H9C2 Cells through Modulating Akt-P53/Mdm2 Signaling Pathway

    PubMed Central

    Li, Chun; Wang, Qiyan; Lu, Linghui; Zhang, Qian

    2016-01-01

    Introduction. Luteolin, a falconoid compound in many Chinese herbs and formula, plays important roles in cardiovascular diseases. The underlying mechanism of luteolin remains to be further elaborated. Methods. A model of hydrogen peroxide- (H2O2-) induced H9C2 cells apoptosis was established. Cell viabilities were examined with an MTT assay. 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) and flow cytometry were used to detect ROS level and apoptosis rate, respectively. The expressions of signaling proteins related to apoptosis were analyzed by western blot and mRNA levels were detected by real-time polymerase chain reaction (PCR). Quercetin was applied as positive drug. Results. Incubation with various concentrations of H2O2 (0, 50, 100, and 200 μM) for 1 h caused dose-dependent loss of cell viability and 100 μM H2O2 reduced the cell viability to approximately 50%. Treatments with luteolin and quercetin protected cells from H2O2-induced cytotoxicity and reduced cellular ROS level and apoptosis rate. Moreover, luteolin could downregulate the expressions of Bax, caspase-8, cleaved-caspase-3, and p53 in apoptotic signaling pathway. Further study showed that the expressions of Akt, Bcl-2, and Mdm2 were upregulated by luteolin. Conclusion. Luteolin protects H9C2 cells from H2O2-induced apoptosis. The protective and antiapoptotic effects of luteolin could be mediated by regulating the Akt-P53/Mdm2 apoptotic pathway. PMID:27525270

  15. Ling Zhi-8 reduces lung cancer mobility and metastasis through disruption of focal adhesion and induction of MDM2-mediated Slug degradation.

    PubMed

    Lin, Tung-Yi; Hsu, Hsien-Yeh

    2016-06-01

    We recently reported that recombinant Ling Zhi-8 (rLZ-8), a medicinal mushroom Ganoderma lucidum recombinant protein, effectively prevents lung cancer cells proliferation in vivo mice model. In our current study, we demonstrated that rLZ-8 suppressed tumor metastasis and increased the survival rate in Lewis lung carcinoma cell-bearing mice. The epithelial to mesenchymal transition (EMT) process is regarded as the critical event in tumor metastasis. Herein, we showed that rLZ-8 effectively induced changes in EMT by interfering with cell adhesion and focal adhesion kinase (FAK) functions in lung cancer cells. Slug, a transcription factor, represses E-cadherin transcription and is regarded as a critical event in EMT and tumor metastasis. Functional studies revealed that downregulation of Slug as a result of rLZ-8-induced FAK inactivation enhanced E-cadherin expression and repressed cancer cell mobility. Moreover, we found that rLZ-8 enhanced the ubiquitination proteasome pathway (UPP)-mediated degradation of Slug in CL1-5 cells. Mechanistically, we demonstrated that rLZ-8 promoted the interaction between MDM2 and Slug, resulting in Slug degradation; however, MDM2-shRNA abolished rLZ-8-enhanced Slug degradation. This study is the first to determine anti-metastatic activity of rLZ-8 and its potential mechanism, with how the regulation of EMT and cell mobility is via the negative modulation of FAK, and thereby leading to the ubiquitination and degradation of Slug. Our findings suggest that the targets of FAK play a key role in metastasis. Moreover, rLZ-8 may be useful as a chemotherapeutic agent for treating lung cancer. PMID:26992741

  16. HBP1-mediated Regulation of p21 Protein through the Mdm2/p53 and TCF4/EZH2 Pathways and Its Impact on Cell Senescence and Tumorigenesis*

    PubMed Central

    Chen, Yifan; Pan, Kewu; Wang, Pingzhang; Cao, Zhengyi; Wang, Weibin; Wang, Shuya; Hu, Ningguang; Xue, Junhui; Li, Hui; Jiang, Wei; Li, Gang; Zhang, Xiaowei

    2016-01-01

    The activity of the CDK inhibitor p21 is associated with diverse biological activities, including cell proliferation, senescence, and tumorigenesis. However, the mechanisms governing transcription of p21 need to be extensively studied. In this study, we demonstrate that the high-mobility group box-containing protein 1 (HBP1) transcription factor is a novel activator of p21 that works as part of a complex mechanism during senescence and tumorigenesis. We found that HBP1 activates the p21 gene through enhancing p53 stability by inhibiting Mdm2-mediated ubiquitination of p53, a well known positive regulator of p21. HBP1 was also found to enhance p21 transcription by inhibiting Wnt/β-catenin signaling. We identified histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2, as a target of Wnt/β-catenin signaling. HBP1-mediated repression of EZH2 through Wnt/β-catenin signaling decreased the level of trimethylation of histone H3 at lysine 27 of overall and specific histone on the p21 promoter, resulting in p21 transactivation. Although intricate, the reciprocal partnership of HBP1 and p21 has exceptional importance. HBP1-mediated elevation of p21 through the Mdm2/p53 and TCF4/EZH2 pathways contributes to both cellular senescence and tumor inhibition. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence and tumorigenesis with an impact on protein ubiquitination and overall histone methylation state. PMID:27129219

  17. Evaluation of MDM2 and CDK4 amplification by real-time PCR on paraffin wax-embedded material: a potential tool for the diagnosis of atypical lipomatous tumours/well-differentiated liposarcomas.

    PubMed

    Hostein, I; Pelmus, M; Aurias, A; Pedeutour, F; Mathoulin-Pélissier, S; Coindre, J M

    2004-01-01

    Atypical lipomatous tumours/well-differentiated liposarcomas and dedifferentiated liposarcomas are characterized by 12q13-15 region amplification. In contrast, this molecular event has not been reported in benign lipomas. Within the 12q13-15 chromosomal region, the MDM2, SAS, HMGA2, and CDK4 genes are the most frequent targets of amplification. A series of lipomas (36 cases) and liposarcomas (48 cases) was analysed for MDM2 and CDK4 gene amplification by real-time PCR. MDM2 and CDK4 gene amplification was detected in 2.8% and 5.6% of lipomas and 98.2% and 82.4% of liposarcomas, respectively. Moreover, co-amplification of the two genes as well as a higher-level amplification was observed more frequently in dedifferentiated liposarcomas than in atypical lipomatous tumours/well-differentiated liposarcomas. Real-time PCR proved to be a fast and reliable method to characterize lipomas and liposarcomas by quantification of MDM2 and CDK4 gene amplification. It is applicable to paraffin wax-embedded tissues and could be useful when histological diagnosis is difficult.

  18. The ubiquitin ligase Mdm2 controls oligodendrocyte maturation by intertwining mTOR with G protein-coupled receptor kinase 2 in the regulation of GPR17 receptor desensitization.

    PubMed

    Fumagalli, Marta; Bonfanti, Elisabetta; Daniele, Simona; Zappelli, Elisa; Lecca, Davide; Martini, Claudia; Trincavelli, Maria L; Abbracchio, Maria P

    2015-12-01

    During oligodendrocyte precursor cell (OPC) differentiation, defective control of the membrane receptor GPR17 has been suggested to block cell maturation and impair remyelination under demyelinating conditions. After the immature oligodendrocyte stage, to enable cells to complete maturation, GPR17 is physiologically down-regulated via phosphorylation/desensitization by G protein-coupled receptor kinases (GRKs); conversely, GRKs are regulated by the "mammalian target of rapamycin" mTOR. However, how GRKs and mTOR are connected to each other in modulating GPR17 function and oligodendrogenesis has remained elusive. Here we show, for the first time, a role for Murine double minute 2 (Mdm2), a ligase previously involved in ubiquitination/degradation of the onco-suppressor p53 protein. In maturing OPCs, both rapamycin and Nutlin-3, a small molecule inhibitor of Mdm2-p53 interactions, increased GRK2 sequestration by Mdm2, leading to impaired GPR17 down-regulation and OPC maturation block. Thus, Mdm2 intertwines mTOR with GRK2 in regulating GPR17 and oligodendrogenesis and represents a novel actor in myelination.

  19. Altered expression of the cell cycle regulatory molecules pRb, p53 and MDM2 exert a synergetic effect on tumor growth and chromosomal instability in non-small cell lung carcinomas (NSCLCs).

    PubMed Central

    Gorgoulis, V. G.; Zacharatos, P.; Kotsinas, A.; Mariatos, G.; Liloglou, T.; Vogiatzi, T.; Foukas, P.; Rassidakis, G.; Garinis, G.; Ioannides, T.; Zoumpourlis, V.; Bramis, J.; Michail, P. O.; Asimacopoulos, P. J.; Field, J. K.; Kittas, C.

    2000-01-01

    BACKGROUND: Recent in vitro studies provide evidence that the cell cycle molecules pRb, p53 and MDM2 form a tightly regulated protein network. In this study, we examined the relationship of this protein network in a series of non-small cell lung carcinomas (NSCLCs), with the kinetic parameters, including proliferative activity or proliferation index (PI) and apoptotic index (AI), and ploidy status of the tumors. MATERIAL AND METHODS: A total of 87 NSCLCs were examined using immunohistochemical and molecular methods in order to estimate the status of the pRb-p53-MDM2 network. The kinetic parameters and the ploidy status of the tumors were assessed by in situ assays. The possible associations between alterations of the network, kinetic parameters and ploidy status of the carcinomas were assessed with a series of statistical methods. RESULTS: Aberrant expression of pRb (Ab) and overexpression of p53 (P) and MDM2 (P) proteins were observed in 39%, 57%, and 68% of the carcinomas, respectively. The comprehensive analysis revealed that concurrent alterations in all three cell cycle regulatory molecules were the most frequent pattern, pRb(Ab)/p53(P)/MDM2(P); this "full abnormal" phenotype represented approximately 27% of the cases. This immunoprofile obtained the highest PI/AI value; whereas, the "normal" phenotype was the lowest one (p = 0.004). Furthermore, the pattern pRb(Ab)/p53(P)/MDM2(P) acquired the highest PI (p < 0.001) and lowest AI (p < 0.001) scores. Interestingly, the groups of carcinomas with impaired expression of one or two molecules attained PI/AI ratio values clustered in a narrow range placed in the middle of the scores exhibited by the "normal" and "full abnormal" phenotypes. These tumors had significantly lower AI, but similar PI values, compared with those noticed in the normal pattern. In addition, it was observed that the pRb(Ab)/p53(P)/MDM2(P) phenotype was also significantly associated with aneuploidy (p = 0.002) and a tendency was observed when

  20. The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway.

    PubMed

    Bailly, A; Perrin, A; Bou Malhab, L J; Pion, E; Larance, M; Nagala, M; Smith, P; O'Donohue, M-F; Gleizes, P-E; Zomerdijk, J; Lamond, A I; Xirodimas, D P

    2016-01-28

    The ubiquitin-like molecule NEDD8 is essential for viability, growth and development, and is a potential target for therapeutic intervention. We found that the small molecule inhibitor of NEDDylation, MLN4924, alters the morphology and increases the surface size of the nucleolus in human and germline cells of Caenorhabditis elegans in the absence of nucleolar fragmentation. SILAC proteomics and monitoring of rRNA production, processing and ribosome profiling shows that MLN4924 changes the composition of the nucleolar proteome but does not inhibit RNA Pol I transcription. Further analysis demonstrates that MLN4924 activates the p53 tumour suppressor through the RPL11/RPL5-Mdm2 pathway, with characteristics of nucleolar stress. The study identifies the nucleolus as a target of inhibitors of NEDDylation and provides a mechanism for p53 activation upon NEDD8 inhibition. It also indicates that targeting the nucleolar proteome without affecting nucleolar transcription initiates the required signalling events for the control of cell cycle regulators.

  1. Detection of MDM2-CDK4 amplification by fluorescence in situ hybridization in 200 paraffin-embedded tumor samples: utility in diagnosing adipocytic lesions and comparison with immunohistochemistry and real-time PCR.

    PubMed

    Sirvent, Nicolas; Coindre, Jean-Michel; Maire, Georges; Hostein, Isabelle; Keslair, Frédérique; Guillou, Louis; Ranchere-Vince, Dominique; Terrier, Philippe; Pedeutour, Florence

    2007-10-01

    Atypical lipomatous tumor/well-differentiated liposarcomas and dedifferentiated liposarcomas are characterized by the amplification of MDM2 and CDK4 genes. To evaluate the accuracy of fluorescence in situ hybridization (FISH) analysis in the differential diagnosis of adipose tissue tumors, we investigated MDM2-CDK4 status by FISH, real-time polymerase chain reaction (PCR) [quantitative PCR (Q-PCR)] and immunohistochemistry (IHC) in a series of 200 adipose tumors. First, we evaluated MDM2-CDK4 amplification and expression in a series of 94 well-defined adipose tissue tumors. Results showed that FISH was interpretable in 45 of 50 cases (90%), and was more specific and sensitive than Q-PCR and IHC. We then used the same techniques as complementary diagnostic tools in a series of 106 adipose and soft tissue tumors of unclear diagnosis to distinguish between (i) lipomas and atypical lipomatous tumor/well-differentiated liposarcomas, (ii) malignant undifferentiated tumors and dedifferentiated liposarcomas, and (iii) a variety of benign tumors and liposarcomas. Our results indicate that although helpful, IHC alone is often insufficient to solve diagnostic problems. FISH and Q-PCR methods gave concordant results and were equally informative in most cases. However, the proportion of noninterpretable cases was slightly higher with FISH than with Q-PCR. When tumor cells represented a minor component of the tumor tissue, such as with inflammatory tumors, FISH was more powerful than Q-PCR by allowing visualization of individual cells. In conclusion, we recommend that the evaluation of MDM2-CDK4 amplification using FISH or Q-PCR be used to supplement IHC analysis when diagnosis of adipose tissue tumors is not possible based on clinical and histologic information alone.

  2. The viral non-structural protein 1 alpha (Nsp1α) inhibits p53 apoptosis activity by increasing murine double minute 2 (mdm2) expression in porcine reproductive and respiratory syndrome virus (PRRSV) early-infected cells.

    PubMed

    Wang, Xiaodu; Shao, Chunyan; Wang, Luyan; Li, Qunjing; Song, Houhui; Fang, Weihuan

    2016-02-29

    Apoptosis is one of the most important mechanisms of pathogenesis in porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells. The tumor suppressor p53 plays a critical role in apoptotic induction in viral infections. In the present study, we found that p53 activity was inhibited at the early stage of PRRSV infection in both the highly pathogenic (HP) and lowly pathogenic (LP) PRRSV isolates. Bax expression showed a similar change pattern to that of p53. Murine double minute 2 (mdm2) expressed higher in PRRSV-infected cells than in uninfected cells at the early stage of infection and promoted p53 degradation. We show for the first time that the non-structural protein 1 alpha (Nsp1α) of PRRSV is a negative regulator of p53 activity through increasing mdm2 expression and p53 ubiquitination, while p53 is inhibitory to PRRSV replication at the early stage of infection. We conclude that PRRSV manipulates the host factors mdm2 and p53 via its Nsp1α for increased replication at the early stage of infection. These provide a novel perspective to understand the interaction between apoptosis and replication of PRRSV.

  3. Long non-coding RNA LOC572558 inhibits bladder cancer cell proliferation and tumor growth by regulating the AKT-MDM2-p53 signaling axis.

    PubMed

    Zhu, Yiping; Dai, Bo; Zhang, Hailiang; Shi, Guohai; Shen, Yijun; Ye, Dingwei

    2016-10-01

    Long non-coding RNAs (lncRNAs) have been suggested to play important roles in the progression of many cancers such as bladder cancer. However, the detailed mechanism has not been fully understood. We have previously identified a collection of aberrantly expressed lncRNAs in bladder cancer using microarray gene profiling assay. In the current study, we aim to further explore the expression profile and the function of LOC572558, one of the most deregulated lncRNAs in bladder cancer. A large cohort of human bladder cancer tissue samples with benign controls, as well as established human bladder cancer cell lines, has been examined for the expression of LOC572558. The biological functions of LOC572558 were examined by CCK-8 assay, flow cytometry analysis, and wound healing and transwell assays. Using a high-throughput phospho-proteome array, we identified proteins that were ectopic phosphorylated in bladder cancer cells where LOC572558 expression was upregulated. We demonstrated that LOC572558 expression was markedly decreased in bladder cancer tissues and bladder cancer cell lines. Moreover, ectopic expression of LOC572558 inhibited cell proliferation and motility, induced S phase arrest of the cell cycle and promoted cell apoptosis in T24 and 5637 bladder cancer cell lines. We further verified that overexpression of LOC572558 was associated with dephosphorylation of AKT, MDM2 and phosphorylation of p53 protein. Our data clearly demonstrated that LOC572558 is a tumor suppressor and regulates the p53 signaling pathway in bladder cancer. Thus, it may serve as a promising new diagnostic marker and therapeutic target in bladder cancer.

  4. Association of genetic polymorphisms in GADD45A, MDM2, and p14{sup ARF} with the risk of chronic benzene poisoning in a Chinese occupational population

    SciTech Connect

    Sun Pin; Zhang Zhongbin; Wan Junxiang; Zhao Naiqing; Jin Xipeng; Xia Zhaolin

    2009-10-01

    Benzene reactive metabolites can lead to DNA damage and trigger the p53-dependent defense responses to maintain genomic stability. We hypothesized that the p53-dependent genes may play a role in the development of chronic benzene poisoning (CBP). In a case-control study of 303 patients with benzene poisoning and 295 workers occupationally exposed to benzene in south China, we investigated associations between the risk of CBP and polymorphisms in three p53-dependent genes. Potential interactions of these polymorphisms with lifestyle factors were also explored. We found p14{sup ARF} rs3731245 polymorphism was associated with risk of CBP (P = 0.014). Compared with those carrying the GG genotype, individuals carrying p14{sup ARF} rs3731245 GA+AA genotypes had a reduced risk of CBP ([adjusted odds ratio (OR{sub adj}) = 0.57, 95%CI = 0.36-0.89]. Further analysis showed p14{sup ARF} TGA/TAG diplotype was associated with an increased risk of CBP (P = 0.0006), whereas p14{sup ARF} TGG/TAA diplotype was associated with a decreased risk of CBP (P = 0.0000001). In addition, we found individuals carrying both MDM2 Del1518 WW genotype and p14{sup ARF} rs3731245 GA+AA genotypes had a lower risk of CBP (OR{sub adj} = 0.25; 95%CI = 0.10-0.62; P = 0.003). Although these results require confirmation and extension, our findings suggest that genetic polymorphisms in p14{sup ARF} may have an impact on the risk of CBP in the study population.

  5. p38 MAPK-induced MDM2 degradation confers paclitaxel resistance through p53-mediated regulation of EGFR in human lung cancer cells

    PubMed Central

    Park, Shin-Hyung; Seong, Myeong-A; Lee, Ho-Young

    2016-01-01

    Paclitaxel (PTX) is a chemotherapeutic agent that is used to treat a variety of cancers, including non-small cell lung cancer (NSCLC). However, the emergence of drug resistance limits the utility of PTX. This study determined the signaling pathway that contributes to PTX resistance. We first established PTX resistant cell lines (H460/R and 226B/R) using a dose-escalating maintenance of PTX. We found that p38 MAPK and epidermal growth factor receptor (EGFR) were constitutively activated in these cell lines. The inhibition of p38 MAPK activity by SB203580 treatment or the transfection of dominant-negative p38 MAPK sensitized both cell lines to PTX treatment. Erlotinib, an EGFR inhibitor, also increased PTX-induced apoptosis in PTX resistant cells, which suggests a role for p38 MAPK and EGFR in the development of PTX resistance. We demonstrated that p38 MAPK enhanced EGFR expression via the induction of the rapid degradation of mouse double-minute 2 homolog (MDM2) and the consequent stabilization of p53, a transcription factor of EGFR. These results suggest for the first time that the p38 MAPK/p53/EGFR axis is crucial for the facilitation of PTX resistance in NSCLCs. We also propose a mechanism for the role of the tumor-suppressor p53 in drug resistance. These results provide a foundation for the future development of potential therapeutic strategies to regulate the p38 MAPK/p53/EGFR pathway for the treatment of lung cancer patients with PTX resistance. PMID:26799187

  6. Efficient Atomistic Simulation of Pathways and Calculation of Rate Constants for a Protein-Peptide Binding Process: Application to the MDM2 Protein and an Intrinsically Disordered p53 Peptide.

    PubMed

    Zwier, Matthew C; Pratt, Adam J; Adelman, Joshua L; Kaus, Joseph W; Zuckerman, Daniel M; Chong, Lillian T

    2016-09-01

    The characterization of protein binding processes - with all of the key conformational changes - has been a grand challenge in the field of biophysics. Here, we have used the weighted ensemble path sampling strategy to orchestrate molecular dynamics simulations, yielding atomistic views of protein-peptide binding pathways involving the MDM2 oncoprotein and an intrinsically disordered p53 peptide. A total of 182 independent, continuous binding pathways were generated, yielding a kon that is in good agreement with experiment. These pathways were generated in 15 days using 3500 cores of a supercomputer, substantially faster than would be possible with "brute force" simulations. Many of these pathways involve the anchoring of p53 residue F19 into the MDM2 binding cleft when forming the metastable encounter complex, indicating that F19 may be a kinetically important residue. Our study demonstrates that it is now practical to generate pathways and calculate rate constants for protein binding processes using atomistic simulation on typical computing resources. PMID:27532687

  7. Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation

    PubMed Central

    Sørensen, Belinda Halling; Nielsen, Dorthe; Thorsteinsdottir, Unnur Arna; Hoffmann, Else Kay

    2016-01-01

    The leucine-rich repeat containing 8A (LRRC8A) protein is an essential component of the volume-sensitive organic anion channel (VSOAC), and using pharmacological anion channel inhibitors (NS3728, DIDS) and LRRC8A siRNA we have investigated its role in development of Cisplatin resistance in human ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21Waf1/Cip1, Bax, Noxa, MDM2, and activation of Caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter apoptosis, i.e., their p53 and downstream signaling are reduced and caspase activity unaltered following Cisplatin exposure. Reduced LRRC8A expression and VSOAC activity are previously shown to correlate with Cisplatin resistance, and here we demonstrate that pharmacological inhibition and transient knockdown of LRRC8A reduce the protein level of p53, MDM2, and p21Waf1/Cip1 as well as Caspase-9/-3 activation in Cisplatin-sensitive cells. Cisplatin resistance is accompanied by reduction in total LRRC8A expression (A2780) or LRRC8A expression in the plasma membrane (A549). Activation of Caspase-3 dependent apoptosis by TNFα-exposure or hyperosmotic cell shrinkage is almost unaffected by pharmacological anion channel inhibition. Our data indicate 1) that expression/activity of LRRC8A is essential for Cisplatin-induced increase in p53 protein level and its downstream signaling, i.e., Caspase-9/-3 activation, expression of p21Waf1/Cip1 and MDM2; and 2) that downregulation of LRRC8A-dependent osmolyte transporters contributes to acquirement of Cisplatin resistance in ovarian and lung carcinoma cells. Activation of LRRC8A-containing channels is upstream to apoptotic volume decrease as hypertonic cell shrinkage induces apoptosis independent of the presence of LRRC8A. PMID:26984736

  8. An EBV recombinant deleted for residues 130-159 in EBNA3C can deregulate p53/Mdm2 and Cyclin D1/CDK6 which results in apoptosis and reduced cell proliferation

    PubMed Central

    El-Naccache, Darine W.; Robertson, Erle S.

    2016-01-01

    Epstein-Barr virus (EBV), a gamma herpes virus is associated with B-cell malignancies. EBNA-3C is critical for in vitro primary B-cell transformation. Interestingly, the N terminal domain of EBNA3C which contains residues 130–159, interacts with various cellular proteins, such as p53, Mdm2, CyclinD1/Cdk6 complex, and E2F1. In the current reverse genetics study, we deleted the residues 130-159 aa within EBNA3C open reading frame (ORF) by BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the 130-159 aa showed a reduction in cell proliferation. Also, this recombinant virus showed with higher infectivity of human peripheral blood mononuclear cells (PBMCs) compared to wild type EBV. PBMCs- infected with recombinant EBV deleted for 130-159 residues have differential expression patterns for the p53/Mdm2, CyclinD1/Cdk6 and pRb/E2F1 pathways compared to wild type EBV-infected PBMCs. PBMCs infected with recombinant virus showed increased apoptotic cell death which further resulted in activation of polymerase 1 (PARP1), an important contributor to apoptotic signaling. Interestingly, cells infected with this recombinant virus showed a dramatic decrease in chromosomal instability, indicated by the presence of increased multinucleation and micronucleation. In addition infection with recombinant virus have increased cells in G0/G1 phase and decreased cells in S-G2M phase when compared to wild type infected cells. Thus, these differences in signaling activities due to 29 amino acid residues of EBNA3C is of particular significance in deregulation of cell proliferation in EBV-infected cells. PMID:26908453

  9. Epithelial cell-derived periostin functions as a tumor suppressor in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14ARF/Mdm2 signaling pathway.

    PubMed

    Lv, Hongjun; Liu, Rui; Fu, Jiao; Yang, Qi; Shi, Jing; Chen, Pu; Ji, Meiju; Shi, Bingyin; Hou, Peng

    2014-01-01

    Periostin is usually considered as an oncogene in diverse human cancers, including breast, prostate, colon, esophagus, and pancreas cancers, whereas it acts as a tumor suppressor in bladder cancer. In gastric cancer, it has been demonstrated that periglandular periostin expression is decreased whereas stromal periostin expression is significantly increased as compared with normal gastric tissues. Moreover, periostin produced by stromal myofibroblasts markedly promotes gastric cancer cell growth. These observations suggest that periostin derived from different types of cells may play distinct biological roles in gastric tumorigenesis. The aim of this study was to explore the biological functions and related molecular mechanisms of epithelial cell-derived periostin in gastric cancer. Our data showed that periglandular periostin was significantly down-regulated in gastric cancer tissues as compared with matched normal gastric mucosa. In addition, its expression in metastatic lymph nodes was significantly lower than that in their primary cancer tissues. Our data also demonstrated that periglandular periostin expression was negatively associated with tumor stage. More importantly, restoration of periostin expression in gastric cancer cells dramatically suppressed cell growth and invasiveness. Elucidation of the mechanisms involved revealed that periostin restoration enhanced Rb phosphorylation and sequentially activated the transcription of E2F1 target gene p14(ARF), leading to Mdm2 inactivation and the stabilization of p53 and E-cadherin proteins. Strikingly, these effects of periostin were abolished upon Rb deletion. Collectively, we have for the first time demonstrated that epithelial cell-derived periostin exerts tumor-suppressor activities in gastric cancer through stabilizing p53 and E-cadherin proteins via the Rb/E2F1/p14(ARF)/Mdm2 signaling pathway. PMID:25486483

  10. Diaryl- and triaryl-pyrrole derivatives: inhibitors of the MDM2-p53 and MDMX-p53 protein-protein interactions†Electronic supplementary information (ESI) available: Experimental details for compound synthesis, analytical data for all compounds and intermediates. Details for the biological evaluation. Further details for the modeling. Table of combustion analysis data. See DOI: 10.1039/c3md00161jClick here for additional data file.

    PubMed

    Blackburn, Tim J; Ahmed, Shafiq; Coxon, Christopher R; Liu, Junfeng; Lu, Xiaohong; Golding, Bernard T; Griffin, Roger J; Hutton, Claire; Newell, David R; Ojo, Stephen; Watson, Anna F; Zaytzev, Andrey; Zhao, Yan; Lunec, John; Hardcastle, Ian R

    2013-09-21

    Screening identified 2-(3-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)-2,5-dimethyl-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile as an MDM2-p53 inhibitor (IC50 = 12.3 μM). MDM2-p53 and MDMX-p53 activity was seen for 5-((1-(4-chlorophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (MDM2 IC50 = 0.11 μM; MDMX IC50 = 4.2 μM) and 5-((1-(4-nitrophenyl)-2,5-diphenyl-1H-pyrrol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (MDM2 IC50 = 0.15 μM; MDMX IC50 = 4.2 μM), and cellular activity consistent with p53 activation in MDM2 amplified cells. Further SAR studies demonstrated the requirement for the triarylpyrrole moiety for MDMX-p53 activity but not for MDM2-p53 inhibition.

  11. The effect of varying type and volume of sedimenting agents on leukocyte harvesting and labelling in sickle cell patients.

    PubMed

    Webber, D; Nunan, T O; O'Doherty, M J

    1994-09-01

    Leukocyte labelling in patients with sickle cell anaemia has been reported as difficult if not impossible due to the slow erythrocyte sedimentation rate (ESR) in these patients. This study investigated standard sedimentation methods in patients with sickle cell disease (n = 16) and compared the results obtained with those following changes in the amount and type of sedimenting agent used. Labelling with either 111In-oxine or 99Tcm-exametazime was attempted in only five patients. Replacement of the commonly used 6% Hetastarch (Hespan) with Dextran or Haemaccel did not improve leukocyte harvesting, even when the proportions used of these agents were increased. In most cases where standard procedures for leukocyte collection did not lead to harvesting of viable samples, it was possible to obtain adequate leukocyte labelling in the majority of sickle cell patients using a minor modification of standard techniques. In this group of patients a ratio of 8 ml of Hespan to 16 ml of blood should be used for cell separation. If this fails then donor cells, anti-granulocyte antibody labelling or HIG should be considered.

  12. Theaflavin-3, 3′-digallate induces apoptosis and G2 cell cycle arrest through the Akt/MDM2/p53 pathway in cisplatin-resistant ovarian cancer A2780/CP70 cells

    PubMed Central

    TU, YOUYING; KIM, EUNHYE; GAO, YING; RANKIN, GARY O.; LI, BO; CHEN, YI CHARLIE

    2016-01-01

    Ovarian cancer is the most lethal gynecological cancer among women worldwide. Adverse side effects and acquired resistance to conventional platinum based chemotherapy are major impediments in ovarian cancer treatment, and drive the development of more selective anticancer drugs that target cancer-specific defects. In this study, theaflavin-3, 3′-digallate (TF3), the major theaflavin monomer in black tea, exhibited a potent growth inhibitory effect on the cisplatin-resistant ovarian cancer A2780/CP70 cells (IC50, 23.81 μM), and was less cytotoxic to a normal ovarian IOSE-364 cells (IC50, 59.58 μM) than to the cancer cells. Flow cytometry analysis indicated that TF3 induced preferential apoptosis and G2 cell cycle arrest in A2780/CP70 cells with respect to IOSE-364 cells. TF3 induced apoptosis through both the intrinsic and extrinsic apoptotic pathways, and caused G2 cell cycle arrest via cyclin B1 in A2780/CP70 cells. The p53 protein played an important role in TF3-induced apoptosis and G2 cell cycle arrest. TF3 might upregulate the p53 expression via the Akt/MDM2 pathway. Our findings help elucidate the mechanisms by which TF3 may contribute to the prevention and treatment of platinum-resistant ovarian cancer. PMID:27082635

  13. Combination treatment in vitro with Nutlin, a small-molecule antagonist of MDM2, and pegylated interferon-α 2a specifically targets JAK2V617F-positive polycythemia vera cells

    PubMed Central

    Lu, Min; Wang, Xiaoli; Li, Yan; Tripodi, Joseph; Mosoyan, Goar; Mascarenhas, John; Kremyanskaya, Marina; Najfeld, Vesna

    2012-01-01

    Interferon (IFN-α) is effective therapy for polycythemia vera (PV) patients, but it is frequently interrupted because of adverse events. To permit the long-term use of IFN, we propose combining low doses of IFN with Nutlin-3, an antagonist of MDM2, which is also capable of promoting PV CD34+ cell apoptosis. Combination treatment with subtherapeutic doses of Peg IFN-α 2a and Nutlin-3 inhibited PV CD34+ cell proliferation by 50% while inhibiting normal CD34+ cells by 30%. Combination treatment with Nutlin-3 and Peg IFN-α 2a inhibited PV colony formation by 55%-90% while inhibiting normal colony formation by 22%-30%. The combination of these agents also decreased the proportion of JAK2V617F-positive hematopoietic progenitor cells in 6 PV patients studied. Treatment with low doses of Peg IFN-α 2a combined with Nutlin-3 increased phospho-p53 and p21 protein levels in PV CD34+ cells and increased the degree of apoptosis. These 2 reagents affect the tumor suppressor p53 through different pathways with Peg IFN-α 2a activating p38 MAP kinase and STAT1, leading to increased p53 transcription, whereas Nutlin-3 prevents the degradation of p53. These data suggest that treatment with low doses of both Nutlin-3 combined with Peg IFN-α 2a can target PV hematopoietic progenitor cells, eliminating the numbers of malignant hematopoietic progenitor cells. PMID:22872685

  14. Stress response of glioblastoma cells mediated by miR-17-5p targeting PTEN and the passenger strand miR-17-3p targeting MDM2.

    PubMed

    Li, Haoran; Yang, Burton B

    2012-12-01

    Tumor development not only destroys the homeostasis of local tissues but also the whole body, and thus the tumor cells have to face the body's defense system, a shortage of nutrition and oxygen, and chemotherapeutic drug treatment. In response to these stresses, tumor cells often alter gene expression and microRNA levels to facilitate survival. We have demonstrated that glioblastoma cells deprived of nutrition or treated with chemotherapeutics drugs expressed increased levels of miR-17. Ectopic transfection of miR-17 prolonged glioblastoma cell survival when the cells were deprived with nutrition or treated with chemotherapeutic drugs. Expression of miR-17 also promoted cell motility, invasion, and tube-like structure formation. We found that these phenotypes were the results of miR-17 targeting PTEN. As a consequence, HIF1α and VEGF were up-regulated. Ectopic expression of miR-17 was found to facilitate enrichment of stem-like tumor cells, since the cells became drug-resistant, showed increased capacity to form colonies and neurospheres, and expressed higher levels of CD133, a phenotype similar to ectopic expression of HIF1α. To further confirm the phenotypic property of stem cells, we demonstrated that glioblastoma cells transfected with miR-17 proliferated slower in different nutritional conditions by facilitating more cells staying in the G1 phase than the control cells. Finally, we demonstrated that miR-17 could repress MDM2 levels, resulting in decreased cell proliferation and drug-resistance. Our results added a new layer of functional mechanism for the well-studied miRNA miR-17.

  15. Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling

    PubMed Central

    Wu, Yuteng; Olsen, Lasse B.; Lau, Yu Heng; Jensen, Claus Hatt; Rossmann, Maxim; Baker, Ysobel R.; Sore, Hannah F.; Collins, Súil

    2016-01-01

    Abstract Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo‐crosslinking motif and a peptide stapling reagent. Using double‐click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo‐crosslinking amino acid. When applied to a p53‐derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull‐down assays, and can be used to investigate the target selectivity of stapled peptides. PMID:26919579

  16. Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling.

    PubMed

    Wu, Yuteng; Olsen, Lasse B; Lau, Yu Heng; Jensen, Claus Hatt; Rossmann, Maxim; Baker, Ysobel R; Sore, Hannah F; Collins, Súil; Spring, David R

    2016-04-15

    Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo-crosslinking motif and a peptide stapling reagent. Using double-click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo-crosslinking amino acid. When applied to a p53-derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull-down assays, and can be used to investigate the target selectivity of stapled peptides. PMID:26919579

  17. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the Food and Drug Administration (FDA) and the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

  18. Genetic analysis of Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes in 2',3'-dideoxycytidine- and 1,3-butadiene-induced lymphomas in B6C3F1 mice.

    PubMed

    Zhuang, S; Söderkvist, P

    2000-07-20

    We have previously identified activation of ras proto-oncogenes and inactivation of tumor suppressor genes including p53, p16(INK4a) and p15(INK4b) in 2',3'-dideoxycytidine (ddC)- and/or 1,3-butadiene (BD)-induced lymphomas derived from B6C3F1 (C57BL/6xC3H/He) mice, indicating that alterations of ras signaling pathway, p53 and pRb growth control pathways are important in the development of these chemically induced lymphomas. However, there is still a subset of tumors that displayed no changes in these genes. Thus, we investigated whether the Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes, which are implicated in the ras or p53 or pRb pathways, are alternative oncogenic target genes. Analyses of gross genomic alterations by Southern blots failed to reveal rearrangement or amplification in any of the tumors examined. Frequent point mutations on the substrate binding domain of the Raf1 gene has been reported in 1-ethyl-1-nitrosourea (ENU)-induced murine lymphomas and lung tumors, along with a conspicuous lack of ras mutations [U. Naumann, I. Eisenmann-Tappe, U.R. Rapp, The role of raf kinases in development and growth of tumors, Recent Results Cancer Res., 143 (1997) 237-244]. To investigate whether Raf1 mutation is involved in our set of tumor especially those without ras mutations, the PCR-based single-strand conformation analyses (SSCA) and direct DNA sequencing were employed. No mutations but four genetic polymorphisms between C57BL/6 and C3H/He were found, with two of them reported as point mutations previously (op. cit.). The polymorphisms were utilized for allelic loss study of Raf1 locus. Losses of heterozygosity were found in six of 31 BD-induced lymphomas. These results indicate that genetic alterations of c-Myc, Cdc25, Raf1 and Mdm2 proto-oncogenes may not be involved in the development of ddC- and BD-induced lymphomas and the inactivation of tumor suppressor gene(s) located close to Raf1 gene might be important in the development of a subset of BD

  19. Mental Labels and Tattoos

    ERIC Educational Resources Information Center

    Hyatt, I. Ralph

    1977-01-01

    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  20. Bar Code Labels

    NASA Technical Reports Server (NTRS)

    1988-01-01

    American Bar Codes, Inc. developed special bar code labels for inventory control of space shuttle parts and other space system components. ABC labels are made in a company-developed anodizing aluminum process and consecutively marketed with bar code symbology and human readable numbers. They offer extreme abrasion resistance and indefinite resistance to ultraviolet radiation, capable of withstanding 700 degree temperatures without deterioration and up to 1400 degrees with special designs. They offer high resistance to salt spray, cleaning fluids and mild acids. ABC is now producing these bar code labels commercially or industrial customers who also need labels to resist harsh environments.

  1. Spanish labeling guide.

    PubMed

    Juliá, A M; García, S V; Breckinridge, M F

    1983-01-01

    A systematic reference of English-Spanish prescription label translations is presented. The purpose of the reference list (which is the most comprehensive published to date) is to enable a pharmacist to write precise, accurate label directions in Spanish for any patient who cannot read English.

  2. Spin-labeled polyribonucleotides.

    PubMed Central

    Petrov, A I; Sukhorukov, B I

    1980-01-01

    Poly (U), poly (C) and poly (A) were spin labeled with N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)-imidazole. This spin label interacts selectively with 2' OH ribose groups of polynucleotides and does not modify the nucleic acid bases. The extent of spin labeling is not dependent upon the nature of the base and is entirely determined by rigidity of the secondary structure of the polynucleotide. The extent of modification for poly (U), poly (C) and poly (A) was 4.2, 1.7 and 1.5 per cent, respectively, the secondary structure of the polynucleotides being practically unchanged. Some physico-chemical properties of the spin-labeled polynucleotides were investigated by ESR spectroscopy. Rotational correlation times of the spin label and activation energy of its motion were calculated. PMID:6253911

  3. Label fusion strategy selection.

    PubMed

    Robitaille, Nicolas; Duchesne, Simon

    2012-01-01

    Label fusion is used in medical image segmentation to combine several different labels of the same entity into a single discrete label, potentially more accurate, with respect to the exact, sought segmentation, than the best input element. Using simulated data, we compared three existing label fusion techniques-STAPLE, Voting, and Shape-Based Averaging (SBA)-and observed that none could be considered superior depending on the dissimilarity between the input elements. We thus developed an empirical, hybrid technique called SVS, which selects the most appropriate technique to apply based on this dissimilarity. We evaluated the label fusion strategies on two- and three-dimensional simulated data and showed that SVS is superior to any of the three existing methods examined. On real data, we used SVS to perform fusions of 10 segmentations of the hippocampus and amygdala in 78 subjects from the ICBM dataset. SVS selected SBA in almost all cases, which was the most appropriate method overall. PMID:22518113

  4. How to Read Drug Labels

    MedlinePlus

    ... and alternative medicine Healthy Aging How to read drug labels Printer-friendly version How to Read Drug ... read drug labels How to read a prescription drug label View a text version of this picture. ...

  5. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1983-07-15

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  6. Behind the Label "Alcoholic."

    ERIC Educational Resources Information Center

    Wright, Deborah M.

    1989-01-01

    Relates individual's personal story of her childhood influenced by her parent's alcoholism, her own alcoholism as a young adult, and her experiences with counseling. Asks others not to reject her because of the label "alcoholic." (ABL)

  7. Like your labels?

    PubMed

    Field, Michele

    2010-01-01

    The descriptive “conventions” used on food labels are always evolving. Today, however, the changes are so complicated (partly driven by legislation requiring disclosures about environmental impacts, health issues, and geographical provenance) that these labels more often baffle buyers than enlighten them. In a light-handed manner, the article points to how sometimes reading label language can be like deciphering runes—and how if we are familiar with the technical terms, we can find a literal meaning, but still not see the implications. The article could be ten times longer because food labels vary according to cultures—but all food-exporting cultures now take advantage of our short attention-span when faced with these texts. The question is whether less is more—and if so, in this contest for our attention, what “contestant” is voted off. PMID:21539053

  8. Routing and Label Space Reduction in Label Switching Networks

    NASA Astrophysics Data System (ADS)

    Solano, Fernando; Caro, Luis Fernando; Stidsen, Thomas; Papadimitriou, Dimitri

    This chapter is devoted to the analysis and modeling of some problems related to the optimal usage of the label space in label switching networks. Label space problems concerning three different technologies and architectures - namely Multi-protocol Label Switching (MPLS), Ethernet VLAN-Label Switching (ELS) and All-Optical Label Switching (AOLS) - are discussed in this chapter. Each of these cases yields to different constraints of the general label space reduction problem. We propose a generic optimization model and, then, we describe some adaptations aiming at modeling each particular case. Simulation results are briefly discussed at the end of this chapter.

  9. Off-Label Drug Use

    MedlinePlus

    ... Your Local Offices Close + - Text Size Off-label Drug Use What is off-label drug use? In the United States new drugs are ... unapproved use of a drug. Is off-label drug use legal? The off-label use of FDA- ...

  10. The effect of forced expiration on the uniformity of 99Tcm-DTPA aerosol ventilation images in patients with excess sputum production.

    PubMed

    Middleton, E; Clout, C; Occleshaw, C; Tindale, W B; Parker, B V; Barber, D C; Barrington, N A

    1990-08-01

    This study examines the use of the forced expiratory technique (FET) as a means of improving the uniformity of radionuclide aerosol ventilation images in patients with excess sputum production. Ventilation images are objectively classified by two computer derived indices to characterize the degree of radioaerosol clumping and overall uniformity. In a series of twenty five patients with a long-standing history of daily sputum production, images acquired before and after forced expiration and again after a second ventilation immediately following FET showed no significant change in either index. The results obtained do not provide evidence to support the routine use of FET in conjunction with radioaerosol scintigraphy. The method of image classification correlates well with a visual assessment of image uniformity and has general application.

  11. Labeling lake water with tritium

    USGS Publications Warehouse

    Frederick, B.J.

    1963-01-01

    A method of packaging tritiated water in a manner that facilitates safe handling in environmental labeling operations, and procedures followed in labeling a large body of water with a small volume of tritiated water are described. ?? 1963.

  12. 99m tc labeled liposomes

    SciTech Connect

    Phillips, W.T.; Klipper, R.W.; Timmons, J.H.; Rudolph, A.S.

    1992-10-27

    This patent describes a method of preparing stable gamma-emitting radionuclide-labeled alkyleneamine oxime, the incubating being for a period of time sufficient to form labeled liposome-encapsulated protein.

  13. Decode the Sodium Label Lingo

    MedlinePlus

    ... For Preschooler For Gradeschooler For Teen Decode the Sodium Label Lingo Published January 24, 2013 Print Email Reading food labels can help you slash sodium. Here's how to decipher them. "Sodium free" or " ...

  14. Learning with imperfectly labeled patterns

    NASA Technical Reports Server (NTRS)

    Chittineni, C. B.

    1979-01-01

    The problem of learning in pattern recognition using imperfectly labeled patterns is considered. The performance of the Bayes and nearest neighbor classifiers with imperfect labels is discussed using a probabilistic model for the mislabeling of the training patterns. Schemes for training the classifier using both parametric and non parametric techniques are presented. Methods for the correction of imperfect labels were developed. To gain an understanding of the learning process, expressions are derived for success probability as a function of training time for a one dimensional increment error correction classifier with imperfect labels. Feature selection with imperfectly labeled patterns is described.

  15. Label and Label-Free Detection Techniques for Protein Microarrays

    PubMed Central

    Syahir, Amir; Usui, Kenji; Tomizaki, Kin-ya; Kajikawa, Kotaro; Mihara, Hisakazu

    2015-01-01

    Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano-biological events.

  16. Principles of protein labeling techniques.

    PubMed

    Obermaier, Christian; Griebel, Anja; Westermeier, Reiner

    2015-01-01

    Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.

  17. Appliance energy labeling takes effect

    SciTech Connect

    Not Available

    1980-06-01

    Consumers buying household appliances will be helped by energy-efficiency labels and minimum efficiency standards required for refrigerators and refrigerator/freezers, freezers, dishwashers, water heaters, clothes washers, room air conditioners, and furnaces. The ENERGYGUIDE labels must be displayed in the store and in catalogs. Two voluntary efficiency programs were combined in the Energy Policy and Conservation Act (EPCA) requiring labels by 1980. Shoppers may compare the efficiencies of appliances and compute the actual cost differential over the lifetime of the equipment. Manufacturers have responded with more-efficient models, but the impact of efficient appliances on energy consumption will be small. A sample label with the required information is illustrated. (DCK)

  18. Labeled Cocaine Analogs

    SciTech Connect

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-03-30

    Novel methods for positron emission tomography or single photon emission spectroscopy using tracer compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)napthyl Y in .beta. configuration is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, The compounds bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  19. 78 FR 66826 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-07

    ... the Agency (76 FR 75809). FSIS also proposed to combine the regulations that provide for the approval... preamble (76 FR 75814), FSIS wrote: . . . statements on labels that are defined in FSIS's regulations or... ``Product Labeling: Definition of the Term ``Natural'' and related materials (71 FR 70503, Dec. 5, 2006)...

  20. Laser labeling, a safe technology to label produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Labeling of the produce has gained marked attention in recent years. Laser labeling technology involves the etching of required information on the surface using a low energy CO2 laser beam. The etching forms alphanumerical characters by pinhole dot matrix depressions. These openings can lead to wat...

  1. Laser labeling, a safe technology to label produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Laser labeling of fruits and vegetables is an alternative means to label produce. Low energy CO2 laser beams etch the surface showing the contrasting underlying layer. These etched surfaces can promote water loss and potentially allow for entry of decay organisms. The long-term effects of laser labe...

  2. 76 FR 75809 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ... poultry products will take effect January 1, 2012 (75 FR 82148, Dec. 29, 2010). These mandatory features... limited types of labels (e.g., labels for raw, single ingredient meat and poultry products) (48 FR 11410... Agency. On March 25, 1992, FSIS published an Advance Notice of Proposed Rulemaking (ANPRM) (57 FR...

  3. Dose rate measurements from radiopharmaceuticals: implications for nuclear medicine staff and for children with radioactive parents.

    PubMed

    Greaves, C D; Tindale, W B

    1999-02-01

    Following the introduction of a number of radiopharmaceuticals, we assessed the dose received by staff working in the nuclear medicine department and also by children who may be in close contact with a radioactive parent. We measured departure dose rates (microSv.h-1) at distances of 0.1, 0.5 and 1.0 m from the skin surface at the level of the thyroid, chest and bladder of patients undergoing the following nuclear medicine procedures: MUGA scans using 99Tcm-labelled red blood cells, myocardial perfusion scans using 99Tcm-labelled radiopharmaceuticals, lymphoscintigraphy using colloidal 99Tcm (Re) sulphide, bone scans using 99Tcm-labelled oxidronate, 111In-octreotide scans, 111In-labelled leukocyte studies and cardiac reinjection studies using 201Tl. The maximum dose rates at 0.1 m were those from MUGA studies (167.3 microSv.h-1) and myocardial perfusion studies (one-day protocol = 391.7 microSv.h-1, two-day protocol = 121.8 microSv.h-1). The implications of these dose rates on both technical and nursing staff are assessed. Also, the dose received by an infant in close contact with a parent following a nuclear medicine investigation was estimated.

  4. Nutrition Marketing on Food Labels

    ERIC Educational Resources Information Center

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

  5. Meat and Poultry Labeling Terms

    MedlinePlus

    ... Food Standards and Labels: The Facts Labeling and Marketing Information [ Top of Page ] OVEN PREPARED: Product is fully cooked and ready to eat. [ Top of Page ] YOUNG TURKEY: Turkeys of either sex that are less than 8 months of age according to present regulations. [ Top of Page ] Last ...

  6. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  7. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  8. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  9. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Container label. 610.60 Section 610.60 Food and... GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.60 Container label. (a) Full label. The following items shall appear on the label affixed to each container of a product capable of bearing a...

  10. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 16 Commercial Practices 1 2012-01-01 2012-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  11. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 16 Commercial Practices 1 2014-01-01 2014-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  12. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  13. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  14. 16 CFR 460.12 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 16 Commercial Practices 1 2013-01-01 2013-01-01 false Labels. 460.12 Section 460.12 Commercial Practices FEDERAL TRADE COMMISSION TRADE REGULATION RULES LABELING AND ADVERTISING OF HOME INSULATION § 460.12 Labels. If you are a manufacturer, you must label all packages of your insulation. The labels...

  15. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  16. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  17. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium...

  18. 9 CFR 317.4 - Labeling approval.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... labeling of such final labeling has been submitted for approval to the Food Labeling Division, Regulatory... Secretary upon request. (b) The Food Labeling Division shall permit submission for approval of only sketch... Food Labeling Division, Regulatory Programs, Food Safety and Inspection Service, U.S. Department...

  19. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  20. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  1. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  2. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  3. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  4. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  5. MDM2 antagonist nutlin-3a sensitizes tumors to V-ATPase inhibition.

    PubMed

    Schneider, Lina S; Ulrich, Melanie; Lehr, Thorsten; Menche, Dirk; Müller, Rolf; von Schwarzenberg, Karin

    2016-08-01

    Treating cancer is one of the big challenges of this century and it has become evident that single chemotherapeutic treatment is rarely effective. As tumors often carry multiple mutations using combination therapy which addresses different targets seems therefore more beneficial. One of the most frequently mutated genes in tumors is the tumor suppressor p53. Significant work has been put in the development of p53 activators, which are now in clinical studies against diverse cancers. Recently, we could show that inhibition of V-ATPase, a multisubunit proton pump, by archazolid induces p53 protein levels in cancer cells. In this study, we provide evidence that the combination of archazolid with the p53 activator nutlin-3a is synergistically inducing cell death in different p53 wild type tumor cell lines. Mechanistically, this effect could presumably be attributed to reduction of glycolysis as TIGAR mRNA levels were increased and glucose uptake and Glut1 protein levels were reduced. In addition, combination treatment highly activated pro-apoptotic pathways including IGFBP3 and Bax inducing caspase-9 and PARP cleavage. Remarkably, combination of archazolid and nutlin-3a was more efficient in reducing tumor growth compared to single dose treatment in a U87MG mouse model in vivo. Hence, our findings suggest the combination of archazolid and nutlin-3a as a highly promising strategy for the treatment of p53 wild type tumors.

  6. Period2 downregulation inhibits glioma cell apoptosis by activating the MDM2-TP53 pathway

    PubMed Central

    Hechun, Xia; Jun, Wang; Lijian, Zhang; Dede, Ma; Wenbin, Liu; Lei, Yin

    2016-01-01

    The Period2 (Per2) gene is an essential component of the mammalian circadian clock and is strongly linked to glioma occurrence and its response to radiotherapy. Here, we examined the role of Per2 in the response to X-ray-induced DNA damage in U343 glioma cells and in a mouse cancer model. Following low dose X-ray irradiation, we observed that lowering Per2 expression using RNAi reduces DNA damage and cell death in U343 cells and glioma tissue. Additionally, Per2 was associated with increased TP53 activity and was involved in the DNA damage during TP53-mediated apoptosis. These findings suggest that Per2, a core circadian gene, is not only a tumor suppressor gene but can also be regarded as an upstream regulator of TP53. It thus appears that Per2 is an important inhibitor of tumor growth that acts by increasing TP53 expression, DNA damage repair, and apoptosis. PMID:27036047

  7. MDM2 antagonist nutlin-3a sensitizes tumors to V-ATPase inhibition.

    PubMed

    Schneider, Lina S; Ulrich, Melanie; Lehr, Thorsten; Menche, Dirk; Müller, Rolf; von Schwarzenberg, Karin

    2016-08-01

    Treating cancer is one of the big challenges of this century and it has become evident that single chemotherapeutic treatment is rarely effective. As tumors often carry multiple mutations using combination therapy which addresses different targets seems therefore more beneficial. One of the most frequently mutated genes in tumors is the tumor suppressor p53. Significant work has been put in the development of p53 activators, which are now in clinical studies against diverse cancers. Recently, we could show that inhibition of V-ATPase, a multisubunit proton pump, by archazolid induces p53 protein levels in cancer cells. In this study, we provide evidence that the combination of archazolid with the p53 activator nutlin-3a is synergistically inducing cell death in different p53 wild type tumor cell lines. Mechanistically, this effect could presumably be attributed to reduction of glycolysis as TIGAR mRNA levels were increased and glucose uptake and Glut1 protein levels were reduced. In addition, combination treatment highly activated pro-apoptotic pathways including IGFBP3 and Bax inducing caspase-9 and PARP cleavage. Remarkably, combination of archazolid and nutlin-3a was more efficient in reducing tumor growth compared to single dose treatment in a U87MG mouse model in vivo. Hence, our findings suggest the combination of archazolid and nutlin-3a as a highly promising strategy for the treatment of p53 wild type tumors. PMID:27157929

  8. Labeled Cocaine Analogs

    SciTech Connect

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-01-26

    Novel compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)naphthyl Y in .beta. configuration is Y.sub.1 or Y.sub.2, where Y.sub.1 is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, and Y.sub.2 is 2-methanesulfonyloxy ethoxy, 3-methanesulfonyloxy propoxy, 4-methanesulfonyloxy butoxy, 2-methanesulfonyloxy cyclopropoxy, 2 or 3-methanesulfonyloxy cyclobutoxy, 1'methanesulfonyloxy isopropoxy, 1'-fluoro, 3'-methanesulfonyloxy isopropoxy, 1'-methanesulfonyloxy, 3'-fluoro isopropoxy, 1'-methanesulfonyloxy isobutoxy, or 4'-methanesulfonyloxy isobutoxy bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  9. Synthesis Of Labeled Metabolites

    DOEpatents

    Martinez, Rodolfo A.; Silks, III, Louis A.; Unkefer, Clifford J.; Atcher, Robert

    2004-03-23

    The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

  10. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 9 2012-04-01 2012-04-01 false Location and size of symbol on label and labeling. 1302.04 Section 1302.04 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE LABELING... and labeling. The symbol shall be prominently located on the label or the labeling of the...

  11. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 9 2013-04-01 2013-04-01 false Location and size of symbol on label and labeling. 1302.04 Section 1302.04 Food and Drugs DRUG ENFORCEMENT ADMINISTRATION, DEPARTMENT OF JUSTICE LABELING... and labeling. The symbol shall be prominently located on the label or the labeling of the...

  12. Homosexual Labeling by University Youths

    ERIC Educational Resources Information Center

    Nyberg, Kenneth L.; Alston, Jon P.

    1977-01-01

    Details the responses of young, urban, college-educated people on their attitudes toward homosexuals, specifically focusing on issues of public identification and negative labeling as it effects homosexual persons and their behaviors. (Author/RK)

  13. How to read food labels

    MedlinePlus

    ... 1 serving. You should also pay attention to trans fats on any food label. These fats raise "bad" ... foods and desserts. Many fast food restaurants use trans fats for frying. If a food has these fats, ...

  14. Dietary Supplement Label Database (DSLD)

    MedlinePlus

    ... Print Report Error T he Dietary Supplement Label Database (DSLD) is a joint project of the National ... participants in the latest survey in the DSLD database (NHANES): The search options: Quick Search, Browse Dietary ...

  15. Food Labels Tell the Story!

    MedlinePlus

    ... Environment Kids Health Topics Environment & Health Healthy Living Pollution Reduce, Reuse, Recycle Science – How It Works The ... Pay close attention to serving sizes. Products labeled "light" or "lite" must have 1/3 fewer calories ...

  16. Electrothermal branding for embryo labeling.

    PubMed

    Wang, L; Beebe, D J; Williams, A R; Easley, K D

    1997-11-01

    A novel embryo labeling technique based on electrothermal branding is developed. Two types of micro branding irons are fabricated and tested. One utilizes 25 microns tungsten wire as the heating element. The other utilizes surface micromachining techniques to fabricate polysilicon branding irons. The thermal behavior of the branding irons and the heat distributions in the embryos are analytically modeled. Micron-scale labels on unfertilized bovine embryos are achieved.

  17. Multi-focus cluster labeling.

    PubMed

    Eikvil, Line; Jenssen, Tor-Kristian; Holden, Marit

    2015-06-01

    Document collections resulting from searches in the biomedical literature, for instance, in PubMed, are often so large that some organization of the returned information is necessary. Clustering is an efficient tool for organizing search results. To help the user to decide how to continue the search for relevant documents, the content of each cluster can be characterized by a set of representative keywords or cluster labels. As different users may have different interests, it can be desirable with solutions that make it possible to produce labels from a selection of different topical categories. We therefore introduce the concept of multi-focus cluster labeling to give users the possibility to get an overview of the contents through labels from multiple viewpoints. The concept for multi-focus cluster labeling has been established and has been demonstrated on three different document collections. We illustrate that multi-focus visualizations can give an overview of clusters along axes that general labels are not able to convey. The approach is generic and should be applicable to any biomedical (or other) domain with any selection of foci where appropriate focus vocabularies can be established. A user evaluation also indicates that such a multi-focus concept is useful.

  18. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Container label. 610.60 Section 610.60 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.60 Container label. (a) Full label....

  19. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  20. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  1. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  2. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  3. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  4. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  5. 9 CFR 381.132 - Labeling approval.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... been submitted for approval to the Food Labeling Division, Regulatory Programs, Food Safety and... request. (b) The Food Labeling Division shall permit submission for approval of only sketch labeling, as... Labeling Division, Regulatory Programs, Food Safety and Inspection Service, U.S. Department of...

  6. 9 CFR 381.132 - Labeling approval.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... product unless the sketch labeling of such final labeling has been submitted for approval to the Food Labeling Division, Regulatory Programs, Food Safety and Inspection Service, and approved by such division... authorized representative of the Secretary upon request. (b) The Food Labeling Division shall...

  7. 9 CFR 381.132 - Labeling approval.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... been submitted for approval to the Food Labeling Division, Regulatory Programs, Food Safety and... request. (b) The Food Labeling Division shall permit submission for approval of only sketch labeling, as... Labeling Division, Regulatory Programs, Food Safety and Inspection Service, U.S. Department of...

  8. 9 CFR 381.132 - Labeling approval.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... been submitted for approval to the Food Labeling Division, Regulatory Programs, Food Safety and... request. (b) The Food Labeling Division shall permit submission for approval of only sketch labeling, as... Labeling Division, Regulatory Programs, Food Safety and Inspection Service, U.S. Department of...

  9. 9 CFR 381.132 - Labeling approval.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... been submitted for approval to the Food Labeling Division, Regulatory Programs, Food Safety and... request. (b) The Food Labeling Division shall permit submission for approval of only sketch labeling, as... Labeling Division, Regulatory Programs, Food Safety and Inspection Service, U.S. Department of...

  10. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  11. 21 CFR 895.25 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... eliminated by labeling or a change in labeling, or change in advertising if the device is a restricted device... person(s) responsible for the labeling or advertising of the device specifying: (1) The deception or risk... labeling, or change in advertising if the device is a restricted device, necessary to correct the...

  12. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... edges of the label. If you wish to change the format of this single component label, you must petition... no closer than 3/16″ (.48 cm) from the side edges of the label. If you wish to change the format of.... All labels must be capable of withstanding extremes of weather conditions for a period of at least...

  13. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... edges of the label. If you wish to change the format of this single component label, you must petition... no closer than 3/16″ (.48 cm) from the side edges of the label. If you wish to change the format of.... All labels must be capable of withstanding extremes of weather conditions for a period of at least...

  14. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... edges of the label. If you wish to change the format of this single component label, you must petition... no closer than 3/16″ (.48 cm) from the side edges of the label. If you wish to change the format of.... All labels must be capable of withstanding extremes of weather conditions for a period of at least...

  15. Optimal policy for labeling training samples

    NASA Astrophysics Data System (ADS)

    Lipsky, Lester; Lopresti, Daniel; Nagy, George

    2013-01-01

    Confirming the labels of automatically classified patterns is generally faster than entering new labels or correcting incorrect labels. Most labels assigned by a classifier, even if trained only on relatively few pre-labeled patterns, are correct. Therefore the overall cost of human labeling can be decreased by interspersing labeling and classification. Given a parameterized model of the error rate as an inverse power law function of the size of the training set, the optimal splits can be computed rapidly. Projected savings in operator time are over 60% for a range of empirical error functions for hand-printed digit classification with ten different classifiers.

  16. 21 CFR 201.70 - Calcium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... product is more than 3.2 grams: “Ask a doctor before use if you have 1 kidney stones a calcium-restricted... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Calcium labeling. 201.70 Section 201.70 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.70 Calcium labeling. (a) The labeling...

  17. 21 CFR 201.70 - Calcium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... product is more than 3.2 grams: “Ask a doctor before use if you have 1 kidney stones a calcium-restricted... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Calcium labeling. 201.70 Section 201.70 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.70 Calcium labeling. (a) The labeling...

  18. 21 CFR 201.70 - Calcium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... product is more than 3.2 grams: “Ask a doctor before use if you have 1 kidney stones a calcium-restricted... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Calcium labeling. 201.70 Section 201.70 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.70 Calcium labeling. (a) The labeling...

  19. 21 CFR 201.70 - Calcium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... product is more than 3.2 grams: “Ask a doctor before use if you have 1 kidney stones a calcium-restricted... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Calcium labeling. 201.70 Section 201.70 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.70 Calcium labeling. (a) The labeling...

  20. 21 CFR 201.70 - Calcium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... product is more than 3.2 grams: “Ask a doctor before use if you have 1 kidney stones a calcium-restricted... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Calcium labeling. 201.70 Section 201.70 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.70 Calcium labeling. (a) The labeling...

  1. Nutrition Labeling Using a Computer Program

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    The 1990 Nutrition Labeling and Education Act mandated nutritional labeling of most foods. As a result, a large portion of food analysis is performed for nutritional labeling purposes. A food labeling guide and links to the complete nutritional labeling regulations are available online at http://vm.cfsan.fda.gov/˜dms/flg-toc.html. However, interpretation of these regulations and the appropriate usage of rounding rules, available nutrient content claims, reference amounts, and serving size can be difficult.

  2. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  3. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 2 2011-10-01 2011-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  4. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 2 2013-10-01 2013-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  5. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 2 2012-10-01 2012-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  6. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 2 2014-10-01 2014-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  7. Metrics for Labeled Markov Systems

    NASA Technical Reports Server (NTRS)

    Desharnais, Josee; Jagadeesan, Radha; Gupta, Vineet; Panangaden, Prakash

    1999-01-01

    Partial Labeled Markov Chains are simultaneously generalizations of process algebra and of traditional Markov chains. They provide a foundation for interacting discrete probabilistic systems, the interaction being synchronization on labels as in process algebra. Existing notions of process equivalence are too sensitive to the exact probabilities of various transitions. This paper addresses contextual reasoning principles for reasoning about more robust notions of "approximate" equivalence between concurrent interacting probabilistic systems. The present results indicate that:We develop a family of metrics between partial labeled Markov chains to formalize the notion of distance between processes. We show that processes at distance zero are bisimilar. We describe a decision procedure to compute the distance between two processes. We show that reasoning about approximate equivalence can be done compositionally by showing that process combinators do not increase distance. We introduce an asymptotic metric to capture asymptotic properties of Markov chains; and show that parallel composition does not increase asymptotic distance.

  8. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  9. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  10. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  11. Denture labeling: A new approach.

    PubMed

    Bansal, Pardeep K; Sharma, Akshey; Bhanot, Rajesh

    2011-04-01

    The need for denture labeling is important for forensic and social reasons in case patients need to be identified individually. The importance of denture marking has long been acknowledged by the dental profession. Over the years, various denture marking systems have been reported in the literature, but none till date fulfills all the prescribed ADA specifications. A simple, easy, inexpensive procedure for marking accurate identification marks on dentures with a lead foil is described here. The label caring the patient information is incorporated in the acrylic resin during the denture processing.

  12. Rock Music Gets a Label.

    ERIC Educational Resources Information Center

    Cutietta, Robert

    1986-01-01

    A group called Parents Music Resource Center (PMRC) has captured the media spotlight with a proposal to have warning labels placed on music albums containing sexually explicit or violent lyrics. Major record companies have agreed to a version of the PMRC's demands for a one-year trial period, beginning in 1986. (RM)

  13. Nutrition Marketing on Food Labels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nutrition marketing may influence purchasing behavior and thereby be a factor in the obesity epidemic. Very little peer-reviewed research has been published which investigates the relationship between nutrition marketing on food labels and consumer behavior. The purpose of this paper was to give an ...

  14. Revisiting Labels: "Hearing" or Not?

    ERIC Educational Resources Information Center

    Rhoades, Ellen A.

    2010-01-01

    This position paper briefly presents evidence-based findings pertaining to the language of labels for people with hearing loss that relate to stigma, expectation levels, stereotypes, and self-fulfilling prophecies. These constructs are important for auditory-based practitioners, administrators, policymakers, students, families, and persons with…

  15. Psychological effectiveness of carbon labelling

    NASA Astrophysics Data System (ADS)

    Beattie, Geoffrey

    2012-04-01

    Despite the decision by supermarket-giant Tesco to delay its plan to add carbon-footprint information onto all of its 70,000 products, carbon labelling, if carefully designed, could yet change consumer behaviour. However, it requires a new type of thinking about consumers and much additional work.

  16. The Labelling Approach to Deviance.

    ERIC Educational Resources Information Center

    Rains, Prudence M.; Kitsuse, John L.; Duster, Troy; Freidson, Eliot

    2003-01-01

    This reprint of one chapter from the 1975 text, "Issues in the Classification of Children" by Nicholas Hobbs and others, addresses the theoretical, methodological, and empirical issues involved in the "labeling" approach to the sociology of deviance. It examines the social process of classification, the use of classification in social agencies,…

  17. The labeling debate in the United States.

    PubMed

    Marchant, Gary E; Cardineau, Guy A

    2013-01-01

    The mandatory labeling of genetically modified (GM) food has become the predominant policy issue concerning biotechnology in the United States. The controversy over GM labeling is being debated at several different levels and branches of government. At the federal level, the Food and Drug Administration, which has primary jurisdiction over food safety and labeling, has steadfastly refused to require labeling of GM foods since 1992 based on its conclusion that GM foods as a category present no unique or higher risks than other foods. Proposed legislation has been repeatedly introduced in the US. Congress over the years to mandate GM labeling, but has made very little progress. With federal labeling requirements apparently stalled, the main activity has switched to the state level, where numerous individual states are considering mandatory GM labeling, either through legislation or proposition. The debate over GM labeling, at both the federal and state levels, has focused on five issues: (1) public opinion; (2) the legality of labeling requirements; (3) the risks and benefits of GM foods; (4) the costs and burdens of GM labeling; and (5) consumer choice. While the pro-labeling forces argue that all of these factors weigh in favor of mandatory GM labeling, a more careful evaluation of the evidence finds that all five factors weigh decisively against mandatory GM labeling requirements.

  18. 21 CFR 211.125 - Labeling issuance.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... identity and conformity to the labeling specified in the master or batch production records. (c) Procedures... discrepancies shall be investigated in accordance with § 211.192. Labeling reconciliation is waived for cut...

  19. 21 CFR 211.125 - Labeling issuance.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... identity and conformity to the labeling specified in the master or batch production records. (c) Procedures... discrepancies shall be investigated in accordance with § 211.192. Labeling reconciliation is waived for cut...

  20. 21 CFR 211.125 - Labeling issuance.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... identity and conformity to the labeling specified in the master or batch production records. (c) Procedures... discrepancies shall be investigated in accordance with § 211.192. Labeling reconciliation is waived for cut...

  1. 21 CFR 211.125 - Labeling issuance.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... identity and conformity to the labeling specified in the master or batch production records. (c) Procedures... discrepancies shall be investigated in accordance with § 211.192. Labeling reconciliation is waived for cut...

  2. 21 CFR 211.125 - Labeling issuance.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... identity and conformity to the labeling specified in the master or batch production records. (c) Procedures... discrepancies shall be investigated in accordance with § 211.192. Labeling reconciliation is waived for cut...

  3. 40 CFR 205.158 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... TRANSPORTATION EQUIPMENT NOISE EMISSION CONTROLS Motorcycles § 205.158 Labeling requirements. (a)(1) The... information: (i) The label heading: Motorcycle Noise Emission Control Information; (ii) The statement: This ___ (model year) ___ (model specific code) motorcycle, ___ (serial number), meets EPA noise...

  4. 40 CFR 1033.135 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... THAT IT IS REMANUFACTURED, EXCEPT AS ALLOWED BY 40 CFR 1033.750.” (3) Label diesel-fueled locomotives... locomotives certified for use with both LSD and ULSD. (c) Engine labels. (1) For engines not...

  5. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  6. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  7. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  8. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  9. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  10. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  11. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  12. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  13. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  14. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  15. 27 CFR 19.437 - Labels.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... affix a label showing the following information: (1) The proprietor's name and plant number; (2) The... paragraph (a) of this section is not required when the sample container bears a label approved under part...

  16. 27 CFR 19.704 - Labels.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... containers bear an approved label pursuant to 27 CFR Part 5 and subpart S of this part and the sample is... spirits to be withdrawn under the provisions of § 19.701, the proprietor shall affix a label showing...

  17. 27 CFR 19.437 - Labels.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... affix a label showing the following information: (1) The proprietor's name and plant number; (2) The... paragraph (a) of this section is not required when the sample container bears a label approved under part...

  18. 27 CFR 19.437 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... affix a label showing the following information: (1) The proprietor's name and plant number; (2) The... paragraph (a) of this section is not required when the sample container bears a label approved under part...

  19. 27 CFR 19.437 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... affix a label showing the following information: (1) The proprietor's name and plant number; (2) The... paragraph (a) of this section is not required when the sample container bears a label approved under part...

  20. 49 CFR 172.407 - Label specifications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Register citations affecting § 172.407, see the List of CFR Sections Affected, which appears in the Finding... the CORROSIVE label. (iii) White may be used for the symbol for the ORGANIC PEROXIDE label. (3)...

  1. 21 CFR 225.80 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... labeling, including placards, upon receipt from the printer shall be proofread against the Master Record... responsible individual, and kept for 1 year after all the labels from that batch have been used. (3) In...

  2. 21 CFR 225.80 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... labeling, including placards, upon receipt from the printer shall be proofread against the Master Record... responsible individual, and kept for 1 year after all the labels from that batch have been used. (3) In...

  3. 99mTc: Labeling Chemistry and Labeled Compounds

    NASA Astrophysics Data System (ADS)

    Alberto, R.; Abram, U.

    This chapter reviews the radiopharmaceutical chemistry of technetium related to the synthesis of perfusion agents and to the labeling of receptor-binding biomolecules. To understand the limitations of technetium chemistry imposed by future application of the complexes in nuclear medicine, an introductory section analyzes the compulsory requirements to be considered when facing the incentive of introducing a novel radiopharmaceutical into the market. Requirements from chemistry, routine application, and market are discussed. In a subsequent section, commercially available 99mTc-based radiopharmaceuticals are treated. It covers the complexes in use for imaging the most important target organs such as heart, brain, or kidney. The commercially available radiopharmaceuticals fulfill the requirements outlined earlier and are discussed with this background. In a following section, the properties and perspectives of the different generations of radiopharmaceuticals are described in a general way, covering characteristics for perfusion agents and for receptor-specific molecules. Technetium chemistry for the synthesis of perfusion agents and the different labeling approaches for target-specific biomolecules are summarized. The review comprises a general introduction to the common approaches currently in use, employing the N x S4-x , [3+1] and 2-hydrazino-nicotinicacid (HYNIC) method as well as more recent strategies such as the carbonyl and the TcN approach. Direct labeling without the need of a bifunctional chelator is briefly reviewed as well. More particularly, recent developments in the labeling of concrete targeting molecules, the second generation of radiopharmaceuticals, is then discussed and prominent examples with antibodies/peptides, neuroreceptor targeting small molecules, myocardial imaging agents, vitamins, thymidine, and complexes relevant to multidrug resistance are given. In addition, a new approach toward peptide drug development is described. The section

  4. Automated labeling in document images

    NASA Astrophysics Data System (ADS)

    Kim, Jongwoo; Le, Daniel X.; Thoma, George R.

    2000-12-01

    The National Library of Medicine (NLM) is developing an automated system to produce bibliographic records for its MEDLINER database. This system, named Medical Article Record System (MARS), employs document image analysis and understanding techniques and optical character recognition (OCR). This paper describes a key module in MARS called the Automated Labeling (AL) module, which labels all zones of interest (title, author, affiliation, and abstract) automatically. The AL algorithm is based on 120 rules that are derived from an analysis of journal page layouts and features extracted from OCR output. Experiments carried out on more than 11,000 articles in over 1,000 biomedical journals show the accuracy of this rule-based algorithm to exceed 96%.

  5. Homosexual Labeling and the Male Role.

    ERIC Educational Resources Information Center

    Karr, Rodney G.

    1978-01-01

    In this study, men were perceived to be less masculine and less preferred as fellow participants if they were labeled homosexual. The man responsible for the primary labeling of the homosexual was perceived as more masculine and more sociable when he labeled the homosexual than when he did not. (Author/WI)

  6. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  7. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  8. 40 CFR 600.301 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ECONOMY AND GREENHOUSE GAS EXHAUST EMISSIONS OF MOTOR VEHICLES Fuel Economy Labeling § 600.301 Labeling... each dealer shall maintain or cause to be maintained on each automobile: (1) A general fuel economy... vehicle for which a specific label is requested which has a combined FTP/HFET-based fuel economy value,...

  9. 40 CFR 600.301 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... ECONOMY AND GREENHOUSE GAS EXHAUST EMISSIONS OF MOTOR VEHICLES Fuel Economy Labeling § 600.301 Labeling... each dealer shall maintain or cause to be maintained on each automobile: (1) A general fuel economy... vehicle for which a specific label is requested which has a combined FTP/HFET-based fuel economy value,...

  10. 40 CFR 600.301 - Labeling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ECONOMY AND GREENHOUSE GAS EXHAUST EMISSIONS OF MOTOR VEHICLES Fuel Economy Labeling § 600.301 Labeling... each dealer shall maintain or cause to be maintained on each automobile: (1) A general fuel economy... vehicle for which a specific label is requested which has a combined FTP/HFET-based fuel economy value,...

  11. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  12. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  13. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  14. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Required by U.S. EPA regulation 40 CFR part 211, subpart ___.” EC01FE92.055 ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Label content. 211.104 Section 211.104... LABELING General Provisions § 211.104 Label content. The following data and information must be on...

  15. 40 CFR 1036.135 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ....135 Labeling. Label your engines as described in 40 CFR 86.007-35(a)(3), with the following additional information: (a) (b) Identify the emission control system. Use terms and abbreviations as described in 40 CFR... labeling requirement to be consistent with the intent of 40 CFR part 1037....

  16. 40 CFR 1036.135 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ....135 Labeling. Label your engines as described in 40 CFR 86.007-35(a)(3), with the following additional information: (a) (b) Identify the emission control system. Use terms and abbreviations as described in 40 CFR... labeling requirement to be consistent with the intent of 40 CFR part 1037....

  17. 16 CFR 306.12 - Labels.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... biodiesel, biomass-based diesel, biodiesel blends, and biomass-based diesel blends. The label is 3 inches (7... the black band. Directly underneath the black band, the label shall read “contains biomass-based... the side edges of the label. (5) For biomass-based diesel blends containing more than 5 percent and...

  18. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Device labeling. 820.120 Section 820.120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES QUALITY SYSTEM REGULATION Labeling and Packaging Control § 820.120 Device labeling. Each...

  19. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... TREASURY ALCOHOL PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container of concentrate will have affixed thereto, before transfer, a label identifying the product and... 27 Alcohol, Tobacco Products and Firearms 1 2014-04-01 2014-04-01 false Label. 18.55 Section...

  20. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... following items shall appear on the label affixed to each container of a product capable of bearing a full label: (1) The proper name of the product; (2) The name, address, and license number of manufacturer; (3... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Container label. 610.60 Section 610.60 Food...

  1. 27 CFR 19.604 - Caution label.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Caution label. 19.604... OF THE TREASURY LIQUORS DISTILLED SPIRITS PLANTS Containers and Marks Marks § 19.604 Caution label... denaturer may be printed on such label, but no other extraneous matter will be permitted thereon without...

  2. 40 CFR 763.171 - Labeling requirements.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... cannot be removed without defacing or destroying them. Product labels shall appear as in paragraph (d)(2... packaging, the label must be attached to the innermost layer adjacent to the product. If the innermost layer... product's innermost layer of product wrapping or packaging, or a label must be attached to the next...

  3. 40 CFR 211.105 - Label format.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Label format. 211.105 Section 211.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.105 Label format. (a) Unless specified otherwise in other...

  4. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... TREASURY ALCOHOL PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container of concentrate will have affixed thereto, before transfer, a label identifying the product and... 27 Alcohol, Tobacco Products and Firearms 1 2013-04-01 2013-04-01 false Label. 18.55 Section...

  5. 27 CFR 18.55 - Label.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... TREASURY LIQUORS PRODUCTION OF VOLATILE FRUIT-FLAVOR CONCENTRATE Operations § 18.55 Label. Each container of concentrate will have affixed thereto, before transfer, a label identifying the product and... 27 Alcohol, Tobacco Products and Firearms 1 2012-04-01 2012-04-01 false Label. 18.55 Section...

  6. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... following items shall appear on the label affixed to each container of a product capable of bearing a full label: (1) The proper name of the product; (2) The name, address, and license number of manufacturer; (3... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Container label. 610.60 Section 610.60 Food...

  7. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    .... Each label shall be identified as to: (1) Name and product code number as it appears on the product... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Label records. 116.3 Section 116.3..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label...

  8. 40 CFR 211.105 - Label format.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Label format. 211.105 Section 211.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.105 Label format. (a) Unless specified otherwise in other...

  9. 40 CFR 211.105 - Label format.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Label format. 211.105 Section 211.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.105 Label format. (a) Unless specified otherwise in other...

  10. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    .... Each label shall be identified as to: (1) Name and product code number as it appears on the product... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Label records. 116.3 Section 116.3..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label...

  11. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    .... Each label shall be identified as to: (1) Name and product code number as it appears on the product... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Label records. 116.3 Section 116.3..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label...

  12. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... following items shall appear on the label affixed to each container of a product capable of bearing a full label: (1) The proper name of the product; (2) The name, address, and license number of manufacturer; (3... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Container label. 610.60 Section 610.60 Food...

  13. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    .... Each label shall be identified as to: (1) Name and product code number as it appears on the product... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Label records. 116.3 Section 116.3..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label...

  14. 21 CFR 1271.250 - Labeling controls.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Labeling controls. 1271.250 Section 1271.250 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.250 Labeling controls. (a) General. You must establish and maintain procedures to control the labeling of HCT/Ps. You must...

  15. 10 CFR 61.57 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Labeling. 61.57 Section 61.57 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Technical Requirements for Land Disposal Facilities § 61.57 Labeling. Each package of waste must be clearly labeled...

  16. 10 CFR 61.57 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false Labeling. 61.57 Section 61.57 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Technical Requirements for Land Disposal Facilities § 61.57 Labeling. Each package of waste must be clearly labeled...

  17. 10 CFR 61.57 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false Labeling. 61.57 Section 61.57 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Technical Requirements for Land Disposal Facilities § 61.57 Labeling. Each package of waste must be clearly labeled...

  18. 10 CFR 61.57 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false Labeling. 61.57 Section 61.57 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Technical Requirements for Land Disposal Facilities § 61.57 Labeling. Each package of waste must be clearly labeled...

  19. 10 CFR 61.57 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false Labeling. 61.57 Section 61.57 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Technical Requirements for Land Disposal Facilities § 61.57 Labeling. Each package of waste must be clearly labeled...

  20. 9 CFR 317.4 - Labeling approval.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... approval to the Food Labeling Division, Regulatory Programs, Food Safety and Inspection Service, and... approval as set forth in § 317.4(a) shall be submitted in duplicate to the Food Labeling Division... deemed deficient in some particular may be granted by the Food Labeling Division. Temporary approvals...

  1. 9 CFR 317.4 - Labeling approval.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... approval to the Food Labeling Division, Regulatory Programs, Food Safety and Inspection Service, and... approval as set forth in § 317.4(a) shall be submitted in duplicate to the Food Labeling Division... deemed deficient in some particular may be granted by the Food Labeling Division. Temporary approvals...

  2. 9 CFR 317.4 - Labeling approval.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... approval to the Food Labeling Division, Regulatory Programs, Food Safety and Inspection Service, and... approval as set forth in § 317.4(a) shall be submitted in duplicate to the Food Labeling Division... deemed deficient in some particular may be granted by the Food Labeling Division. Temporary approvals...

  3. 75 FR 81943 - Appliance Labeling Rule

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-29

    ... for its new light bulb labeling requirements (published on July 19, 2010, 75 FR 41696) to January 1... (75 FR 41696), the Commission published amendments to the Appliance Labeling Rule (Rule) creating new... new labeling requirements. \\5\\ 59 FR 25176 (May 13, 1994). \\6\\ Pursuant to the revised rule, after...

  4. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  5. Cigarette Warning Labels as Educational Devices.

    ERIC Educational Resources Information Center

    Newman, Ian M.; And Others

    This paper reports an investigation on the educational impact of warning labels on cigarette packages on adolescents. Subjects were asked to identify the locations of warning labels on cigarette packages and advertising and to restate the warning label. Results indicated that official warnings may be well known in general terms but poorly known in…

  6. 21 CFR 701.11 - Identity labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Identity labeling. 701.11 Section 701.11 Food and... COSMETIC LABELING Package Form § 701.11 Identity labeling. (a) The principal display panel of a cosmetic in package form shall bear as one of its principal features a statement of the identity of the commodity....

  7. 40 CFR 205.158 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Labeling requirements. 205.158 Section 205.158 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS... color that contrasts with the background of the label. (5) The label must contain the...

  8. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  9. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Required by U.S. EPA regulation 40 CFR part 211, subpart ___.” EC01FE92.055 ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Label content. 211.104 Section 211.104... LABELING General Provisions § 211.104 Label content. The following data and information must be on...

  10. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 3 2012-01-01 2012-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  11. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 3 2013-01-01 2013-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  12. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 3 2014-01-01 2014-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  13. 21 CFR 349.80 - Professional labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... labeling. The labeling of any OTC ophthalmic demulcent drug product provided to health professionals (but... 21 Food and Drugs 5 2011-04-01 2011-04-01 false Professional labeling. 349.80 Section 349.80 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS...

  14. 21 CFR 349.80 - Professional labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... labeling. The labeling of any OTC ophthalmic demulcent drug product provided to health professionals (but... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Professional labeling. 349.80 Section 349.80 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS...

  15. 21 CFR 357.280 - Professional labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 5 2013-04-01 2013-04-01 false Professional labeling. 357.280 Section 357.280 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS... Drug Products § 357.280 Professional labeling. The labeling provided to health professionals (but...

  16. Learning Words from Labeling and Directive Speech

    ERIC Educational Resources Information Center

    Callanan, Maureen A.; Akhtar, Nameera; Sussman, Lisa

    2014-01-01

    Despite the common intuition that labeling may be the best way to teach a new word to a child, systematic testing is needed of the prediction that children learn words better from labeling utterances than from directive utterances. Two experiments compared toddlers' label learning in the context of hearing words used in directive versus labeling…

  17. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Label contents. 47.42 Section 47.42 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.42 Label contents. When an operator...

  18. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment. The... hazardous substance if furnished with: (a) Complete labeling or proposed labeling, which may be in...

  19. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment. The... hazardous substance if furnished with: (a) Complete labeling or proposed labeling, which may be in...

  20. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment. The... hazardous substance if furnished with: (a) Complete labeling or proposed labeling, which may be in...

  1. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment. The... hazardous substance if furnished with: (a) Complete labeling or proposed labeling, which may be in...

  2. 16 CFR 1500.128 - Label comment.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Commercial Practices CONSUMER PRODUCT SAFETY COMMISSION FEDERAL HAZARDOUS SUBSTANCES ACT REGULATIONS HAZARDOUS SUBSTANCES AND ARTICLES; ADMINISTRATION AND ENFORCEMENT REGULATIONS § 1500.128 Label comment. The... hazardous substance if furnished with: (a) Complete labeling or proposed labeling, which may be in...

  3. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be completely... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling....

  4. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be completely... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling....

  5. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... manufacturer or private labeler, pursuant to § 431.36(f), and applicable to that motor. Such CC number must be... EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1) Required information. The permanent nameplate of an electric motor for which standards are prescribed in § 431.25...

  6. Gender, status, and psychiatric labels.

    PubMed

    Kroska, Amy; Harkness, Sarah K; Brown, Ryan P; Thomas, Lauren S

    2015-11-01

    We examine a key modified labeling theory proposition-that a psychiatric label increases vulnerability to competence-based criticism and rejection-within task- and collectively oriented dyads comprised of same-sex individuals with equivalent education. Drawing on empirical work that approximates these conditions, we expect the proposition to hold only among men. We also expect education, operationalized with college class standing, to moderate the effects of gender by reducing men's and increasing women's criticism and rejection. But, we also expect the effect of education to weaken when men work with a psychiatric patient. As predicted, men reject suggestions from teammates with a psychiatric history more frequently than they reject suggestions from other teammates, while women's resistance to influence is unaffected by their teammate's psychiatric status. Men also rate psychiatric patient teammates as less powerful but no lower in status than other teammates, while women's teammate assessments are unaffected by their teammate's psychiatric status. Also as predicted, education reduces men's resistance to influence when their teammate has no psychiatric history. Education also increases men's ratings of their teammate's power, as predicted, but has no effect on women's resistance to influence or teammate ratings. We discuss the implications of these findings for the modified labeling theory of mental illness and status characteristics theory.

  7. 27 CFR 19.642 - Statements required on labels under an exemption from label approval.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... meaning given, and be stated in the manner provided in 27 CFR part 5. (Sec. 201, Pub. L. 85-859, 72 Stat... labels under an exemption from label approval. 19.642 Section 19.642 Alcohol, Tobacco Products and... PLANTS Liquor Bottle and Label Requirements Bottle Label Requirements § 19.642 Statements required...

  8. Abandoning a label doesn’t make it disappear: The perseverance of labeling effects

    PubMed Central

    Foroni, Francesco; Rothbart, Myron

    2012-01-01

    Labels exert strong influence on perception and judgment. The present experiment examines the possibility that such effects may persist even when labels are abandoned. Participants judged the similarity of pairs of silhouette drawings of female body types, ordered on a continuum from very thin to very heavy, under conditions where category labels were, and were not, superimposed on the ordered stimuli. Consistent with earlier research, labels had strong effects on perceived similarity, with silhouettes sharing the same label judged as more similar than those having different labels. Moreover, when the labels were removed and no longer present, the effect of the labels, although diminished, persisted. It did not make any difference whether the labels were simply abandoned or, in addition, had their validity challenged. The results are important for our understanding of categorization and labeling processes. The potential theoretical and practical implications of these results for social processes are discussed. PMID:23105148

  9. Stigma of a label: educational expectations for high school students labeled with learning disabilities.

    PubMed

    Shifrer, Dara

    2013-01-01

    Poorer outcomes for youth labeled with learning disabilities (LDs) are often attributed to the student's own deficiencies or cumulative disadvantage; but the more troubling possibility is that special education placement limits rather than expands these students' opportunities. Labeling theory partially attributes the poorer outcomes of labeled persons to stigma related to labels. This study uses data on approximately 11,740 adolescents and their schools from the Education Longitudinal Survey of 2002 to determine if stigma influences teachers' and parents' educational expectations for students labeled with LDs and labeled adolescents' expectations for themselves. Supporting the predictions of labeling theory, teachers and parents are more likely to perceive disabilities in, and hold lower educational expectations for labeled adolescents than for similarly achieving and behaving adolescents not labeled with disabilities. The negative effect of being labeled with LDs on adolescents' educational expectations is partially mechanized through parents' and particularly teachers' lower expectations.

  10. Stigma of a label: educational expectations for high school students labeled with learning disabilities.

    PubMed

    Shifrer, Dara

    2013-01-01

    Poorer outcomes for youth labeled with learning disabilities (LDs) are often attributed to the student's own deficiencies or cumulative disadvantage; but the more troubling possibility is that special education placement limits rather than expands these students' opportunities. Labeling theory partially attributes the poorer outcomes of labeled persons to stigma related to labels. This study uses data on approximately 11,740 adolescents and their schools from the Education Longitudinal Survey of 2002 to determine if stigma influences teachers' and parents' educational expectations for students labeled with LDs and labeled adolescents' expectations for themselves. Supporting the predictions of labeling theory, teachers and parents are more likely to perceive disabilities in, and hold lower educational expectations for labeled adolescents than for similarly achieving and behaving adolescents not labeled with disabilities. The negative effect of being labeled with LDs on adolescents' educational expectations is partially mechanized through parents' and particularly teachers' lower expectations. PMID:24311756

  11. Use of Symbols in Labeling. Final rule.

    PubMed

    2016-06-15

    The Food and Drug Administration (FDA or the Agency) is issuing this final rule revising its medical device and certain biological product labeling regulations to explicitly allow for the optional inclusion of graphical representations of information, or symbols, in labeling (including labels) without adjacent explanatory text (referred to in this document as "stand-alone symbols") if certain requirements are met. The final rule also specifies that the use of symbols, accompanied by adjacent explanatory text continues to be permitted. FDA is also revising its prescription device labeling regulations to allow the use of the symbol statement "Rx only" or "[rx] only" in the labeling for prescription devices.

  12. Use of Symbols in Labeling. Final rule.

    PubMed

    2016-06-15

    The Food and Drug Administration (FDA or the Agency) is issuing this final rule revising its medical device and certain biological product labeling regulations to explicitly allow for the optional inclusion of graphical representations of information, or symbols, in labeling (including labels) without adjacent explanatory text (referred to in this document as "stand-alone symbols") if certain requirements are met. The final rule also specifies that the use of symbols, accompanied by adjacent explanatory text continues to be permitted. FDA is also revising its prescription device labeling regulations to allow the use of the symbol statement "Rx only" or "[rx] only" in the labeling for prescription devices. PMID:27311137

  13. 46 CFR 160.133-17 - Marking and labeling.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Marking and labeling. (a) Each hook body of a release mechanism must be marked with a plate or label...) The plate or label must be in English, but may also be in other languages. (c) The plate or label...

  14. 27 CFR 4.32 - Mandatory label information.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... October 6, 1984. (d) (e) Declaration of sulfites. There shall be stated on a front label, back label, strip label or neck label, the statement “Contains sulfites” or “Contains (a) sulfiting agent(s)” or...

  15. Hemoglobin Labeled by Radioactive Lysine

    DOE R&D Accomplishments Database

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  16. Label-free cell profiling.

    PubMed

    Schasfoort, Richard B M; Bentlage, Arthur E H; Stojanovic, Ivan; van der Kooi, Alex; van der Schoot, Ellen; Terstappen, Leon W M M; Vidarsson, Gestur

    2013-08-01

    A surface plasmon resonance (SPR) array imaging method is outlined for label-free cell profiling. Red blood cells (RBCs) were injected into a flow chamber on top of a spotted sensor surface. Spots contained antibodies to various RBC membrane antigens. A typical sensorgram showed an initial response corresponding to cell sedimentation (S) followed by a specific upward response (T) corresponding to specific binding of cells during a critical wash step. The full analysis cycle for RBC profiling was less than 6 min. The sensor surface could be regenerated at least 100 times, allowing the determination of a cell surface antigen profile of RBCs.

  17. Particle-based labeling: Fast point-feature labeling without obscuring other visual features.

    PubMed

    Luboschik, Martin; Schumann, Heidrun; Cords, Hilko

    2008-01-01

    In many information visualization techniques, labels are an essential part to communicate the visualized data. To preserve the expressiveness of the visual representation, a placed label should neither occlude other labels nor visual representatives (e.g., icons, lines) that communicate crucial information. Optimal, non-overlapping labeling is an NP-hard problem. Thus, only a few approaches achieve a fast non-overlapping labeling in highly interactive scenarios like information visualization. These approaches generally target the point-feature label placement (PFLP) problem, solving only label-label conflicts. This paper presents a new, fast, solid and flexible 2D labeling approach for the PFLP problem that additionally respects other visual elements and the visual extent of labeled features. The results (number of placed labels, processing time) of our particle-based method compare favorably to those of existing techniques. Although the esthetic quality of non-real-time approaches may not be achieved with our method, it complies with practical demands and thus supports the interactive exploration of information spaces. In contrast to the known adjacent techniques, the flexibility of our technique enables labeling of dense point clouds by the use of non-occluding distant labels. Our approach is independent of the underlying visualization technique, which enables us to demonstrate the application of our labeling method within different information visualization scenarios.

  18. Enzyme-gold affinity labelling of cellulose.

    PubMed

    Berg, R H; Erdos, G W; Gritzali, M; Brown, R D

    1988-04-01

    The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Label-free photoacoustic nanoscopy

    PubMed Central

    Danielli, Amos; Maslov, Konstantin; Garcia-Uribe, Alejandro; Winkler, Amy M.; Li, Chiye; Wang, Lidai; Chen, Yun; Dorn, Gerald W.; Wang, Lihong V.

    2014-01-01

    Abstract. Super-resolution microscopy techniques—capable of overcoming the diffraction limit of light—have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules. PMID:25104412

  20. Chemical kin label in seabirds.

    PubMed

    Célérier, Aurélie; Bon, Cécile; Malapert, Aurore; Palmas, Pauline; Bonadonna, Francesco

    2011-12-23

    Chemical signals yield critical socio-ecological information in many animals, such as species, identity, social status or sex, but have been poorly investigated in birds. Recent results showed that chemical signals are used to recognize their nest and partner by some petrel seabirds whose olfactory anatomy is well developed and which possess a life-history propitious to olfactory-mediated behaviours. Here, we investigate whether blue petrels (Halobaena caerulea) produce some chemical labels potentially involved in kin recognition and inbreeding avoidance. To overcome methodological constraints of chemical analysis and field behavioural experiments, we used an indirect behavioural approach, based on mice olfactory abilities in discriminating odours. We showed that mice (i) can detect odour differences between individual petrels, (ii) perceive a high odour similarity between a chick and its parents, and (iii) perceive this similarity only before fledging but not during the nestling developmental stage. Our results confirm the existence of an individual olfactory signature in blue petrels and show for the first time, to our knowledge, that birds may exhibit an olfactory kin label, which may have strong implications for inbreeding avoidance. PMID:21525047

  1. Inulin determination for food labeling.

    PubMed

    Zuleta, A; Sambucetti, M E

    2001-10-01

    Inulin and oligofructose exhibit valuable nutritional and functional attributes, so they are used as supplements as soluble fiber or as macronutrient substitutes. As classic analytical methods for dietary fiber measurement are not effective, several specific methods have been proposed. These methods measure total fructans and are based on one or more enzymatic sample treatments and determination of released sugars. To determine inulin for labeling purposes, we developed an easy and rapid anion-exchange high-performance liquid chromatography (HPLC) method following water extraction of inulin. HPLC conditions included an Aminex HPX- 87C column (Bio-Rad), deionized water at 85 degrees C as the mobile phase and a refractive index detector. The tested foods included tailor-made food products containing known amounts of inulin and commercial products (cookies, milk, ice creams, cheese, and cereal bars). The average recovery was 97%, and the coefficient of variation ranged from 1.1 to 5% in the food matrixes. The obtained results showed that this method provides an easier, faster and cheaper alternative than previous techniques for determining inulin with enough accuracy and precision for routine labeling purposes by direct determination of inulin by HPLC with refractive index detection. PMID:11599989

  2. Label-free photoacoustic nanoscopy.

    PubMed

    Danielli, Amos; Maslov, Konstantin; Garcia-Uribe, Alejandro; Winkler, Amy M; Li, Chiye; Wang, Lidai; Chen, Yun; Dorn, Gerald W; Wang, Lihong V

    2014-08-01

    Super-resolution microscopy techniques - capable of overcoming the diffraction limit of light - have opened new opportunities to explore subcellular structures and dynamics not resolvable in conventional far-field microscopy. However, relying on staining with exogenous fluorescent markers, these techniques can sometimes introduce undesired artifacts to the image, mainly due to large tagging agent sizes and insufficient or variable labeling densities. By contrast, the use of endogenous pigments allows imaging of the intrinsic structures of biological samples with unaltered molecular constituents. Here, we report label-free photoacoustic (PA) nanoscopy, which is exquisitely sensitive to optical absorption, with an 88 nm resolution. At each scanning position, multiple PA signals are successively excited with increasing laser pulse energy. Because of optical saturation or nonlinear thermal expansion, the PA amplitude depends on the nonlinear incident optical fluence. The high-order dependence, quantified by polynomial fitting, provides super-resolution imaging with optical sectioning. PA nanoscopy is capable of super-resolution imaging of either fluorescent or nonfluorescent molecules.

  3. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... accuracy including, where applicable, the correct unique device identifier (UDI) or universal product code... manner that provides proper identification and is designed to prevent mixups. (d) Labeling...

  4. Simultaneous segmentation and statistical label fusion

    NASA Astrophysics Data System (ADS)

    Asman, Andrew J.; Landman, Bennett A.

    2012-02-01

    Labeling or segmentation of structures of interest in medical imaging plays an essential role in both clinical and scientific understanding. Two of the common techniques to obtain these labels are through either fully automated segmentation or through multi-atlas based segmentation and label fusion. Fully automated techniques often result in highly accurate segmentations but lack the robustness to be viable in many cases. On the other hand, label fusion techniques are often extremely robust, but lack the accuracy of automated algorithms for specific classes of problems. Herein, we propose to perform simultaneous automated segmentation and statistical label fusion through the reformulation of a generative model to include a linkage structure that explicitly estimates the complex global relationships between labels and intensities. These relationships are inferred from the atlas labels and intensities and applied to the target using a non-parametric approach. The novelty of this approach lies in the combination of previously exclusive techniques and attempts to combine the accuracy benefits of automated segmentation with the robustness of a multi-atlas based approach. The accuracy benefits of this simultaneous approach are assessed using a multi-label multi-atlas whole-brain segmentation experiment and the segmentation of the highly variable thyroid on computed tomography images. The results demonstrate that this technique has major benefits for certain types of problems and has the potential to provide a paradigm shift in which the lines between statistical label fusion and automated segmentation are dramatically blurred.

  5. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... AND PACKAGING REQUIREMENTS FOR CONTROLLED SUBSTANCES § 1302.04 Location and size of symbol on label... substance. The symbol on labels shall be clear and large enough to afford easy identification of...

  6. Labeled nucleotide phosphate (NP) probes

    SciTech Connect

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2009-02-03

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  7. F-18 labeled 3-fluorodiazepam

    SciTech Connect

    Luxen, A.; Barrio, J.R.; Bida, G.T.; Satyamurthy, N.; Phelps, M.E.

    1985-05-01

    3-Fluorodiazepam is a new and potent antianxiety agent with prolonged action. The authors found that molecular fluorine (0.5% in Ne) reacts cleanly with diazepam in freon or chloroform at room temperature to produce 3-fluorodiazepam in good yields. Successful syntheses have employed 2:1 to 5:1 molar ratios diazepam: fluorine to minimize the formation of byproducts. (/sup 18/F) 3-Fluorodiazepam, a potential candidate for PET studies, (specific activity 3-5 Ci/mmol) has been synthesized from /sup 18/F-F/sub 2/ using the same procedure, followed by column chromatographic purification (Silicagel, dichloromethane: ethyl acetate, 5:1) with a radiochemical yield of 12-20% (50% maximum) and a chemical and radiochemical purity >99% as judged by reversed-phase high pressure liquid chromatography analysis (Ultrasyl octyl column, 10 ..mu.. m, 4.6 x 250 mm i.d., 60% MeOH 40% water; flow rate, 1.0 ml/min; retention time for (/sup 18/F) fluorodiazepam, 11.4 min; for diazepam, 13.5 min; radioactivity and ultraviolet detectors). Lower radiochemical yields (5-7%), and significant formation of by-products were observed when (/sup 18/F)acetylhypofluorite, prepared in the gasphase, was used as the reagent. Readily accessible routes to /sup 18/F-labeled benzodiazepines of higher specific activity were also investigated. Approaches to the synthesis of high specific activity (>200 Ci/mmol) (/sup 18/F)3-fluorodiazepam involve nucleophilic displacement at carbon-3 (e.g. from 3-chlorodiazepam) with (/sup 18/F)fluoride ion. The results presented here demonstrate the synthetic accessibility of /sup 18/F-labeled benzodiazepines for application in neurotransmitter ligand studies with PET.

  8. Diagnostic enteral gastrointestinal radiopaque human prescription drugs class labeling guideline for professional labeling. Final report

    SciTech Connect

    Chun, M.Y.

    1982-09-21

    The Food and Drug Administration (FDA) announces the availability of a class labeling guideline for the professional labeling of diagnostic enteral gastrointestinal radiopaque agents. Class labeling is appropriate for these products because they are all closely related in chemical structure, pharmacology, therapeutic activity, and adverse reactions. The guideline is intended to promote the use of identical professional labeling for each member of the diagnostic enteral gastrointestinal radiopaque drug class.

  9. The reappropriation of stigmatizing labels: the reciprocal relationship between power and self-labeling.

    PubMed

    Galinsky, Adam D; Wang, Cynthia S; Whitson, Jennifer A; Anicich, Eric M; Hugenberg, Kurt; Bodenhausen, Galen V

    2013-10-01

    We present a theoretical model of reappropriation--taking possession of a slur previously used exclusively by dominant groups to reinforce another group's lesser status. Ten experiments tested this model and established a reciprocal relationship between power and self-labeling with a derogatory group term. We first investigated precursors to self-labeling: Group, but not individual, power increased participants' willingness to label themselves with a derogatory term for their group. We then examined the consequences of such self-labeling for both the self and observers. Self-labelers felt more powerful after self-labeling, and observers perceived them and their group as more powerful. Finally, these labels were evaluated less negatively after self-labeling, and this attenuation of stigma was mediated by perceived power. These effects occurred only for derogatory terms (e.g., queer, bitch), and not for descriptive (e.g., woman) or majority-group (e.g., straight) labels. These results suggest that self-labeling with a derogatory label can weaken the label's stigmatizing force.

  10. 76 FR 46671 - Food Labeling; Gluten-Free Labeling of Foods; Reopening of the Comment Period

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-03

    ... could use to establish a gluten threshold level to define the food labeling term ``gluten-free'' (72 FR... HUMAN SERVICES Food and Drug Administration 21 CFR Part 101 RIN 0910-ZA26 Food Labeling; Gluten-Free Labeling of Foods; Reopening of the Comment Period AGENCY: Food and Drug Administration, HHS....

  11. Portion Size Labeling and Intended Soft Drink Consumption: The Impact of Labeling Format and Size Portfolio

    ERIC Educational Resources Information Center

    Vermeer, Willemijn M.; Steenhuis, Ingrid H. M.; Leeuwis, Franca H.; Bos, Arjan E. R.; de Boer, Michiel; Seidell, Jacob C.

    2010-01-01

    Objective: To assess what portion size labeling "format" is most promising in helping consumers selecting appropriate soft drink sizes, and whether labeling impact depends on the size portfolio. Methods: An experimental study was conducted in fast-food restaurants in which 2 labeling formats (ie, reference portion size and small/medium/large…

  12. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... antibody or antibodies present as set forth in paragraph (d) of this section. (ii) Name, address (including... antibody designation on the labels of a final container with a capacity of less than 5 milliliters shall be not less than 12 point. The type size for the specificity of the antibody designations on the label...

  13. 40 CFR 1033.135 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM LOCOMOTIVES Emission Standards and Related Requirements § 1033.135 Labeling. As described in... information: (A) The label heading: “ORIGINAL LOCOMOTIVE EMISSION CONTROL INFORMATION.”...

  14. 40 CFR 1033.135 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM LOCOMOTIVES Emission Standards and Related Requirements § 1033.135 Labeling. As described in... information: (A) The label heading: “ORIGINAL LOCOMOTIVE EMISSION CONTROL INFORMATION.”...

  15. 40 CFR 1033.135 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS CONTROL OF EMISSIONS FROM LOCOMOTIVES Emission Standards and Related Requirements § 1033.135 Labeling. As described in... information: (A) The label heading: “ORIGINAL LOCOMOTIVE EMISSION CONTROL INFORMATION.”...

  16. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...

  17. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...

  18. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...

  19. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...

  20. 21 CFR 201.313 - Estradiol labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... Pharmacopeia under the designation “Alpha Estradiol.” The substance should no longer be referred to in drug labeling as “Alpha Estradiol.” The Food and Drug Administration would not object to label references to the... referred to the presence of “Estradiol (formerly known as Alpha Estradiol).”...