Journal of Biological Chemistry · 2009 · 33 citations · 25 references
Life SciencesCancer BiologyOncologyApoptosisCell SignalingMolecular OncologyApoptosis In CancerApoptosis ModulationApoptosis RegulationApoptosis ResistanceCancer Cell SignalingTumor Suppressor GenesCell Cycle Arrest MechanismsCell Division ArrestP53-mediated ApoptosisP53-mediated Cell Cycle RegulationP53-mediated Cell DeathProtein Expression LevelsRetinoblastoma Family ProteinsRetinoblastoma Protein Signaling
The retinoblastoma protein (Rb) plays a pivotal role in regulating cell proliferation and apoptosis. Nutlin-3, a small molecule MDM2 antagonist blocking interaction between MDM2 and p53, activates p53 resulting in cell growth arrest or apoptosis in various cancer cells. However, the molecular basis for the different cellular responses upon nutlin-3 treatment is not fully understood. In this study, we show that nutlin-3 activates p53 resulting in a dramatic increase in MDM2 expression and a marked reduction in total Rb protein levels. Interestingly, nutlin-3 reduces the levels of hypophosphorylated Rb and induces massive apoptosis in SJSA-1 cells, which can be largely rescued by knockdown of MDM2 or by expression of constitutively active Rb. By contrast, nutlin-3 treatment of several human cancer cells, including A549, U2-OS, and HCT116, results in an accumulation of hypophosphorylated Rb and cell cycle arrest but not apoptosis. Furthermore, we show that down-regulation of Rb by nutlin-3 does not lead to E2F1 activation nor does E2F1 play a critical role for nutlin-3-induced apoptosis in SJSA-1 cells. Taken together, these results suggest that Rb plays a critical role in influencing cellular response to activation of p53 pathway by nutlin-3. The retinoblastoma protein (Rb) plays a pivotal role in regulating cell proliferation and apoptosis. Nutlin-3, a small molecule MDM2 antagonist blocking interaction between MDM2 and p53, activates p53 resulting in cell growth arrest or apoptosis in various cancer cells. However, the molecular basis for the different cellular responses upon nutlin-3 treatment is not fully understood. In this study, we show that nutlin-3 activates p53 resulting in a dramatic increase in MDM2 expression and a marked reduction in total Rb protein levels. Interestingly, nutlin-3 reduces the levels of hypophosphorylated Rb and induces massive apoptosis in SJSA-1 cells, which can be largely rescued by knockdown of MDM2 or by expression of constitutively active Rb. By contrast, nutlin-3 treatment of several human cancer cells, including A549, U2-OS, and HCT116, results in an accumulation of hypophosphorylated Rb and cell cycle arrest but not apoptosis. Furthermore, we show that down-regulation of Rb by nutlin-3 does not lead to E2F1 activation nor does E2F1 play a critical role for nutlin-3-induced apoptosis in SJSA-1 cells. Taken together, these results suggest that Rb plays a critical role in influencing cellular response to activation of p53 pathway by nutlin-3. Retinoblastoma protein (Rb) 2The abbreviations used are: Rbretinoblastoma proteinPARPpoly ADP-ribose polymeraseFACSfluorescent-activated cell sorting. has been shown to play a pivotal role in regulating cell proliferation, DNA damage response, apoptosis, and differentiation. One major function of Rb is to interact with E2F transcription factors in assembly of transcription repressor complexes to repress expression of E2F downstream target genes involved in cell cycle progression and apoptosis (1Stevaux O. Dyson N.J. Curr. Opin. Cell Biol. 2002; 14: 684-691Crossref PubMed Scopus (352) Google Scholar, 2La Thangue N.B. Nat. Cell Biol. 2003; 5: 587-589Crossref PubMed Scopus (102) Google Scholar). retinoblastoma protein poly ADP-ribose polymerase fluorescent-activated cell sorting. The Rb tumor suppressor function is critically regulated by cyclin/CDK-dependent phosphorylation (3Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4326) Google Scholar, 4Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5159) Google Scholar). Notably, there are multiple cyclin/CDK phosphorylation sites throughout the sequence of Rb protein (5Zarkowska T. Mittnacht S. J. Biol. Chem. 1997; 272: 12738-12746Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar) and mutation of those sites, especially the seven Ser/Thr-Pro sites in Rb C terminus (Rb C-pocket) confers Rb with constitutively active growth suppression function to block G1-S transition as well as the S-phase progression (6Knudsen E.S. Wang J.Y. Mol. Cell Biol. 1997; 17: 5771-5783Crossref PubMed Scopus (238) Google Scholar, 7Knudsen E.S. Buckmaster C. Chen T.T. Feramisco J.R. Wang J.Y. Genes Dev. 1998; 12: 2278-2292Crossref PubMed Scopus (190) Google Scholar). Hypophosphorylated Rb has been shown to possess growth suppression function through interaction with a set of cellular proteins including the E2F transcription factors. Notably, MDM2 preferentially binds to hypophosphorylated Rb and facilitates proteasome-mediated Rb protein degradation (8Sdek P. Ying H. Zheng H. Margulis A. Tang X. Tian K. Xiao Z.X. J. Biol. Chem. 2004; 279: 53317-53322Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Sdek P. Ying H. Chang D.L. Qiu W. Zheng H. Touitou R. Allday M.J. Xiao Z.X. Mol. Cell. 2005; 20: 699-708Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Recently, several potent small molecule MDM2 antagonists, the nutlins, have been identified (10Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E.A. Science. 2004; 303: 844-848Crossref PubMed Scopus (3825) Google Scholar). Nutlin-3 specifically binds to MDM2 in the p53-binding pocket and blocks MDM2-p53 interaction, resulting in a dramatic stabilization of p53 and activation of the p53 pathway. In response to nutlin-3 treatment, p53+ cancer cells undergo either cell cycle arrest or apoptosis (11Tovar C. Rosinski J. Filipovic Z. Higgins B. Kolinsky K. Hilton H. Zhao X. Vu B.T. Qing W. Packman K. Myklebost O. Heimbrook D.C. Vassilev L.T. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1888-1893Crossref PubMed Scopus (585) Google Scholar, 12Carvajal D. Tovar C. Yang H. Vu B.T. Heimbrook D.C. Vassilev L.T. Cancer Res. 2005; 65: 1918-1924Crossref PubMed Scopus (172) Google Scholar, 13Vassilev L.T. Trends Mol. Med. 2007; 13: 23-31Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). In addition, nutlin-3 can induce differentiation (14Maimets T. Neganova I. Armstrong L. Lako M. Oncogene. 2008; 27: 5277-5287Crossref PubMed Scopus (111) Google Scholar) and cellular senescence (15Kumamoto K. Spillare E.A. Fujita K. Horikawa I. Yamashita T. Appella E. Nagashima M. Takenoshita S. Yokota J. Harris C.C. Cancer Res. 2008; 68: 3193-3203Crossref PubMed Scopus (185) Google Scholar). It has been shown that an array of factors affects the outcome of nutlin-3 treatment, including the single nucleotide polymorphism of MDM2 (16Arva N.C. Talbott K.E. Okoro D.R. Brekman A. Qiu W.G. Bargonetti J. Ethn. Dis. 2008; 18: S2-1-8Google Scholar), MDM4 (17Patton J.T. Mayo L.D. Singhi A.D. Gudkov A.V. Stark G.R. Jackson M.W. Cancer Res. 2006; 66: 3169-3176Crossref PubMed Scopus (174) Google Scholar, 18Hu B. Gilkes D.M. Farooqi B. Sebti S.M. Chen J. J. Biol. Chem. 2006; 281: 33030-33035Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), p73 (19Lau L.M. Nugent J.K. Zhao X. Irwin M.S. Oncogene. 2008; 27: 997-1003Crossref PubMed Scopus (161) Google Scholar), ATM (20Kojima K. Konopleva M. McQueen T. O'Brien S. Plunkett W. Andreeff M. Blood. 2006; 108: 993-1000Crossref PubMed Scopus (200) Google Scholar), and E2F1 (21Ambrosini G. Sambol E.B. Carvajal D. Vassilev L.T. Singer S. Schwartz G.K. Oncogene. 2007; 26: 3473-3481Crossref PubMed Scopus (145) Google Scholar, 22Kitagawa M. Aonuma M. Lee S.H. Fukutake S. McCormick F. Oncogene. 2008; 27: 5303-5314Crossref PubMed Scopus (79) Google Scholar). Because activation of p53 up-regulates p21 and MDM2, both of which are important regulators for Rb, we investigated whether Rb is affected upon nutlin-3 treatment and whether Rb plays a role in cellular response to nutlin-3. In this study, we show that nultin-3 affects both Rb protein levels and Rb phosphorylation, which significantly impact the cellular responses to nutlin-3. Human IMR90, WI-38, A549, MCF-7, SJSA-1, U2-OS, H1299, and HCT116 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and 1% penicillin/streptomycin in a humidified incubator at 37 °C and 5% CO2. HCT116, HCT116-p53−/−, and HCT116-p21−/− cell lines were kindly provided by Dr. Vogelstein (John Hopkins University). Stock solutions were prepared as follows: Nutlin-3 (Cayman chemical), 10 mm in DMSO; camptothecin (Sigma), 10 mm in DMSO; MG132 (Peptide Institute), 20 mm in DMSO. Exponentially growing cells were treated with either DMSO or nutlin-3 as indicated. Retrovirus infection was performed as described previously (23Chang D.L. Qiu W. Ying H. Zhang Y. Chen C.Y. Xiao Z.X. Oncogene. 2007; 26: 4627-4634Crossref PubMed Scopus (13) Google Scholar). Briefly, 293FT cells were transfected using retroviral vector or plasmid encoding p53shRNA (kindly provided by Dr. Scott Lowe, Cold Spring Harbor Laboratory) and accessory plasmids by Lipofectamine2000. At 48 h after transfection, the media were collected and filtered through a 0.45-μm filter to remove debris. The retroviral particles were then concentrated by ultra-centrifugation (27,000 rpm, 1 h 45 min at 4 °C), resuspended in fresh medium supplemented with polybrene (10 μg/ml) and used to infect cells. 48 h after infection, cells were selected in growth medium supplemented with puromycin (4 μg/ml). Cells were collected, washed with phosphate-buffered saline, and resuspended in EBC250 lysis buffer (250 mm NaCl, 50 mm Tris, pH 8.0, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, and 2 μg/ml leupeptin). Protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad). An equal amount of protein was loaded, separated on a 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore), and hybridized to an appropriate primary antibody and horseradish peroxidase-conjugated secondary antibody for subsequent detection by ECL. Monoclonal antibody DO-1 specific for p53 (Santa Cruz Biotechnology) was used at a dilution of 1:250. Monoclonal antibody SXM30 specific for p21 (PharMingen) was used at 1:200 and goat polyclonal C-11 for actin (Santa Cruz Biotechnology) was used at 1: 1000. Monoclonal antibody SMP14 specific or MDM2 (Santa Cruz Biotechnology) was used at 1:200 dilutions. Cleaved PARP-specific monoclonal antibodies (Cell Signaling Technology) were used at 1:1000. Rb antibodies: monoclonal antibodies G3–245 (BD PharMingen) were used at 1:400; monoclonal antibodies for hypophosphorylated Rb (BD PharMingen) were used at 1: 200. Polyclonal antibodies specific for hyperphosphorylated Rb were a mixture (1:1000) of antibodies specific for phospho-Rb Ser-780, Ser-795, or Ser-807/811 (Cell Signaling). SJSA-1 cells were transiently transfected with siRNA oligos (Qiagen Inc) using Lipofectamine2000 (Invitrogen) for 16 h. Cells were then treated with 10 μm nutlin-3 for 48 h. Cells were subjected to FACS analysis or Western blot analysis. The siRNA oligonucleotide sequence for human MDM2: 5′-r(GCCUGGCUCUGUGUGUAAU)dTdT-3′ control (lamin) siRNA oligonucleotide sequence; 5′-r(CUGGACUUCCAGAAGAACA)dTdT-3′; E2F1, 5′-r(GGCCCGAUCGAUGUUUUCC)dTdT-3′. AllStars Neg. control siRNA were purchased from Qiagen. Cells were trypsinized, washed with cold phosphate-buffered saline, and fixed in 70% at 4 °C 1 cells were with μg/ml) supplemented with μg/ml) at in for 1 h. Cells were then subjected to FACS analysis by were using the Cell It is well that nutlin-3 activates p53 to accumulation of the MDM2 has been shown to play a role in of Rb (8Sdek P. Ying H. Zheng H. Margulis A. Tang X. Tian K. Xiao Z.X. J. Biol. Chem. 2004; 279: 53317-53322Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Sdek P. Ying H. Chang D.L. Qiu W. Zheng H. Touitou R. Allday M.J. Xiao Z.X. Mol. Cell. 2005; 20: 699-708Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, Z.X. Chen J. N. J. D.M. 1995; PubMed Scopus Google Scholar, J.K. Mittnacht S. S. X. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, C. S. K. T. T. S. T. H. T. K. H. M. J. 2005; PubMed Scopus Google Scholar). to whether nutlin-3 has an impact on Rb. we selected p53 cancer HCT116, A549, and SJSA-1 or cells. cells were treated with and the expression levels of p53, MDM2, and Rb protein were with (11Tovar C. Rosinski J. Filipovic Z. Higgins B. Kolinsky K. Hilton H. Zhao X. Vu B.T. Qing W. Packman K. Myklebost O. Heimbrook D.C. Vassilev L.T. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1888-1893Crossref PubMed Scopus (585) Google Scholar), treatment of nutlin-3 to and p53, with a dramatic increase in p21 and MDM2 Nutlin-3 on p53, MDM2, and p21 in cells, as nutlin-3 significantly Rb protein levels in the p53+ cancer cells The nutlin-3-induced down-regulation of Rb is on p53 nutlin-3 on Rb protein levels in cells Because Rb function is critically regulated by phosphorylation, and the hypophosphorylated Rb is active in to E2F transcription we investigated whether nutlin-3 affects Rb human cancer cells U2-OS, A549, MCF-7, SJSA-1, and or cells and were treated with nutlin-3 and subjected to Western blot analysis using antibodies specific for either or total Rb. In these cancer cells, the protein levels of total Rb as well as hyperphosphorylated Rb were upon treatment with and Interestingly, the levels of hypophosphorylated Rb were significantly in of these p53+ cancer cells with the of SJSA-1, which of MDM2 (11Tovar C. Rosinski J. Filipovic Z. Higgins B. Kolinsky K. Hilton H. Zhao X. Vu B.T. Qing W. Packman K. Myklebost O. Heimbrook D.C. Vassilev L.T. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1888-1893Crossref PubMed Scopus (585) Google Scholar). In contrast, nutlin-3 to a down-regulation of hypophosphorylated Rb in SJSA-1 cells nutlin-3 not Rb protein levels or Rb phosphorylation in cells that nutlin-3 affects both Rb phosphorylation and Rb protein levels. in as to nutlin-3 affects Rb phosphorylation in these cancer cells. the of nutlin-3 on Rb in cells to that of SJSA-1 cells. shown in nutlin-3 on activation of p53 in SJSA-1 cells, as by a of MDM2 and at or 2 of nutlin-3. hypophosphorylated Rb is in SJSA-1 cells in to of hypophosphorylated Rb in cells. The of Rb phosphorylation and protein levels upon nutlin-3 treatment is on p53 Rb was not affected in the cells 1 and this we cells specific for p53 and the vector control cells In we used HCT116 and the HCT116-p53−/−, which treated these and and with nutlin-3 and Rb protein nultin-3 regulated total Rb protein levels hypophosphorylated Rb in both HCT116 and cells. However, nutlin-3 either or on Rb in cells with p53 In addition, hyperphosphorylated Rb was in cells p53 or p21 in the of as nutlin-3 was potent in accumulation of hypophosphorylated Rb in HCT116-p21−/− cells, that of p21 significantly to the accumulation of hypophosphorylated Rb in response to nutlin-3. However, hypophosphorylated Rb was to a Interestingly, total Rb protein levels were in cells p53 or Because hypophosphorylated Rb is for proteasome-mediated degradation J. A. P. S. Oncogene. 20: PubMed Scopus Google Scholar), is that hyperphosphorylated Rb is to to to MDM2 (8Sdek P. Ying H. Zheng H. Margulis A. Tang X. Tian K. Xiao Z.X. J. Biol. Chem. 2004; 279: 53317-53322Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Sdek P. Ying H. Chang D.L. Qiu W. Zheng H. Touitou R. Allday M.J. Xiao Z.X. Mol. Cell. 2005; 20: 699-708Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Taken together, these suggest that nutlin-3 affects the levels of hypophosphorylated Rb in a in which p21 plays a major suggest that nutlin-3 Rb protein levels. Because MDM2 can Rb degradation through a pathway P. Ying H. Chang D.L. Qiu W. Zheng H. Touitou R. Allday M.J. Xiao Z.X. Mol. Cell. 2005; 20: 699-708Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, C. S. K. T. T. S. T. H. T. K. H. M. J. 2005; PubMed Scopus Google Scholar), we whether Rb is the pathway. shown in nutlin-3 treatment of and HCT116 cells total Rb protein which was in the of a MG132 and nutlin-3 hypophosphorylated Rb in those cells, which was by In SJSA-1 cells, of the to an in reduction of hypophosphorylated Rb these suggest that proteasome-mediated degradation of Rb is an important with which Rb. Because nutlin-3 hypophosphorylated Rb in different cancer cells, we whether this as a cell in response to nutlin-3. and SJSA-1 cells were treated with nutlin-3 for 48 h. Cells were then subjected to FACS analysis or Western blot analysis. with nutlin-3 cell cycle arrest of cells and of cancer cell lines including U2-OS, HCT116, and not nutlin-3 massive apoptosis in SJSA-1 cells as shown by FACS analysis. In with the from FACS nutlin-3 of poly ADP-ribose polymerase a for apoptosis, in SJSA-1 cells but not in or cells Notably, the nutlin-3-induced with of hypophosphorylated Rb in which hypophosphorylated Rb was in cells in SJSA-1 cells upon nutlin-3 treatment 2 and the of nultin-3 on cancer cells, which have been shown to undergo apoptosis upon nutlin-3 treatment (11Tovar C. Rosinski J. Filipovic Z. Higgins B. Kolinsky K. Hilton H. Zhao X. Vu B.T. Qing W. Packman K. Myklebost O. Heimbrook D.C. Vassilev L.T. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1888-1893Crossref PubMed Scopus (585) Google Scholar). to SJSA-1 cells, nutlin-3 significantly apoptosis in cells, as by the of with to down-regulation of hypophosphorylated Rb. are with a that of Rb by siRNA with nutlin-3 apoptosis M. Aonuma M. Lee S.H. Fukutake S. McCormick F. Oncogene. 2008; 27: 5303-5314Crossref PubMed Scopus (79) Google Scholar). these suggest that of hypophosphorylated Rb protein levels be critical for cell in response to nutlin-3. SJSA-1 cells MDM2 that results in MDM2 protein Because MDM2 Rb, we investigated whether pathway is important for nutlin-3 using siRNA oligos to knockdown MDM2 expression to nutlin-3 Cells were then subjected to FACS analysis or Western blot analysis. In SJSA-1 cells transfected with siRNA oligos specific for MDM2, p53 and p21 and MDM2, as However, nutlin-3 was to fully hypophosphorylated Rb and to induce apoptosis and that MDM2 is critical for down-regulation of hypophosphorylated Rb upon the role of hypophosphorylated Rb in cellular response to we used a constitutively active Rb that is to phosphorylation Z.X. M. J. Mol. Biol. 1995; Google Scholar). SJSA-1 cells were transiently transfected with by nutlin-3 Cell were then subjected to Western blot analysis. Nutlin-3 p53 in SJSA-1 cells However, significantly apoptosis of SJSA-1, as shown by of these suggest that down-regulation of hypophosphorylated Rb is for nutlin-3 to apoptosis in SJSA-1 cells. In addition, expression of Rb was to block nutlin-3-induced apoptosis in SJSA-1 cells the camptothecin apoptosis in cells. shown in knockdown of Rb significantly apoptosis as by a increase of that Rb play an important role in cancer It has been well that Rb with E2F transcription factors and E2F transcription Notably, activation of E2F1 can induce expression of genes involved in apoptosis. investigated whether E2F1 is for nutlin-3-induced apoptosis in of nutlin-3 on E2F1 protein levels and E2F shown in several p53+ cancer cells down-regulation of E2F1 protein levels upon treatment of in with a M. Aonuma M. Lee S.H. Fukutake S. McCormick F. Oncogene. 2008; 27: 5303-5314Crossref PubMed Scopus (79) Google Scholar). In addition, nutlin-3 E2F transcription in both SJSA-1 and cells as by assay Furthermore, an important downstream target of E2F1 involved in was not in these cancer cells both SJSA-1 and cells apoptosis upon treatment of nutlin-3. we the of knockdown of E2F1 by specific siRNA oligos on nutlin-3-induced apoptosis. SJSA-1 cells were transiently transfected with E2F1 siRNA oligos or control oligos to nutlin-3 shown in nutlin-3 p53 and MDM2, and the expression of hypophosphorylated Rb and E2F1, with and apoptosis of the or of E2F1 FACS analysis that knockdown of E2F1 was to block nutlin-3-induced apoptosis in SJSA-1 cells that E2F1 is not for nutlin-3-induced apoptosis. Because the is with MDM2 in we whether nutlin-3 affects the expression levels of shown in protein expression was in SJSA-1 cells, as However, nutlin-3 not significantly protein levels. of by siRNA not of Rb nor apoptosis upon nutlin-3 treatment and Nutlin-3 is a small molecule that blocks MDM2-p53 interaction, p53, and activates p53 target expression L.T. Trends Mol. Med. 2007; 13: 23-31Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). Interestingly, the cellular response to nultin-3 to be in a cancer cell nutlin-3 a potent in nutlin-3 induces cell cycle It has been in to p53 and MDM2, (17Patton J.T. Mayo L.D. Singhi A.D. Gudkov A.V. Stark G.R. Jackson M.W. Cancer Res. 2006; 66: 3169-3176Crossref PubMed Scopus (174) Google Scholar, 18Hu B. Gilkes D.M. Farooqi B. Sebti S.M. Chen J. J. Biol. Chem. 2006; 281: 33030-33035Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), p73 (19Lau L.M. Nugent J.K. Zhao X. Irwin M.S. Oncogene. 2008; 27: 997-1003Crossref PubMed Scopus (161) Google Scholar), ATM (20Kojima K. Konopleva M. McQueen T. O'Brien S. Plunkett W. Andreeff M. Blood. 2006; 108: 993-1000Crossref PubMed Scopus (200) Google Scholar), and in E2F1 (21Ambrosini G. Sambol E.B. Carvajal D. Vassilev L.T. Singer S. Schwartz G.K. Oncogene. 2007; 26: 3473-3481Crossref PubMed Scopus (145) Google Scholar, 22Kitagawa M. Aonuma M. Lee S.H. Fukutake S. McCormick F. Oncogene. 2008; 27: 5303-5314Crossref PubMed Scopus (79) Google Scholar) to of cell upon nutlin-3 treatment in various tumor cells. In this study, we show that nutlin-3 significantly affects Rb protein levels and Rb phosphorylation, in which p21 and MDM2 play Nutlin-3 induces p21 expression through p53, to accumulation of active hypophosphorylated Rb. p21 is for accumulation of hypophosphorylated Rb, HCT116 cells p21 accumulation of hypophosphorylated Rb that nutlin-3 can Rb of Nutlin-3 induces proteasome-mediated degradation of Rb. In the of cancer cell lines as cancer cells, nutlin-3 hypophosphorylated Rb in SJSA-1 cells, nutlin-3 hypophosphorylated Rb. It is to a of Rb phosphorylation, nutlin-3 induces p21 well in these cells. It is that the accumulation of MDM2 facilitates protein of hypophosphorylated Rb. from (11Tovar C. Rosinski J. Filipovic Z. Higgins B. Kolinsky K. Hilton H. Zhao X. Vu B.T. Qing W. Packman K. Myklebost O. Heimbrook D.C. Vassilev L.T. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1888-1893Crossref PubMed Scopus (585) Google Scholar) and from this that MDM2 is by p53 upon nutlin-3 treatment in SJSA-1 cells, which MDM2 with cancer cells a single MDM2 the nutlin-3 significantly reduces hypophosphorylated Rb and induces apoptosis in cells cells not have MDM2 Furthermore, nutlin-3 does not the levels of hypophosphorylated Rb and does not significantly induce apoptosis of cells that MDM2 M. Aonuma M. Lee S.H. Fukutake S. McCormick F. Oncogene. 2008; 27: 5303-5314Crossref PubMed Scopus (79) Google Scholar). these the of in hypophosphorylated Rb to in apoptosis. It is that the of Rb protein and the of Rb phosphorylation by p21 the outcome of the active Rb which to cell cycle progression and apoptosis. In SJSA-1 cells, massive activation of MDM2 by nutlin-3 facilitates degradation of hypophosphorylated Rb that cells for apoptosis. is by the that knockdown of MDM2 in SJSA-1 cells to the of hypophosphorylated Rb upon nutlin-3 treatment, which to of In addition, expression of Rb significantly nutlin-3 apoptosis in SJSA-1 cells. However, nutlin-3 the levels of hypophosphorylated Rb in cancer cells, as and for It is that MDM2 expression or MDM2 are in cells as single nucleotide polymorphism of MDM2 (16Arva N.C. Talbott K.E. Okoro D.R. Brekman A. Qiu W.G. Bargonetti J. Ethn. Dis. 2008; 18: S2-1-8Google Scholar), or factors Rb as J.T. Stark G.R. Jackson M.W. Cancer Res. 2007; PubMed Scopus Google Scholar), A. Mol. Cancer Res. 2007; 5: PubMed Scopus Google Scholar), and p21 Mol. Cell Biol. 2007; 27: PubMed Scopus Google Scholar), of which have on the outcome of nutlin-3 In addition, factors as MDM4 (17Patton J.T. Mayo L.D. Singhi A.D. Gudkov A.V. Stark G.R. Jackson M.W. Cancer Res. 2006; 66: 3169-3176Crossref PubMed Scopus (174) Google Scholar, 18Hu B. Gilkes D.M. Farooqi B. Sebti S.M. Chen J. J. Biol. Chem. 2006; 281: 33030-33035Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), p73 (19Lau L.M. Nugent J.K. Zhao X. Irwin M.S. Oncogene. 2008; 27: 997-1003Crossref PubMed Scopus (161) Google Scholar) to the of nutlin-3-induced apoptosis in the of of does reduction in hypophosphorylated Rb in cells lead to It is that hypophosphorylated Rb binds E2F1 with of Rb expression or Rb phosphorylation to of E2F1 from Rb and that E2F1 It is that E2F1 with the response to nutlin-3 M. Aonuma M. Lee S.H. Fukutake S. McCormick F. Oncogene. 2008; 27: 5303-5314Crossref PubMed Scopus (79) Google Scholar). However, that E2F1 is to be to nutlin-3-induced apoptosis. nutlin-3 significantly reduces E2F1 protein levels and E2F In addition, knockdown of E2F1 does not block apoptosis. reduction of hypophosphorylated Rb to apoptosis of E2F1 upon nutlin-3 treatment is not In this that upon activation of the p53 pathway by Rb is a critical influencing cellular In p53+ cancer cells, nutlin-3 induces Rb and cells undergo cell cycle in a of cancer cells, nutlin-3 hypophosphorylated Rb and cells undergo apoptosis. Rb cancer cell to to a and specific Dr. Vogelstein for cell lines and Dr. Scott for are to from of with
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