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Small Molecule Inhibition of HDM2 Leads to p53-Mediated Cell Death in Retinoblastoma Cells
Jasmine R. Elison, MD;
David Cobrinik, MD, PhD;
Nidia Claros, BS;
David H. Abramson, MD;
Thomas C. Lee, MD
Arch Ophthalmol. 2006;124:1269-1275.
ABSTRACT
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Purpose To determine the efficacy of inducing p53-mediated cell death in retinoblastoma cells by Nutlin 3A, a small molecule HDM2 inhibitor.
Methods Retinoblastoma cell lines WERI-RB-1 and Y79 were treated with Nutlin 3A. Cell viability assays, Western blot analyses, confocal microscopy, and flow cytometry were performed to measure cell survival, p53 protein levels, activation of downstream targets, and apoptosis. To determine whether the effects of Nutlin 3A were p53-dependent, cell viability assays were performed on Y79 cells expressing short interfering RNA (siRNA) against p53.
Results Nutlin 3A induced cell death in Y79 and WERI-RB-1 in the 5- to 10-µM dose range. Treated cells demonstrated increased protein levels of p53 and the p53 targets p21 and HDM2. Phosphorylation of p53-serine-15, a marker for activation of p53 via genotoxic mechanisms, was absent. Y79 cells expressing siRNA against p53 demonstrated resistance to Nutlin 3A.
Conclusions Nutlin 3A induced p53-mediated apoptosis in a dose-dependent, nongenotoxic fashion in 2 retinoblastoma cell lines.
Clinical Relevance Nutlin 3A is effective against retinoblastoma cells in a nongenotoxic manner. Because the mutagenic effects of radiation and chemotherapy may increase risks of secondary tumor formation, targeted p53 activation may be a safer alternative treatment for retinoblastoma in the future.
INTRODUCTION
Retinoblastoma is due in part to mutations of both copies of the retinoblastoma (RB1) gene. Forty percent of patients carry a germline mutation in RB1 and are therefore at risk for developing multiple tumors in both eyes as well as nonocular tumors.1-2 The rate of nonocular tumor formation may be as high as 1% per year, and exposure to mutagenic agents such as radiation increases this risk.3 Studies have suggested that external beam radiation delivered prior to the age of 1 year increases the risk of new tumors approximately 2-fold.4-5 Chemotherapy is often offered as primary treatment in conjunction with focal measures in an effort to avoid the development of future nonocular tumors.6 Chemotherapy, however, also has adverse effects, and long-term consequences regarding its impact on nonocular tumors are still unknown. An ideal treatment would be as efficacious as radiation but would lack the risks caused by radiation-induced DNA damage.
Chemotherapy and radiation function in large part by inducing DNA damage to activate the tumor suppressor p53. A cascade of events follows, leading to either cell cycle arrest or cell death via activation of genes such as p21 or BAX.7 p53 is tightly autoregulated by transcriptionally activating its own inhibitor, HDM2. HDM2 protein binds to p53, blocks its transactivation domain, escorts it into the cytoplasm, and targets it for proteolysis.8-11 Loss of HDM2 can lead to sufficient accumulation of p53 to induce apoptosis in tumor cells that contain wild-type p53.12 Thus, strategies that suppress HDM2-mediated inactivation of p53 may be effective for the treatment of p53 wild-type tumors. Several methods have been described, including antisense oligonucleotides and peptide inhibitors of HDM2.13-15 Recently, small molecule inhibitors of the p53-HDM2 interaction have been investigated.16-19 RITA (reactivation of p53 and induction of tumor cell apoptosis), a molecule identified by screening a chemical library for compounds suppressing tumor growth, demonstrated p53 binding and induced apoptosis in tumor cells.17
A structure-based approach by Vassilev et al18 resulted in the identification of Nutlin 3A, a small molecule that fits into a hydrophobic pocket within the p53-HDM2 binding site, inhibiting the interaction between p53 and HDM2. Nutlin 3A treatment led to activation of the p53 pathway, apoptosis in various p53 wild-type tumor cell lines, as well as oral efficacy with minimal systemic adverse effects in an osteosarcoma model. Furthermore, Nutlin 3A was not associated with phosphorylation of p53 on serine residues that are phosphorylated as part of the cellular stress response, suggesting that the drug is not genotoxic.19 In this article, we examine the efficacy of Nutlin 3A to induce p53-mediated cell death in retinoblastoma cells.
METHODS
CELLS AND DRUG TREATMENT
Y79, WERI-RB-1, and MDA-MB-435 cells were grown in the recommended media supplemented with 10% fetal bovine serum at 37°C in a humidified environment with 5% carbon dioxide. Nutlin 3A and 3B, gifts from Dr Lubomir Vassilev (Hoffman-La Roche, Nutley, NJ), were dissolved in dimethyl sulfoxide (DMSO) and kept at –20°C as 10-mM stocks. Doxorubicin (Sigma, St Louis, Mo) was dissolved in DMSO to 10 mM and stored at +4°C. For cell viability assays, cells were grown in 12 well plates (at initial concentrations of 1 x 105 cells per mL for WERI-RB-1, 2 x 104 cells per mL for Y79, and 5 x 104 cells per mL for MDA-MB-435) and counted using the trypan blue exclusion assay.
IMMUNOCYTOCHEMISTRY
WERI-RB-1 and Y79 cells were incubated with the drug or with a DMSO vehicle for 8 hours. Cells were centrifuged and resuspended in 50 µL of Accutase (Innovative Cell Technology, Inc, San Diego, Calif). They were centrifuged again, resuspended in fresh media, and incubated for 30 minutes at 37°C on poly-L-lysine slides to attach. Cells were washed with phosphate-buffered saline, incubated for 10 minutes in 500 µL 4% paraformaldehyde/phosphate-buffered saline, and rewashed. Slides were incubated with p53 antibody 1:100 (SC263; Santa Cruz Biotechnology, Santa Cruz, Calif) for 24 hours, followed by the nuclear stain 4"6-diamidino-2-phenylindole 1:10 000 (catalog #D1036; Molecular Probes, Invitrogen, Carlsbad, Calif) and the fluorescent secondary antibody Alexa 488 1:500 (A11029; Invitrogen). Slides were then examined with the Leica TCS SP confocal microscope (Leica Microsystems, Bannockburn, Ill).
WESTERN BLOTTING
Y79 cells were lysed in RIPA (radioimmunoprecipitation assay) lysis buffer containing 150 mM sodium chloride; 50 mM Tris at pH 7.5; 1 mM EDTA; 1% nonidet-P-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate; and protease inhibitors aprotinin (10 µg/mL), leupeptin (2.5 µg/mL), and 1 mM phenylmethylsulfonyl fluoride. Aliquots of lysates containing 10 µg/mL of protein were separated on 12% Tris-glycine polyacrylamide gels (Invitrogen). Proteins were detected using enhanced chemiluminescence reagents (Amersham, Piscataway, NJ) using antibodies specific for human p53 (SC263; Santa Cruz Biotechnology), Phospho-p53 (PC386; Calbiochem, San Diego), p21 (OP64; Oncogene Research Products, San Diego), MDM2 (SC695; Santa Cruz Biotechnology), and  -actin (Sigma).
ANNEXIN V STAINING
Y79 cells were incubated with 10 µM of Nutlin 3A for 36 hours or the equivalent volume of DMSO. Cells were washed twice with phosphate-buffered saline and stained with propidium iodide and Annexin V-FITC (Annexin V-FITC Apoptosis Detection Kit I; BD Pharmingen, San Diego) according to the manufacturer's instructions. This was followed by flow cytometry.
PRODUCTION OF CELLS EXPRESSING P53 SHORT INTERFERING RNA
pRETRO-SUPER and pRETRO-SUPER-p53 virus,20 gifts from Dr Reuven Agami, were produced in GP2 cells (Clontech, Mountain View, Calif). Supernatant was filtered and then added to Y79 cells in the presence of 4 mg/mL Polybrene (hexadimethrine bromide; Sigma) followed by selection for 7 days with 1 µg/mL puromycin.
RESULTS
NUTLIN 3A UP-REGULATES LEVELS OF P53, HDM2, AND P21
We first examined the effects of Nutlin 3A on p53 protein levels. Under normal growth conditions, WERI-RB-1 cells and Y79 cells expressed minimal p53 protein. Treatment with 5 µM of Nutlin 3A resulted in both cell lines accumulating nuclear and cytoplasmic p53 at 8 hours based on confocal microscopy (Figure 1A). Incubation with Nutlin 3B, the less potent enantiomer, had no discernable effect. We performed Western blot analysis on Y79 cells to determine whether Nutlin 3A affected levels of p53 and 2 of its transcriptional targets, p21 and HDM2. Nutlin 3A increased p53, p21, and HDM2 protein levels in a dose-dependent fashion at 8 hours. Incubation with Nutlin 3B appeared to have a minimal effect (Figure 1B).
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Figure 1. Activation of p53 cascade in WERI-RB-1 and Y79 cells. A, Confocal indirect immunofluorescence microscopy showing minimal p53 in WERI-RB-1 cells (left) and Y79 cells (right). There is increased nuclear and cytoplasmic p53 (green) after 8 hours of exposure to 5 µM of Nutlin 3A in both cell lines, but no discernable response to Nutlin 3B. B, Western blot analysis of Y79 cells after 8 hours of exposure to both Nutlin 3A and 3B at 0 (dimethyl sulfoxide vehicle), 1, 5, and 10 µM. After Nutlin 3A treatment, p53 levels rise in association with increases in both HDM2 and p21. There are minimal effects after Nutlin 3B treatment.
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NUTLIN 3A INDUCES CELL DEATH BY APOPTOSIS
Cell viability assays were performed on exponentially growing WERI-RB-1 and Y79 cells exposed to DMSO or Nutlin 3A at varying concentrations. Nutlin 3A–treated cells exhibited a dose-dependent reduction in cell viability with cell death in the 5- to 10-µM range (Figure 2). To determine whether cell death occurred via apoptosis, Annexin V staining was performed on WERI-RB-1 cells exposed to either DMSO or 10 µM of Nutlin 3A for 36 hours. Cells treated with Nutlin 3A demonstrated a significant increase in Annexin V but not propidium iodide staining, indicating early cell death by apoptosis (Figure 3).
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Figure 2. Nutlin 3A causes cell death in a dose-dependent manner. Y79 and WERI-RB-1 cells were incubated with 0 (dimethyl sulfoxide vehicle), 1, 5, or 10 µM of Nutlin 3A. Viable cells were identified by trypan blue exclusion, counted over 5 days, and expressed graphically as cells per high-power field (HPF). Experiments were done in duplicate and Y error bars correspond to standard deviations from the mean. In both experiments, Nutlin 3A at 10 µM results in near complete cell death.
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Figure 3. Apoptosis in Nutlin 3A–treated WERI-RB-1 cells. A, WERI-RB-1 cells treated with dimethyl sulfoxide (DMSO) for 36 hours demonstrate little Annexin V and propidium iodide (PI) staining. B, WERI-RB-1 cells treated with 10 µM of Nutlin 3A for 36 hours demonstrate a shift to the lower-right quadrant, indicating Annexin V but not PI positivity, which is consistent with early apoptotic activity.
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NUTLIN 3A ACTIVITY IS P53-DEPENDENT
Cell viability assays were performed on MDA-MB-435 cells, which contain mutated p53, in parallel with WERI-RB-1 and Y79 cells (Figure 4). MDA-MB-435 cells were resistant to Nutlin 3A. To determine specifically whether retinoblastoma cells required p53 for Nutlin 3A sensitivity, we developed a Y79 cell line that stably expressed short interfering RNA (siRNA) directed against p53 and a control line infected with a control siRNA vector. Of note, multiple p53 bands were detected on the Western blot, which may represent previously described p53 splice variants.21 The retinoblastoma cell line containing p53 siRNA had an approximately 70% reduction in p53 protein levels, with reduction of all 3 isoforms (Figure 5A). After incubation with Nutlin 3A, cells expressing siRNA to p53 exhibited significant resistance to the drug as compared with vector control (Figure 5B and C). This resistance was not complete but correlated with the approximately 70% reduction in the amount of p53 protein detected by Western blotting.
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Figure 4. p53-deficient cell line demonstrates resistance to Nutlin 3A. MDA-MB-435, Y79, and WERI-RB-1 cells were incubated with 0 (dimethyl sulfoxide [DMSO] vehicle), 1, or 10 µM of Nutlin 3A, and viability was assessed on day 5 by the trypan blue exclusion assay. Viability was expressed as a percentage of viable cells as compared with the DMSO control. MDA-MB-435 cells demonstrated resistance to Nutlin 3A–induced cell death. This experiment was done as a single experiment; therefore, there are no error bars.
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Figure 5. Y79 with short interfering RNA (siRNA) to p53 is resistant to Nutlin 3A. A, Western blots demonstrating decreased p53 expression in siRNA infected cells vs vector control and Y79 cells. B, Y79 cells with vector alone were plated and incubated with 0 (dimethyl sulfoxide vehicle), 1, 5, or 10 µM of Nutlin 3A for 5 days. Viable cells were counted using the trypan blue exclusion assay. C, Y79 cells with siRNA to p53 were incubated with Nutlin 3A as described and found to have an attenuated response to the small molecule. HPF indicates high-power field.
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NUTLIN 3A DOES NOT INDUCE PHOSPHORYLATION OF P53-SERINE-15
We next evaluated whether activation of p53 could occur via a mechanism independent of Nutlin 3A's effects on HDM2. Known genotoxic drugs such as doxorubicin lead to stabilization of p53 via phosphorylation of specific serine residues on the protein, most commonly serine 15. We performed Western blot experiments on cells exposed to DMSO, Nutlin 3A, or doxorubicin and assessed levels of p53-serine-15 phosphorylation using an antibody specific to this modified residue (Figure 6). Neither Nutlin 3A nor DMSO induced phosphorylation whereas doxorubicin induced phosphorylation of p53-serine-15.
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Figure 6. Absence of phosphorylation at serine-15 p53. Western blot analysis on protein lysate from WERI-RB-1 and Y79 cells treated with dimethyl sulfoxide (DMSO), 5 µM of Nutlin 3A, or 1 µM of doxorubicin (Dox) for a period of 24 hours. Nutlin 3A showed no evidence of inducing phosphorylation of Serine-15 p53 in contrast to doxorubicin, implying that p53 activation by Nutlin 3A does not involve DNA damage.
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COMMENT
p53 is a transcription factor that plays a critical role in the defense against tumorgenesis. It prevents uncontrolled cell division through 2 mechanisms. The first is by inducing a cell cycle arrest through expression of p21, a protein that can prevent entry into the S phase. p21 regulates cell division indirectly by maintaining the Rb protein in an active hypophosphorylated state. The second mechanism is to initiate apoptosis, resulting in a caspase-mediated cell death. Either action can be triggered by oncogenic stimuli or DNA damage.22-24 Because of its powerful effects, many cancers acquire the ability to inhibit the p53 pathway with almost 50% of human cancers having mutations within the p53 gene.25 Many other cancers will evolve alternative mechanisms to circumvent p53 such as overexpression of HDM2.
Prior studies have demonstrated that human retinoblastoma tumors are the exception to this pattern and rarely contain p53 mutations.26-27 One reason may be that the tumor can circumvent a cell cycle arrest since it does not have functional Rb. This in turn would allow unrestricted entry into cell division. However, it is not clear whether the tumor still needs to inactivate or attenuate the p53 pathway to prevent the other option of cell death. Some animal models of retinoblastoma suggest that this is required for tumor formation in the mouse retina. In the initial retinoblastoma mouse models, loss of both the Rb and p53 pathways were required for tumorgenesis and inactivation of Rb alone resulted in photoreceptor cell death.28-29 More recent mouse models, however, have shown that inactivation of p53 is not required if both Rb and its relative p107 or p130 are inactivated together during retinal development.30-32
The role of p53 in the development of human retinoblastoma remains unclear. There is empirical evidence that suggests p53 is active based on expression patterns in poorly differentiated primary human retinoblastoma tumors.27, 33 In 2 studies, the highest expression of nuclear p53 occurred in tumor cells that were the furthest away from a central blood vessel. This also correlated with the region with the highest rate of apoptosis. Interestingly, in poorly differentiated tumors, regions with p53 expression had the lowest p21 expression, implying that the p53 pathway was driving the cell toward apoptosis as opposed to differentiation.
There is some functional evidence to suggest that the pathway is intact based on the observation that retinoblastoma cell death resulting from either radiation or chemotherapy is associated with increased levels of p53.34-35 Nork et al33 were the first to show that specific up-regulation of p53 induces retinoblastoma cell death. They transiently transfected retinoblastoma cells with a p53 expression vector and observed elevated p53 levels and cell death. Harbour et al36 provide the best evidence to date that it is possible to activate p53 and achieve retinoblastoma cell death without overexpressing the gene. They developed transducible peptides to interfere with HDM2, which allowed p53 to accumulate in a more physiologic range. They observed effective killing of retinoblastoma cells in the 200-µM dose range in association with an up-regulation of the p53 target Bax. Their data was the first to suggest that inhibition of the HDM2-p53 interaction could be a potential therapeutic option in retinoblastoma.36
We sought to build on this work and investigate whether Nutlin 3A, a small-molecule inhibitor of the p53-HDM2 interaction, could induce accumulation of p53 protein and consequent apoptosis in retinoblastoma cells. We first examined Nutlin 3A's effects on p53 levels by immunocytochemistry and confocal microscopy. Previous studies have suggested that p53's presence in the cytoplasm may not be sufficient for activation of the apoptotic pathway, presumably because the protein is unable to act in its capacity as a transcriptional activator. Nuclear exclusion has been proposed as a means of p53 inactivation in retinoblastoma as well as in other tumors, possibly via an HDM2-mediated mechanism.33, 37-38 Our findings are consistent with prior observations and demonstrated low levels of predominantly cytoplasmic p53 protein in untreated Y79 and WERI-RB-1 cells. After Nutlin 3A treatment, there was a significant amount of both nuclear and cytoplasmic p53 protein in both cell lines.
Nutlin 3A treatment also led to decreased viability in both cell lines in the 5- to 10-µM dose range, similar to the dose range needed for up-regulation of p53 transcriptional targets. It was unclear, however, whether this was a function of forced differentiation, necrosis, or apoptosis. Normally, a p21-related forced differentiation occurs via Rb-mediated cell cycle arrest. That scenario seems unlikely in this system given the loss of Rb function, but it has been proposed that related proteins such as p107 or p130 may be able to compensate for Rb.39 To address this issue, we examined phosphatidylserine, which is translocated to the outer leaflet of the cell membrane in cells undergoing apoptosis. The external presence of this phospholipid can be detected by the phospholipid-binding protein Annexin V.40-41 WERI-RB-1 cells exposed to Nutlin 3A for 36 hours demonstrated an increase in Annexin V staining as compared with those treated with the vehicle DMSO. This is direct evidence that Nutlin 3A treatment is associated with apoptosis in these retinoblastoma cells.
The data thus far demonstrate that Nutlin 3A treatment leads to up-regulation of p53 and its downstream targets as well as apoptosis in retinoblastoma cell lines. Nutlin 3A's specific fit within HDM2's binding site for p53 suggests that these actions should be p53-mediated. We next evaluated whether Nutlin 3A associated cell death could occur independently of p53, potentially via nonspecific toxicity or by alternative effects on HDM2.42 We first examined a p53 mutant breast cancer cell line, MDA-MB-435, and found it to be resistant to Nutlin 3A, suggesting that the drug's effects on cell viability are p53-dependent. To further evaluate this specifically in retinoblastoma, we developed a Y79 cell line expressing siRNA to p53, which exhibited an approximately 70% reduction in p53 levels. As expected, these cells exhibited an attenuated response to the drug, but only after an initial lag time during which the growth curves appeared to be blunted. The initial flattening of the growth curve most likely represents an equilibrium between the death of the minority p53-containing cells vs the growth of those with the siRNA knockdown of p53.
The most important aspect of this study is that Nutlin 3A activates p53 in a manner that bypasses the genotoxic effects of radiation and chemotherapy. DNA damage induces p53 via phosphorylation of specific serine residues, most frequently the serine in the 15 position. Retinoblastoma cells treated with Nutlin 3A had no increase in p53-serine-15 phosphorylation. In contrast, cells treated with doxorubicin, a known genotoxic compound, exhibited a significant increase in serine-15 phosphorylation. Thus, as suggested by Nutlin 3A's mechanism of action, the drug appears to selectively activate the p53 pathway without causing DNA damage.
The predisposition of retinoblastoma patients to the development of secondary tumors is important when considering treatment options because the mutagenic effects of traditional therapies such as chemotherapy and radiation may increase a child's risk of developing secondary nonocular tumors. Although the cure rate for retinoblastoma is 98% in the United States, patients with germline disease have a 50% chance of developing a secondary nonocular tumor later in life, and half of these tumors are fatal.43
Nutlin 3A shows great promise as a treatment for retinoblastoma, but it may be susceptible to inactivation by resistance acquired by tumor cells. Because Nutlin 3A's mechanism of action is dependent on activation of the p53 pathway, mutation of p53 could lead to resistance to the drug. Although p53 mutation in retinoblastoma is considered to be extremely rare, it has been reported in a patient with a tumor metastatic to the lung.44 Acquired p53 mutations have been documented in nonocular tumors such as neuroblastoma and lymphoma as a consequence of treatment with DNA-damaging agents.45-46 If the development of these mutations were a result of the mutagenic effects of the treatments, it is possible that their occurrence may be less likely after treatment with a nongenotoxic drug like Nutlin 3A.
This study demonstrates that targeted inactivation of HDM2 by a small molecule can achieve significant death of retinoblastoma cells while bypassing the genotoxic effects associated with traditional therapies. Nutlin 3A may provide a novel treatment option whose specific mechanism of action theoretically portends a narrow adverse effect profile in terms of risk of secondary tumors, bone marrow suppression, and deformity as compared with radiation and chemotherapy. Its small size could be an advantage in the setting of periocular administration for the achievement of effective intravitreal doses. Further studies are needed to address the efficacy of in vivo dosing as well as retinal and systemic adverse effects.
AUTHOR INFORMATION
Correspondence: Thomas C. Lee, MD, Department of Ophthalmology, Weill Medical College of Cornell University, New York Presbyterian Hospital, 520 E 70th St, Starr 807, New York, NY 10021 (tcl2002{at}med.cornell.edu).
Submitted for Publication: March 21, 2006; final revision received May 19, 2006; accepted June 5, 2006.
Author Contributions: Dr Lee had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Financial Disclosure: None reported.
Funding/Support: This study was supported by the Fred W. Gluck Foundation, the C.V. Starr Foundation, and the Knights Templar Eye Foundation.
Acknowledgment: We thank Dr Lubomir Vassilev, who provided Nutlin 3A and 3B for these experiments.
Author Affiliations: Department of Ophthalmology, Weill Medical College of Cornell University, New York Presbyterian Hospital, New York, NY (Drs Elison, Cobrinik, and Lee and Ms Claros); and Ophthalmic Oncology Service, Department of Surgery, Memorial Sloan-Kettering Hospital, New York, NY (Dr Abramson).
REFERENCES
| |
1. Cavenee WK, Hansen MF, Nordenskjold M, et al. Genetic origin of mutations predisposing to retinoblastoma. Science. 1985;228:501-503.
FREE FULL TEXT
2. T’ang A, Varley JM, Chakraborty S, Murphree AL, Fung YK. Structural rearrangement of the retinoblastoma gene in human breast cancer. Science. 1988;242:263-266.
FREE FULL TEXT
3. Abramson DH, Ronner HJ, Ellsworth RM. Second tumors in nonirradiated bilateral retinoblastoma. Am J Ophthalmol. 1979;87:624-627.
WEB OF SCIENCE
| PUBMED
4. Abramson DH, Ellsworth RM, Kitchin FD, Tung G. Second nonocular tumors in retinoblastoma survivors: are they radiation-induced? Ophthalmology. 1984;91:1351-1355.
WEB OF SCIENCE
| PUBMED
5. Kleinerman RA, Tucker MA, Tarone RE, et al. Risk of new cancers after radiotherapy in long-term survivors of retinoblastoma: an extended follow-up. J Clin Oncol. 2005;23:2272-2279.
FREE FULL TEXT
6. Shields CL, Meadows AT, Leahey AM, Shields JA. Continuing challenges in the treatment of retinoblastoma with chemotherapy. Retina. 2004;24:849-862.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
7. Barlow C, Brown KD, Deng CX, Tagle DA, Wynshaw-Boris A. Atm selectively regulates distinct p53-dependent cell-cycle checkpoint and apoptotic pathways. Nat Genet. 1997;17:453-456.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
8. Oliner JD, Kinzler KW, Meltzer PS, George DL, Vogelstein B. Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature. 1992;358:80-83.
FULL TEXT
| PUBMED
9. Picksley SM, Lane DP. The p53-mdm2 autoregulatory feedback loop: a paradigm for the regulation of growth control by p53? Bioessays. 1993;15:689-690.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
10. Wu X, Bayle JH, Olson D, Levine AJ. The p53-mdm2 autoregulatory feedback loop. Genes Dev. 1993;7:1126-1132.
FREE FULL TEXT
11. Michael D, Oren M. The p53-Mdm2 module and the ubiquitin system. Semin Cancer Biol. 2003;13:49-58.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
12. Mendrysa SM, McElwee MK, Michalowski J, O’Leary KA, Young KM, Perry ME. mdm2 is critical for inhibition of p53 during lymphopoiesis and the response to ionizing radiation. Mol Cell Biol. 2003;23:462-472.
FREE FULL TEXT
13. Zhang Z, Li M, Wang H, Agrawal S, Zhang R. Antisense therapy targeting MDM2 oncogene in prostate cancer: effects on proliferation, apoptosis, multiple gene expression, and chemotherapy. Proc Natl Acad Sci U S A. 2003;100:11636-11641.
FREE FULL TEXT
14. Tortora G, Caputo R, Damiano V, et al. A novel MDM2 anti-sense oligonucleotide has anti-tumor activity and potentiates cytotoxic drugs acting by different mechanisms in human colon cancer. Int J Cancer. 2000;88:804-809.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
15. Chene P. Inhibition of the p53-MDM2 interaction: targeting a protein-protein interface. Mol Cancer Res. 2004;2:20-28.
FREE FULL TEXT
16. Yang Y, Ludwig RL, Jensen JP, et al. Small molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate p53 in cells. Cancer Cell. 2005;7:547-559.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
17. Issaeva N, Bozko P, Enge M, et al. Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nat Med. 2004;10:1321-1328.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
18. Vassilev LT, Vu BT, Graves B, et al. In vivo activation of the p53 pathway by small molecule antagonists of MDM2. Science. 2004;303:844-848.
FREE FULL TEXT
19. Thompson T, Tovar C, Yang H, et al. Phosphorylation of p53 on key serines is dispensable for transcriptional activation and apoptosis. J Biol Chem. 2004;279:53015-53022.
FREE FULL TEXT
20. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell. 2002;2:243-247.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
21. Bourdon JC, Fernandes K, Murray-Zmijewski F, et al. p53 isoforms can regulate p53 transcriptional activity. Genes Dev. 2005;19:2122-2137.
FREE FULL TEXT
22. Lowe SW, Cepero E, Evan G. Intrinsic tumor suppression. Nature. 2004;432:307-315.
FULL TEXT
| PUBMED
23. Vogelstein B, Lane A, Levine AJ. Surfing the p53 network. Nature. 2000;408:307-310.
FULL TEXT
| PUBMED
24. Levine AJ. P53, the cellular gatekeeper for growth and division. Cell. 1997;88:323-331.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
25. Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science. 1991;253:49-53.
FREE FULL TEXT
26. Schlamp CL, Poulsen GL, Nork TM, Nickells RW. Nuclear exclusion of wild-type p53 in immortalized human retinoblastoma cells. J Natl Cancer Inst. 1997;89:1530-1537.
FREE FULL TEXT
27. Divan A, Lawry J, Dunsmore I, Parons A, Royds J. p53 regulates apoptosis in human retinoblastoma. Cancer Res. 2001;61:3157-3163.
FREE FULL TEXT
28. Howes KA, Ransom N, Papermaster DS, Lasudry JG, Albert DM, Windle JJ. Apoptosis or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the presence or absence of p53. Genes Dev. 1994;8:1300-1310.
FREE FULL TEXT
29. Windle JJ, Albert DM, O’Brien JM, et al. Retinoblastoma in transgenic mice. Nature. 1990;343:665-669.
FULL TEXT
| PUBMED
30. Chen D, Livne-bar I, Vanderluit JL, Slack RS, Agochiya M, Bremner R. Cell-specific effects of RB or RB/p107 loss on retinal development implicate an intrinsically death-resistant cell-of-origin in retinoblastoma. Cancer Cell. 2004;5:539-551.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
31. Zhang J, Schweers B, Dyer M. The first knockout mouse model of retinoblastoma. Cell Cycle. 2004;3:952-959.
WEB OF SCIENCE
| PUBMED
32. MacPherson D, Sage J, Kin T, et al. Cell type specific effects of Rb deletion in the murine retina. Genes Dev. 2004;18:1681-1694.
FREE FULL TEXT
33. Nork TM, Poulsen GL, Millecchia L, Jantz R, Nickells R. p53 regulates apoptosis in human retinoblastoma. Arch Ophthalmol. 1997;115:213-219.
FREE FULL TEXT
34. Kondo Y, Kondo S, Liu J, Haqqi T, Barnett GH, Barna BP. Involvement of p53 and WAF1/CIP1 in  -irradiation-induced apoptosis of retinoblastoma cells. Exp Cell Res. 1997;236:51-56.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
35. Lauricella M, Giuliano M, Emanuele S, Vento R, Tesoriere G. Apoptotic effects of different drugs on cultured retinoblastoma cells. Tumour Biol. 1998;19:356-363.
FULL TEXT
| PUBMED
36. Harbour JW, Worley L, Ma D, Cohen M. Transducible peptide therapy for uveal melanoma and retinoblastoma. Arch Ophthalmol. 2002;120:1341-1346.
FREE FULL TEXT
37. Moll UM, Riou G, Levine AJ. Two distinct mechanisms alter p53 in breast cancer: mutation and nuclear exclusion. Proc Natl Acad Sci U S A. 1992;89:7262-7266.
FREE FULL TEXT
38. Rodriguez-Lopez AM, Xenaki D, Eden TO, Hickman JA, Chresta CM. MDM2 mediated nuclear exclusion of p53 attenuates etoposide-induced apoptosis in neuroblastoma cells. Mol Pharmacol. 2000;59:135-143.
WEB OF SCIENCE
| PUBMED
39. Choi HH, Jong HS, Hyun Song S, You Kim T, Kyeong Kim N, Bang YJ. p130 mediates TGF-beta-induced cell-cycle arrest in Rb mutant HT-3 cells. Gynecol Oncol. 2002;86:184-189.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
40. O’Brien MC, Bolton WE. Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. Cytometry. 1995;19:243-255.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
41. Vermes I, Haanen C, Steffens-Nakken H, Retulingsperger CA. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V. J Immunol Methods. 1995;184:39-51.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
42. Momand J, Wu H, Dasguta G. MDM2-master regulator of the p53 supressor protein. Gene. 2000;242:15-29.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
43. Abramson DH. Second nonocular cancers in retinoblastoma: a unified hypothesis. The Francheschetti Lecture. Ophthalmic Genet. 1999;20:193-204.
FULL TEXT
| PUBMED
44. Kato MV, Shimizu T, Ishizaki K, et al. Loss of heterozygosity on chromosome 17 and mutation of the p53 gene in retinoblastoma. Cancer Lett. 1996;106:75-82.
FULL TEXT
|
WEB OF SCIENCE
| PUBMED
45. Tweddle DA, Malcolm AJ, Bown N, Pearson AD, Lunec J. Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line. Cancer Res. 2001;61:8-13.
FREE FULL TEXT
46. Sturm I, Bosanquet AG, Hermann S, Guner D, Dorken B, Daniel PT. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy. Cell Death Differ. 2003;10:477-484.
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|
WEB OF SCIENCE
| PUBMED
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