Antisense MDM2 Enhances E2F1-Induced Apoptosis and the Combination Sensitizes Androgen-Dependent and Androgen-Independent Prostate Cancer Cells to Radiation

We have previously shown in separate studies thatMDM2 knockdown via antisense MDM2 (AS-MDM2) andE2F1 overexpression via adenoviral-mediated E2F1(Ad-E2F1) sensitized prostate cancer cells to radiation.Because E2F1 and MDM2 affect apoptosis through bothcommon and independent pathways, we hypothesizedthat coupling these two treatments would result inincreased killing of prostate cancer cells. In this study,the effect of Ad-E2F1 and AS-MDM2 in combination withradiation was investigated in three prostate cancercell lines: LNCaP cells, LNCaP-Res cells [androgeninsensitive with functional p53 and androgen receptor(AR)], and PC3 cells (androgen insensitive,p53, andAR). A supra-additive radiosensitizing effect wasobserved in terms of clonogenic inhibition and inductionof apoptosis (caspase-3 + caspase-7 activity) inresponse to Ad-E2F1 plus AS-MDM2 treatments in allthree cell lines. In LNCaP and LNCaP-Res, thesecombination treatments elevated the levels ofphospho-Ser p53 with significant induction ofp21, phospho-;H2AX, PUMA, and Bax levelsand reduction of AR and bcl-2 expression. Similarly,AR and p53 PC-3 cells showed elevated levels ofBax and phospho-;H2AX expression. These findingsshow that the combination of Ad-E2F1 and AS-MDM2significantly increases cell death in prostate cancercells exposed to radiation and that this effect occursin the presence or absence of AR and p53.(Mol Cancer Res 2008;6(11):1742–54)IntroductionRadiation therapy (RT) is an established and commontreatment for prostate cancer. Yet, for men with high-riskdisease, failure rates are f40% over 5 years (1). Our previousstudies indicate that manipulation of the apoptotic pathway willincrease cell death in response to RT and that E2F1 and MDM2are key regulators of this response (2, 3). E2F1 is a transcriptionfactor with multiple functions, including the regulation ofapoptosis. E2F1 overexpression has been shown to enhance celldeath via apoptosis in certain cell types in response tochemotherapy and radiotherapy (4-6). We recently reportedthat adenoviral E2F1 (Ad-E2F1) caused pronounced radio-sensitization in p53 (LNCaP) and p53 (PC3) prostatecancer cell lines (2).MDM2 is an oncogene and overexpression of this protein islinked to increased cell proliferation and predisposition totumorigenesis (7). Our results show that MDM2 is over-expressed in 30% to 40% of diagnostic prostate tumor tissuefrom men referred for RT treatment (8). MDM2 regulates p53function through a negative feedback loop involving p53ubiquitination (9-12) and is a negative regulator of p21independently of p53 (13-17). MDM2 also mediatesubiquitination and proteolysis of the androgen receptor(AR; refs. 18-20). Abrogating MDM2 expression by antisenseMDM2 (AS-MDM2) is an effective strategy for inducingapoptosis in vitro and in vivo (3, 13, 21, 22).E2F1 and MDM2 are two key proteins that promote apoptosis through common and independent apoptotic path-ways. In p53 wild-type cells, E2F1 also increases endogenousMDM2 levels through ADP ribosylation factor–mediatedp53 induction, which, in turn, stabilizes p53 protein byreducing proteosomal degradation through MDM2 (23, 24).The combination of Ad-E2F1 + AS-MDM2 should, therefore,enhance the killing of prostate cancer cells treated with RT. ResultsAS-MDM2 Inhibits MDM2 Protein Induced in Response toAdenoviral E2F1 TherapyIn our previous studies, we successfully overexpressed E2F1using an adenoviral vector and knocked down MDM2 usingAS-MDM2 as single agents in prostate cancer cell lines (2, 22).In this study, we used a combination approach. Overexpression of E2F1 by Ad-E2F1 and MDM2 suppression by AS-MDM2were confirmed by immunofluorescence staining of E2F1 andReceived 2/20/08; revised 7/14/08; accepted 7/22/08.Grant support: National Cancer Institute grants CA 101984-01 and CA-006927,Department of Defense U.S. ArmyMedical Research Grant PC020427, and VarianMedical Systems (Palo Alto, CA). The contents are solely the responsibility of theauthors and do not necessarily represent the official views of the National CancerInstitute, U.S. Department of Defense, or Varian Medical Systems.The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.Note: Current address for T.S. Udayakumar, P. Hachem, and M.M. Ahmed:Department of Radiation Oncology, University of Miami Miller School ofMedicine, 1475 NW 12 Ave, Suite 1500 (D-31), Miami, FL 33136.Requests for reprints: Alan Pollack, University of Miami Miller School ofMedicine, 1475 Northwest 12 Avenue, Suite 1500 (D31), Miami, FL 33136.Phone: 305-243-4916; Fax: 305-243-4364. E-mail: [email protected] D 2008 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-08-0102 Mol Cancer Res 2008;6(11). November 20081742on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from MDM2 (Fig. 1A and B show the data for LNCaP). Similarresults were observed in Western blot analysis (Fig. 1C and D)in all three cell lines. As a single agent, Ad-E2F1 caused a modest increase in MDM2 protein in the LNCaP-Res cell line(but was weakly induced in LNCaP and PC3), which wasmanifestly reduced when AS-MDM2 was added. Previously, itwas reported that E2F1 overexpression causes an increase inADP ribosylation factor activity leading to increased expressionof p53 protein, which, in turn, will up-regulate MDM2 (23, 24).The cross talk between E2F1 and MDM2 supports a combinedapproach. Effect of Ad-E2F1 + AS-MDM2 + Radiation on OverallCell Death by Clonogenic Cell Survival AssayTo understand the potential cooperative benefit of Ad-E2F1and AS-MDM2 on radiation response, we measured clonogeniccell survival in the three cell lines. Compared with previousstudies (2), a low concentration of Ad-E2F1 was used todetermine the relative gain from adding AS-MDM2. Based, inpart, on previous dose response studies (2), we found thatmultiplicities of infection (MOI) of 10 for LNCaP cells, 20 forLNCaP-Res, and 50 for PC3 cells resulted in significantectopic overexpression of E2F1 with minimal cytotoxicity.These MOI doses were used in combination with AS-MDM2treatments. There were seven treatment groups: (a) adenoviralluciferase (Ad-Luc) alone; (b) Ad-Luc + mismatch oligonucle-otide (MM); (c) Ad-Luc + AS-MDM2; (d) Ad-E2F1 alone;(e) Ad-E2F1 + MM; (f) Ad-E2F1 + AS-MDM2; and (g)AS-MDM2 alone.LNCaP cells were significantly radiosensitized by Ad-E2F1(D0 = 76.9, SF2 = 0.2946, and n = 4.5 with P < 0.045) orAd-E2F1 + AS-MDM2 (D0 = 64.6, SF2 = 0.0536, and n = 1.2FIGURE 1. MDM2 and E2F1 expression in LNCaP cells after Ad-E2F1 and AS-MDM2 treatment. MDM2 (A) and E2F1 (B) were detected in LNCaP cellsgrown on coverslips incubated with Ad-E2F1 (10 MOI) or AS-MDM2 (200 nmol/L) as described in Materials and Methods. The cells were fixed24 h posttransfection, probed with anti-MDM2 or anti-E2F1 antibodies, counterstained with 4¶,6-diamidino-2-phenylindole (DAPI ), and then visualized byimmunofluorescence microscopy. Images shown are representatives of three experiments. Magnification, 40 (MDM2); 20 (E2F1). Same cells are shownin each row and images on the bottom row are formed by superimpositions of the other two rows.C and D. Total cell extract was prepared from LNCaP,LNCaP-Res, and PC3 cells transfected with Ad-E2F1 or MM alone and combined treatments with Ad-E2F1 + MM or AS and LNCaP control (Lipofectin alone)for 24 h. An equal concentration of protein from each reaction was then probed by immunoblot with antibodies against MDM2 or E2F1 and normalized withh-actin. Bottom, densitometry data for C and D.AS-MDM2 and Radiation Enhances E2F1-Induced Apoptosis Mol Cancer Res 2008;6(11). November 20081743 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from with P < 0.0035) when compared with Ad-Luc (D0 = 93, SF2 =0.24, and n = 2.3; Table 1; Fig. 2A). LNCaP-Res cellsdisplayed a slightly greater degree of radiosensitization from Ad-E2F1 (D0 = 65.6, SF2 = 0.1768, and n = 4 with P < 0.001)or Ad-E2F1 + AS-MDM2 (D0 = 45.9, SF2 = 0.0216, andn = 11.7 with P < 0.00021) when compared with Ad-Luc(D0 = 90.7, SF2 = 0.4091, and n = 4; Table 1; Fig. 2B). In PC-3cells, a slight increase in clonogenic cell survival was observedin response to Ad-E2F1 treatment (D0 = 156, SF2 = 0.496,and n = 1.95) when compared with Ad-Luc (D0 = 141.4,SF2 = 0.4182, and n = 1.85), apparently related to the low MOIused. However, significant radiosensitization of PC-3 cells wasobserved with Ad-E2F1 + AS-MDM2 (D0 = 75, SF2 = 0.0853,and n = 1.25; P < 0.00014) when compared with Ad-Luc(D0 = 141.4, SF2 = 0.4182, and n = 1.85; Table 1; Fig. 2C).In response to Ad-E2F1 + AS-MDM2, atSF2, LNCaP-Rescells had the highest radiation enhancement ratio (18.95),followed by PC3 (4.9) and LNCaP cells (4.5). The radiationenhancement ratio for SF2 was modestly increased in responseto Ad-E2F1 in LNCaP-Res (2.3) and was absent in both LNCaP(0.8) and PC-3 cells (0.85). A similar modest radiationenhancement ratio at SF2 was observed in response to AS-MDM2 in all three cell lines (LNCaP, 1.4; LNCaP-Res, 1.3; andPC-3, 1.6; Table 2). These results show that AS-MDM2synergizes the radiosensitizing effect of Ad-E2F1, particularlyin androgen-resistant LNCaP-Res cells. The D0 radiationenhancement ratio was modestly present in all the cell linesexcept in PC-3 cells treated with Ad-E2F1. Ad-E2F1 and AS-MDM2 in Combination with RadiationIncreases ApoptosisApoptosis by Caspase-3/7 Assay. Caspase-3/7 activationprecedes the onset of cell death (25, 26). Caspase-3/7 activitywas highest in the irradiated Ad-E2F1 + AS-MDM2 group inall three cell lines (Fig. 3). The degree of activation was greatestin LNCaP cells (Fig. 3A) followed by LNCaP-Res (Fig. 3B)and then PC-3 cells (Fig. 3C; Table 3). These findings showthat irrespective of androgen sensitivity, p53 function, and ARstatus, the combination of Ad-E2F1 and AS-MDM2 results inhigh levels of radiosensitization via apoptosis. Activation ofcaspase-3/7 was found to parallel apoptosis as assessed byterminal deoxyribonucleotidyl transferase–mediated dUTPnick end labeling (TUNEL).Apoptosis by TUNEL Assay. TUNEL assays were also doneto assess the incidence of apoptosis in the seven treatmentgroups. The control virus or mismatch oligonucleotidetransfection caused a basal increase in apoptosis as assessedby TUNEL in all three cell lines (data not shown). AS-MDM2alone, Ad-E2F1 alone, or Ad-E2F1 + AS-MDM2 increased celldeath when compared with controls in LNCaP and LNCaP-Res,Table 1. Radiation Inactivation Estimates of Prostate Cancer Cell Lines Treated with Ad-E2F1 or Ad-E2F1 + AS Obtained Usinga Single-Hit Multitarget Model Cell LinesInactivation Estimates RT + Ad-LucRT + Ad-E2F RT + Ad-Luc + MM RT + Ad-Luc + AS RT + Ad-E2F + MM RT + Ad-E2F + AS D0(cGy)SF2 nD0(cGy)SF2 nD0(cGy)SF2 nD0(cGy)SF2 nD0(cGy)SF2 nD0(cGy)SF2 n LNCaP 93 0.24 2.3 76.9 0.2946 4.5 95.1 0.2386 2.1 75.8 0.1753 2.6 86.8 0.2707 3 64.6 0.0536 1.2LNCaP-Res 90.7 0.4091 4 65.6 0.1768 4 95.5 0.4095 4 82.3 0.3218 4.2 66.2 0.0863 1.8 45.9 0.0216 1.7PC-3141.4 0.4182 1.85 156 0.496 1.95 135.4 0.37 1.7 84 0.2522 3 146.9 0.2597 1.01 75 0.0853 1.25 FIGURE 2. Clonogenic assays of LNCaP (A), LNCaP-Res (B), and PC3 (C) cells cultured in respective media and transfected with Ad-E2F1 + AS-MDM2for 24 h before radiation (RT) at 2, 4, and 6 Gy as described in Materials and Methods. Points, mean percent surviving cells from three independentexperiments for all three cell lines; bars, SE.Udayakumar et al. Mol Cancer Res 2008;6(11). November 20081744 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from but not in PC-3 cells (data not shown). When radiation wasadded to these treatment groups, significant induction ofapoptosis was observed in all three cell lines compared withthe unirradiated groups. Interestingly, Ad-E2F1 + AS-MDM2 +5 Gy resulted in cell death that was significantly greater(P < 0.0001) than all other treatment groups studied in all celllines, including in PC-3 cells. Ad-E2F1 in Combination with AS-MDM2 EnhancesPhospho-p53 Levels, Increases p53 TransactivationLeading to Up-Regulation of p53 Targets, IncreasesLevels of Phospho-cH2AX, and Down-Regulates Bcl-2and AR in LNCaP and LNCaP-Res CellsTo further understand the mechanism of radiosensitizationby Ad-E2F1 and MDM2, we first ascertained the modulation ofMDM2 levels in response to these treatments and radiation. Asstated earlier and as shown in Fig. 1C, Ad-E2F1 caused a weakto modest elevation of endogenous MDM2. Radiation alsocaused significant up-regulation of MDM2 in LNCaP, LNCaP-Res, and PC-3 cells treated with Ad-Luc or Ad-Luc + MM(Fig. 4A-C). This up-regulation was mitigated when LNCaP,LNCaP-Res, and PC-3 cells were treated with AS-MDM2.These findings show that AS-MDM2 counteracts the action ofE2F1 overexpression and/or irradiation on MDM2 levels.Next, we analyzed the effects of Ad-E2F1 and AS-MDM2on p53 function in LNCaP and LNCaP-Res cells, which bothhave wild-type p53. As expected and described previously(2, 3, 27), a significant increase in p53 protein was observed inunirradiated LNCaP cells, but not LNCaP-Res cells, treatedwith AS-MDM2. However, Ad-E2F1 significantly increasedp53 in both cell lines. The highest relative increases in p53 overthe Ad-Luc controls were with the treatments of Ad-E2F1 +AS-MDM2 + irradiation (Fig. 5A and B). Inhibition of MDM2led to increased p53 stability.Because p53 protein was elevated in response to Ad-E2F1 +AS-MDM2 + irradiation, we reasoned that this treatment wouldenhance p53 transactivation function. Figure 5A and B(24 hours after treatment) and Fig. 5D (6 hours after treatment)show that phosho-p53 levels are increased by Ad-E2F1 + AS-MDM2 in Western blot analysis in LNCaP and LNCaP-Rescells. A significant increase in serine phosphorylation of p53protein was observed in both unirradiated and irradiated cells.Moreover, p53 transactivation function was most pronounced incells exposed to Ad-E2F1 + AS-MDM2 (Fig. 5C).p21 is downstream of p53 and has been reported to play animportant role in cell cycle arrest; however, the role of p21 inapoptosis is still controversial (28). The levels of p21 weresignificantly elevated in unirradiated LNCaP cells treated withAd-E2F1, Ad-E2F1 + MM, or Ad-E2F1 + AS-MDM2. Inirradiated LNCaP cells, the greatest induction of p21 was seenwith Ad-E2F1 + AS-MDM2 (Fig. 5A). In unirradiated andirradiated LNCaP-Res cells, p21 was found to be elevated inresponse to Ad-Luc + AS-MDM2, Ad-E2F1 alone, Ad-E2F1 +MM, and Ad-E2F1 + AS-MDM2, and these levels were furtherincreased in response to irradiation (Fig. 5B). These resultsshow that Ad-E2F1 + AS-MDM2 in the presence or absence ofradiation induces p53 protein that is functionally activated toenhance the levels of effector genes, such as p21.Because AR plays a critical role in the development andprogression of prostate cancer (29) and is a regulator ofapoptosis (30), and AR activation is regulated in part by p53and MDM2 (19, 31), we analyzed AR expression in response toAd-E2F1 + AS-MDM2. Radiation caused a significant increasein AR protein in LNCaP and LNCaP-Res cells that were treatedwith Ad-Luc, Ad-Luc + MM, and Ad-Luc + AS-MDM2.Lower levels of AR protein expression were observed in bothunirradiated and irradiated LNCaP and LNCaP-Res cells treatedwith Ad-E2F1 F AS-MDM2, with the greatest reduction seenafter exposure to Ad-E2F1 and AS-MDM2 (Fig. 5A and B).Ad-E2F1 + AS-MDM2 treatment was effective at down-regulating radiation-induced AR protein.Because p53 transactivation function was robustly observedwith Ad-E2F1 plus AS in both LNCaP and LNCaP-Res cells(Fig. 5C), we further tested the effect of Ad-E2F1 + AS-MDM2induced p53 transactivation function on p53 targets in LNCaPcells. In the LNCaP cells, we found that phospho-p53 increasedas quickly as 6 hours with Ad-E2F1 or Ad-E2F1 + AS-MDM2alone or in combination with radiation (Fig. 5D). This wasassociated with strong increases in PUMA and Bax with nosignificant changes in the Bcl-2 protein, suggesting that therewas activation of proapoptotic events in response to Ad-E2F1or Ad-E2F1 + AS with or without ionizing radiation.E2F1 has been found to stimulate ATM through a uniquemechanism that is distinct from agents that cause DNA double-strand breaks and cause delayed gH2AX phosphorylation (32).Thus, we analyzed phosphorylated forms of gH2AX in LNCaPcells. A significant increase in phospho-gH2AX was observedwith Ad-E2F1 or Ad-E2F1 + AS-MDM2 treatment with andwithout radiation (Fig. 5E). These findings ascertain the functional effect of E2F1 leading to phosphorylation of p53that may involve ATM kinase in both untreated and irradiatedLNCaP cells. Table 2. Radiation Enhancement Ratios for Prostate Cancer Cell Lines Treated with Ad-E2F1 or Ad-E2F1 + AS Cell LinesRadiation Enhancement Ratios RT + Ad-E2FRT + Ad-Luc + MMRT + Ad-Luc + ASRT + Ad-E2F + MMRT + Ad-E2F + AS D0 RER SF2 RER D0 RER SF2 RER D0 RER SF2 RER D0 RER SF2 RER D0 RER SF2 RER LNCaP1.210.80.971.031.231.41.10.91.444.5LNCaP-Res 1.42.30.950.991.11.31.44.7218.95PC-30.90.811.131.71.60.961.61.84.9 Abbreviation: RER, radiation enhancement ratio.AS-MDM2 and Radiation Enhances E2F1-Induced Apoptosis Mol Cancer Res 2008;6(11). November 20081745 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from Ad-E2F1 + AS-MDM2 Induces Phospho-cH2AX, Bax,and p21 Proteins in p53 PC-3 CellsAndrogen-independent and p53 PC-3 cells respondedeffectively to radiation when they were treated with Ad-E2F1 +AS-MDM2. To understand the mechanism of sensitization, weassessed the level of phospho-gH2AX to ascertain whetherincreased E2F1 is associated with phospho-gH2AX in theabsence of wild-type p53 protein. In addition, we assessed the induction of p21 protein, Bcl-2, and Bax to determine whetherthese p53 targets are induced in the absence of p53. Ad-E2F1and Ad-E2F1 + AS-MDM2 caused induction of phospho-gH2AX in PC3 cells; further induction was evident withirradiation (Fig. 6A).Irradiated PC-3 cells were found to have increased elevation of p21 protein when treated with Ad-E2F1 or Ad-E2F1 + AS-MDM2 (Fig. 6B) in the presence or absence of radiation.Slightly higher levels of p21 were seen when Ad-E2F1 and AS-MDM2 were combined. Up-regulation of p21 afterFIGURE 3. Caspase-3/7 levels in LNCaP,LNCaP-Res, and PC3 cells after Ad-E2F1, AS-MDM2, and RT (5 Gy) treatment. LNCaP (A),LNCaP-Res(B), and PC3 (C) cells were culturedin their respective media for 3 d, and then incu-bated with Ad-E2F1 for 1 h (LNCaP, 10 MOI;LNCaP-Res, 20 MOI; PC3, 50 MOI) followed by200 nmol/L of AS-MDM2 or MM for 36 h inthe presence of Lipofectin. Caspase-3/7 activity(relative fluorescent units) was measured byfluorometric assay. Columns, average from threeindependent experiments; bars, SE. *, P <0.0001, compared with respective MM control;x, P < 0.01, compared with Ad-Luc control (one-way ANOVA least significance difference test).Udayakumar et al. Mol Cancer Res 2008;6(11). November 20081746 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from Ad-E2F1 F AS-MDM2 was seen in both p53 andp53 prostate cancer cells, suggesting a role in radiation-induced apoptosis. To further understand the role of p21, wedetermined the effect of p21 inhibition on the induction ofcaspase-3 activity mediated by Ad-E2F1 or Ad-E2F1 + AS-MDM2. No changes in the induction of caspase-3 activity inresponse to Ad-E2F or Ad-E2F + AS-MDM2 was observedwhen endogenous p21 protein was inhibited using p21 smallinterfering RNA (data not shown). These findings suggest thatp21 may not be directly required in the apoptotic response toAd-E2F or Ad-E2F + AS-MDM2.Because p21 was not a factor in eliciting apoptosis in responseto Ad-E2F1 or Ad-E2F + AS-MDM2, we assessed the inductionof bcl-2 and Bax in response to these treatments. Bax was foundto be elevated in response to Ad-E2F1 or Ad-E2F1 + AS-MDM2with or without radiation; a concurrent reduction in Bcl-2 wasobserved in cells treated with the Ad-E2F1 + AS-MDM2 group(Fig. 6C). These findings suggest that Ad-E2F– or Ad-E2F1 +AS-MDM2–mediated death may be due to induction of Bax, andthus we observe an increase in caspase-3 activity (Fig. 3). Normal Human Fibroblast Cells Are Resistant to Ad-E2F1or Ad-E2F1 + AS-MDM2 TreatmentsThe above findings show that Ad-E2F1 or Ad-E2F1 + AS-MDM2 treatments were effective in killing prostate tumor cells.Next, we tested the effect of these treatments on normal humanTable 3. Other Significant Comparisons for All Three Cell Lines Caspase-3 + Caspase-7LNCaPLNCaP-ResPC3 GroupsRT+RTRT+RTRT+RT Ad-E2F1 vs Ad-E2F1 + ASP < 0.003P < 0.001P < 0.0001P < 0.0001P < 0.002P < 0.0001Ad-E2F1 + AS vs Ad-Luc + ASP < 0.001P < 0.0001P < 0.0001P < 0.0001P < 0.0001P < 0.0001Ad-E2F1 + AS vs ASP < 0.002P < 0.0001P < 0.0001P < 0.0001P < 0.0001P < 0.0001 FIGURE 4. E2F1 or MDM2 levels in prostate cells after Ad-E2F1, AS-MDM2, and RT treatments. Cells were cultured for 3 d and then transduced withAd-E2F1 or Ad-Luc with AS-MDM2 as described in Materials and Methods. For groups with RT, cells were irradiated (5 Gy) after 24 h of gene transduction.Total protein was extracted after 24 h for groups without RT and after 48 h for the RT group. Immunoblot assays for E2F1, MDM2, and h-actin were measuredafter Ad-E2F1 + AS-MDM2 and RT treatments in LNCaP, LNCaP + RT(A), LNCaP-Res, LNCaP-Res + RT (B), and PC3, PC3 + RT (C) cells. Right,densitometry data (A-C).AS-MDM2 and Radiation Enhances E2F1-Induced Apoptosis Mol Cancer Res 2008;6(11). November 20081747 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from fibroblast cells. Radiation alone or radiation with or without Ad-Luc, Ad-Luc + AS-MDM2, Ad-E2F1, Ad-E2F1 + MMfailed to cause an increase in caspase-3/7 activity (Fig. 7), aswas observed significantly in LNCaP, LNCaP-Res, and PC-3cells (Fig. 3). Further, with treatment of Ad-E2F1 + AS-MDM2, there was a weak increase in caspase-3/7 activity bothin the unirradiated and irradiated groups (Fig. 7). Thesefindings show that Ad-E2F1 or Ad-E2F1 + AS-MDM2 therapyis weakly toxic to normal fibroblast cells but highly toxic toprostate cancer cells. DiscussionBoth E2F-1 and MDM2 are key determinants of apoptosis,and as we have shown previously, each sensitizes LNCaPcells to radiation (2, 3). We hypothesized that Ad-E2F1 andAS-MDM2 would work in concert to sensitize prostate cancercells to radiation. The data presented here show that Ad-E2F1 +AS-MDM2 significantly enhanced clonogenic and apoptotic celldeath when administered with radiation over either agent appliedindividually. This is the first report describing the radiosensitization of prostate tumor cells with Ad-E2F1 + AS-MDM2.We also investigated the molecular mechanisms involved inthe enhancement of cell death from Ad-E2F1 + AS-MDM2combined with radiation by measuring key proteins in apoptoticpathways. Although the mechanisms by which E2F1 inducesapoptosis are not completely understood, it has been suggestedthat apoptosis results from incompatible signals for prolifera-tion and cell cycle arrest (33). One such set of conflictingsignals is the concomitant stimulation of E2F1 and p53 activity(23, 34). The p53 gene plays a key role in prevention of tumorformation through regulation of downstream targets, leading togrowth arrest and apoptosis (35). High levels of p53 proteinwere observed when either Ad-E2F1 or AS-MDM2 was givenalone. The levels of p53 were highest when Ad-E2F1 + AS-MDM2 were combined with irradiation. Therefore, theconflicting signals from E2F1 and p53 were amplified by adding AS-MDM2.Several studies have shown that E2F1 and p53 cooperate tomediate apoptosis. In fibroblasts, E2F1-induced apoptosis isFIGURE 5. Key apoptotic protein levels in LNCaP and LNCaP-Res cells after Ad-E2F1, AS-MDM2, and RT treatments. Cells were cultured for 3 d andthen transduced with Ad-E2F1 or Ad-Luc with AS-MDM2. For groups with RT, cells were irradiated (5 Gy) after 24 h of gene transduction. Total protein wasextracted after 24 h for groups without RT and after 48 h for the RT group. Immunoblot assays of key related proteins [p53, phospho-Ser p53 (phos-p53 ),p21, AR, and h-actin] were measured after Ad-E2F1 + AS-MDM2 and RT treatments in LNCaP(A) and LNCaP-Res (B) cells. C. For p53 transactivationstudies, LNCaP and LNCaP-Res cells were incubated with Ad-E2F1 or Ad-Luc for 1 h, followed by cotransfection of p53 luciferase reporter constructs(1 Ag/mL) and AS-MDM2 or MM (200 nmol/L) treatment as described. The cells were harvested 48 h posttransfection in 100 AL reporter lysis buffer(Promega). Luciferase and h-galactosidase activities were measured using kits from Promega and Roche, respectively. Luciferase values were normalizedby transfection efficiency as measured by h-galactosidase. Columns, mean of three independent experiments; bars, SE.Udayakumar et al. Mol Cancer Res 2008;6(11). November 20081748 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from potentiated by high levels of endogenous wild-type p53(36, 37). We investigated the phosphorylation status of p53 tounderstand the influence of E2F1 + AS-MDM2 on p53activation. Using Ser phospho-specific antibodies, treatmentwith Ad-E2F1 + AS-MDM2 was confirmed to significantlyactivate p53 above that of the single treatment controls. Shonoand colleagues (38) reported that p53 phosphorylation at Ser is critical for p53-mediated apoptosis. Other studies have shownthat extracellular signal–regulated kinases and p38 kinaseactivation of p53 and apoptosis are mediated throughphosphorylation of p53 at Ser (39). We found thattranscriptional activation, measured by cotransfection with ap53 response element-luciferase reporter construct in LNCaPand LNCaP-Res cells, was maximal in response to Ad-E2F1 +AS-MDM2. These targeted treatments induced a high level ofp53 function (as observed by increased levels of phospho-p53)in p53 LNCaP and LNCaP-Res cells.Although it was originally believed that p53 was essentialfor E2F1-mediated apoptosis, it is now clear that p53 is notalways required (6, 33, 40). Our results using the PC3(p53) model show that treatment with Ad-E2F1 plus AS-MDM2 is effective at inducing apoptosis and increasing celldeath overall, through p53-independent mechanisms. Thetherapeutic applications are, therefore, broad because manyadvanced prostate cancers harbor mutant or no p53 expression(41-47).Up-regulation and activation of p53 by Ad-E2F1 + AS-MDM2 + irradiation in LNCaP and LNCaP-Res cells heraldedincreased p21 protein expression. It is well known thatexpression of p21 is linked to the up-regulation of phospho-p53 in response to DNA-damaging agents (48). In PC-3 cells,E2F1 + AS-MDM2 and radiation also resulted in an increase inp21, despite the absence of functional p53. p21 is a potentinhibitor of cyclin-dependent kinases and induces cell cyclearrest in response to DNA damage both in the presence and inthe absence of functional p53 (49-51). p21 also blocks DNAreplication by inhibiting proliferation cell nuclear antigenactivity (52-54) and is required for or associated with apoptosisin some cell types (28).Elevation of p21 expression by etoposide, a topoisomeraseinhibitor, has been observed in human breast carcinoma cells totrigger apoptosis (55). Similarly, okadaic acid– induced apoptosis is coupled with the cell cycle–independent up-regulationof endogenous p21 (56). Furthermore, vascular smooth muscleFIGURE 5 Continued. D. Equal amounts of protein from the cell lysates were loaded by SDS-PAGE and immunoblotted with phospho-p53, PUMA,Bcl2, BAX, or actin antibodies. E. Phospho-gH2AX or actin expression in LNCaP cells after Ad-E2F1 and AS-MDM2 followed by RT (5 Gy) treatment. Right,densitometry data.AS-MDM2 and Radiation Enhances E2F1-Induced Apoptosis Mol Cancer Res 2008;6(11). November 20081749 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from cells transfected with p21 DNA exhibit the characteristicfeatures of apoptotic cell death (57). In our study, p21 up-regulation by Ad-E2F1 + AS-MDM and radiation was notassociated with greater levels of apoptotic cell death andclonogenic inhibition in p53 PC-3 cells because knockdownof p21 failed to reduce apoptosis in response to thesetreatments. On the other hand, up-regulation of Bax related toincreased caspase-3/7 activity may play a critical role inregulating the response to Ad-E2F1 + AS-MDM2.We also observed that the levels of AR were reduced afterexposure to Ad-E2F1 + AS-MDM2, compared with Ad-E2F1alone or Ad-Luc alone. Both MDM2 and E2F1 have a role inAR-mediated transcription (58). MDM2 indirectly affects AR-mediated signaling through inactivation of p53. For example,when p53 is overexpressed in hormone-refractory tumors(59-62), there is also increased expression of AR (31, 63) andvice versa (64, 65). Recently, Davis and colleagues (66) studiedthe expression patterns of AR and E2F1; increased levels ofE2F1 were associated with decreased expression of AR inmetastatic prostate tissues. They also found that overexpressionof E2F1 caused a decrease in the mRNA, protein, and promoteractivity of the AR, indicating that E2F1 andAR interactions havean opposing critical role in prostate cancer progression. Otherreports have shown that down-regulation of AR is partly FIGURE 6. gH2AX, p21, Bcl2, and BAX protein levels in PC3 cells. Equal amounts of PC3 cell lysates were loaded by SDS-PAGE and immunoblottedwith phospho-gH2AX or actin antibodies after Ad-E2F1 and AS-MDM2, followed by RT (5 Gy)treatment.A. Phospho-gH2AX or actin expression in PC3 cellsafter Ad-E2F1 combined with AS-MDM2 and RT (5 Gy) treatment.B. p21 expression in PC3 cells after Ad-E2F1 and AS-MDM2 treatment. Right,densitometry data(A and B). C. Bcl2 and BAX expression levels in PC3 cells after Ad-E2F1 combined with AS-MDM2 and RT (5 Gy) treatment. Actin wasmeasured as a loading control.Udayakumar et al. Mol Cancer Res 2008;6(11). November 20081750 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from responsible for the induction of apoptosis in prostate cancer cells(30). Thus, the down-regulation of AR protein in response to Ad-E2F1 + AS-MDM2 in LNCaP and LNCaP-Res cells may havecontributed to the apoptotic response observed; however, AR isnot required because apoptosis was still observed in PC-3 cells.The other hallmark of this study is that in both LNCaP andPC-3 cells, phospho-gH2AX was significantly elevated inresponse to Ad-E2F1 or Ad-E2F1 + AS-MDM2 treatments withor without radiation. This observation is in concordance withPowers et al. (32). It is not clear whether the increase inphospho-gH2AX is an indicator of increased double-strandbreaks caused by Ad-E2F1 because the role of phospho-gH2AX in DNA repair and apoptosis induction is controversial.The mode of killing elicited in response to Ad-E2F1 or Ad-E2F1 + AS-MDM2 was primarily through apoptosis andreproductive death because we failed to observe any kind ofmitotic catastrophe such as micronuclei or large nondividingcells. This is confirmed in another study, which found Ad-E2F1to be involved directly in eliciting apoptosis rather than causingother mitotic changes (67).We provide evidence for the first time that adenoviral-mediated overexpression of E2F1 combined with AS-MDM2–mediated suppression of MDM2 significantly induces apoptosisand clonogenic death when combined with radiation. Theenhancement in radiation-induced cell death was observed inthree prostate cancer cell lines with varying molecularresponses and sensitivities to androgen. Radiosensitization byE2F1 expression and inhibition of MDM2 was associated withup-regulation of Bax, phospho-gH2AX, and p21 in all prostatecancer cell lines tested, with down-regulation of functional ARand bcl-2. Thus, adenoviral E2F1 and AS-MDM2 arepromising adjuncts to radiation in the treatment of prostatecancer, with less or no toxicity to normal cells. Materials and MethodsCell CultureLNCaP and PC3 cells were cultured in DMEM-F12containing 10% fetal bovine serum, penicillin-streptomycin,and L-glutamine, as described previously (68). Androgen-insensitive LNCaP-Res cells were grown in androgen-deprived DMEM/F-12 medium. Androgen deprivation wasachieved by culturing the cells in medium containing 10%charcoal-stripped serum for 3 d. LNCaP-Res cells wereestablished by serial passage of LNCaP cells in androgen-deprived medium for 1 y. Normal human embryonic IMR-90fibroblast cell line (ATCC CCL-186) was obtained fromAmerican Type Culture Collection and grown in DMEM/F-12 medium. Oligonucleotides and AntibodiesIdera, Inc., provided the oligonucleotides. The antisenseMDM2 oligonucleotide (AS-MDM2) and its mismatchcontrol oligonucleotide (MM) are 20-mer mixed-backboneoligonucleotides with the following sequences; AS-MDM2,5-UGACACCTGTTCTCACUCAC-3; MM, 5-UGTCACCC-TTTTTCATUCAC-3. They were stored as frozen aliquots at20jC (13). E2F1, p53, p21, MDM2, phospho-p65, PUMA,and h-actin antibodies were obtained from Oncogene; phospho-gH2AX(Ser) antibody from Abcam; and antimousehorseradish peroxidase–conjugated secondary antibody fromAmersham. Viral infection and Transient TransfectionsLNCaP, LNCaP-Res, PC3, or IMR-90 cells (510) weregrown for 3 d, and then infected with adenovirus-5 constructthat harbors full-length E2F1 under the control of cytomega-lovirus promoter (Ad-E2F1) or control adenoviral construct thatharbors full-length luciferase cDNA under the control ofcytomegalovirus promoter (Ad-Luc; control vector). The cellswere incubated with the virus for 1 h followed by transfectionwith 200 nmol/L AS-MDM2 or mismatch (MM; control)oligonucleotide in 2 mL culture medium for 36 to 48 h in thepresence of 7 Ag/mL Lipofectin (Invitrogen). Groups withradiation (5 Gy) treatment were irradiated 24 h after AS-MDM2or MM and reincubated for 24 h. Western Blot AnalysisWestern blot analyses were done to confirm transductionefficiency. Cells were harvested after 24 h posttransfectionwith Ad-E2F1 or AS-MDM2, lysed in buffer [50 mmol/LTris-HCl (pH 6.8), 2% SDS with protease inhibitor cocktailset I (Calbiochem)], and were sonicated. Thirty micrograms of protein from each cell lysate were electrophoresed on a4% to 20% SDS polyacrylamide gel. After transfer ontoa polyvinylidene difluoride membrane (Millipore) in atransblot apparatus and blocking with 5% low-fat dried milk,the blots were incubated overnight at 4jC with specificFIGURE 7. Normal human embryonic IMR-90fibroblast cells were cultured in DMEM-F12 for(10 MOI) followed by 200 nmol/L of AS-MDM2 orMM for 36 h in the presence of Lipofectin.Caspase-3/7 activity (relative fluorescent units)was measured by fluorometric assay. Columns,average from three independent experiments;bars, SE.AS-MDM2 and Radiation Enhances E2F1-Induced Apoptosis Mol Cancer Res 2008;6(11). November 20081751 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from primary antibodies. The membranes were washed and labeledwith an antimouse horseradish peroxidase–conjugated sec-ondary antibody (Amersham Pharmacia Biotech) at roomtemperature for f1 h. Detection by chemiluminescence wasdone according to the manufacturer’s (Amershan) standardprotocol. Immunofluorescence/Confocal MicroscopyLNCaP cells grown on coverslips were fixed in ice-coldacetone/methanol (1:1) for 15 min at 20jC. Cells were thenair-dried, rehydrated in PBS, blocked, and permeabilized in0.2% Triton X-100/PBS. The cells were then washed in PBSand subsequently blocked in 5% bovine serum albumin.Primary antibodies were applied to the coverslips for 1 h andthen overlaid with Alexa Fluor 568–conjugated goat anti-mouse secondary antibody (Molecular Probes) for 30 min.After three washes in PBS, the coverslips were furtherincubated in 4¶,6-diamidino-2-phenylindole (1 Ag/mL) to stainthe nuclei. The coverslips were then mounted using an aqueousmounting medium (anti-fading agents; Biomeda Corp.). Theimages were analyzed and captured using a Bio-Rad Radiance2000 LSCM confocal microscope. Clonogenic AssaysThe techniques for clonogenic survival assays have beendescribed previously (2). For clonogenic survival assays,LNCaP, LNCaP-Res, and PC3 cells were cultured inrespective media as described above for 2 to 3 d. Appropriatedilutions of Ad-E2F1 or Ad-Luc in 1 mL solution to achieveappropriate MOIs were gently placed onto the monolayer of cells in each dish and incubated for 1 h. Control dishes withmedium alone or with Ad-Luc were exposed to identicalconditions. After incubation, 4 mL of control medium withserum were added to each dish and incubated overnight. At24 h after viral exposure, three sets of dishes at each RT doselevel were irradiated with a high-dose rate cesium unit(Csirradiator, Model 81-14R, JL, Shepherd & Associates) for a totalof 2, 4, and 6 Gy. Immediately after irradiation, the cells weretrypsinized, serially diluted, replated into 100-mm dishes, andincubated. After 14 d, colonies were stained with methylene blueand counted. Cell survival was adjusted for plating efficiency.The data represent the average from three independent experi-ments. D0 and n were calculated using a single-hit multitargetmodel (69). Radiation enhancement ratios were calculated usingSF2 or D0 values of radiation alone versus combined treatmentsas described earlier (70). Detection of Apoptosis by TUNEL AssaysTUNEL was done with the fluorescein-FragEL DNAfragmentation detection kit according to the manufacturer’sinstructions (Oncogene Research Products, Biosciences, Inc.).Briefly, cells were fixed with 4% paraformaldehyde at roomtemperature for 10 min. After being washed with PBS, cellswere treated with proteinase K (2 mg/mL) at room temperaturefor 5 min, followed by addition of TdT equilibration buffer for30 min. The cells were then stained with the fluorescein-FragEL-TdT labeling reaction mix and TdT enzyme at 37jC for1 h. Following washing with TBS, the cells were analyzed byflow cytometric analysis on a FACSCalibur flow cytometer(BD Biosciences). A sample population of 10,000 cells wasused for analysis by cell count software. The data wereanalyzed with FlowJo (Tree Star) software. Determination of Caspase-3/7 ActivityCaspase-3/7 activity was measured using a fluorometricsubstrate, z-DEVD-rhodamine (The Apo-ONE HomogeneousCaspase-3/7 Assay kit, Promega). Cells were cultured in therespective media and treated with Ad-E2F1 or Ad-Luc and AS-MDM2 or MM as described above. A total of 2 10 cells in50 AL culture medium were mixed with 50 AL of HomogeneousCaspase-3/7 reagent in 96-well plates and incubated at roomtemperature for 18 h. Substrate cleavage was quantifiedfluorometrically at 485-nm excitation and 538-nm emission.Fluorescence was measured on a fluorescent plate reader(LabSystems, Inc.). p53-Luciferase Reporter AssaysLNCaP, LNCaP-Res, and PC3 cells were incubated with Ad-E2F1 or Ad-Luc for 1 h, followed by cotransfection of p53luciferase reporter constructs (1 Ag/mL) and AS-MDM2 or MMtreatments as described above. Cells were incubated for 24 h at37 C; after 24 h, fresh medium was added to the cells. The cellswere harvested 48 h posttransfection in 100 AL reporter lysisbuffer (Promega). Luciferase and h-galactosidase activitieswere measured using kits from Promega and Roche, respec-tively. Luciferase values were normalized by transfectionefficiency as measured by h-galactosidase. All data representmean values of three independent experiments F SD. Statistical AnalysisStatistics were done using the Statistical Package for theSocial Sciences (SPSS). Statistical significance among exper-imental groups was determined using the one-way ANOVAleast significance difference test. Differences were consideredstatistically significant at P < 0.05. Disclosure of Potential Conflicts of InterestThe Dr. A. Pollack received a commercial grant from Varian Medical Systems. References1. Pollack A, Salem N, Ashoori F, et al. Lack of prostate cancer radio-sensitization by androgen deprivation. Int J Radiat Oncol Biol Phys 2001;51:1002– 7. 2. Nguyen KH, Hachem P, Khor LY, et al. Adenoviral-E2F-1 radiosensitizesp53wild-type and p53null human prostate cancer cells. Int J Radiat Oncol BiolPhys 2005;63:238–46. 3. Mu Z, Hachem P, Agrawal S, Pollack A. Antisense MDM2 sensitizes prostatecancer cells to androgen deprivation, radiation, and the combination. Int J RadiatOncol Biol Phys 2004;58:336–43. 4. Shan B, Lee WH. Deregulated expression of E2F-1 induces S-phase entry andleads to apoptosis. Mol Cell Biol 1994;14:8166 –73. 5. Pruschy M, Wirbelauer C, Glanzmann C, Bodis S, Krek W. E2F-1 hasproperties of a radiosensitizer and its regulation by cyclin A kinase is requiredfor cell survival of fibrosarcoma cells lacking p53. Cell Growth Differ 1999;10:141 –6. 6. Nip J, Strom DK, Fee BE, Zambetti G, Cleveland JL, Hiebert SW. E2F-1 cooperates with topoisomerase II inhibition and DNA damage toselectively augment p53-independent apoptosis. Mol Cell Biol 1997;17:1049– 56.Udayakumar et al. Mol Cancer Res 2008;6(11). November 20081752 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from 7. Finlay CA. The mdm-2 oncogene can overcome wild-type p53 suppression oftransformed cell growth. Mol Cell Biol 1993;13:301 –6. 8. Khor LY, Desilvio M, Al-Saleem T, et al. MDM2 as a predictor of prostatecarcinoma outcome: an analysis of Radiation Therapy Oncology Group Protocol8610. Cancer 2005;104:962 –7. 9. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation ofp53. Nature 1997;387:296 –9. 10. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3for tumor suppressor p53. FEBS Lett 1997;420:25–7. 11. Kubbutat MH, Jones SN, Vousden KH. Regulation of p53 stability by Mdm2.Nature 1997;387:299– 303. 12. Tao W, Levine AJ. Nucleocytoplasmic shuttling of oncoprotein Hdm2 isrequired for Hdm2-mediated degradation of p53. Proc Natl Acad Sci U S A 1999;96:3077 –80. 13. Zhang Z, Wang H, Prasad G, et al. Radiosensitization by antisense anti-MDM2 mixed-backbone oligonucleotide in in vitro and in vivo human cancermodels. Clin Cancer Res 2004;10:1263 –73. 14. Shieh SY, Ikeda M, Taya Y, Prives C. DNA damage-induced phosphorylationof p53 alleviates inhibition by MDM2. Cell 1997;91:325–34. 15. Maki CG. Oligomerization is required for p53 to be efficiently ubiquitinatedby MDM2. J Biol Chem 1999;274:16531– 5. 16. Chehab NH, Malikzay A, Stavridi ES, Halazonetis TD. Phosphorylation ofSer-20 mediates stabilization of human p53 in response to DNA damage. ProcNatl Acad Sci U S A 1999;96:13777– 82. 17. Maya R, Balass M, Kim ST, et al. ATM-dependent phosphorylation of Mdm2on serine 395: role in p53 activation by DNA damage. Genes Dev 2001;15:1067–77. 18. Lin HK, Wang L, Hu YC, Altuwaijri S, Chang C. Phosphorylation-dependentubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3ligase. EMBO J 2002;21:4037– 48. 19. Gaughan L, Logan IR, Neal DE, Robson CN. Regulation of androgenreceptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation. NucleicAcids Res 2005;33:13 –26. 20. Zhang Z, Wang H, Li M, Rayburn ER, Agrawal S, Zhang R. Stabilization ofE2F1 protein by MDM2 through the E2F1 ubiquitination pathway. Oncogene2005;24:7238–47. 21. Zhang Z, Li M, Wang H, Agrawal S, Zhang R. Antisense therapytargeting MDM2 oncogene in prostate cancer: effects on proliferation, apoptosis,multiple gene expression, and chemotherapy. Proc Natl Acad Sci U S A 2003;100:11636–41. 22. Stoyanova R, Hachem P, Hensley H, et al. Antisense-MDM2 sensitizesLNCaP prostate cancer cells to androgen deprivation, radiation, and thecombination in vivo . Int J Radiat Oncol Biol Phys 2007;68:1151 –60. 23. Hiebert SW, Packham G, Strom DK, et al. E2F-1:DP-1 induces p53and overrides survival factors to trigger apoptosis. Mol Cell Biol 1995;15:6864–74. 24. Bates S, Phillips AC, Clark PA, et al. p14ARF links the tumour suppressorsRB and p53. Nature 1998;395:124– 5. 25. Peitsch MC, Muller C, Tschopp J. DNA fragmentation duringapoptosis is caused by frequent single-strand cuts. Nucleic Acids Res 1993;21:4206–9. 26. Yang HL, Dong YB, Elliott MJ, et al. Adenovirus-mediated E2F-1 genetransfer inhibits MDM2 expression and efficiently induces apoptosis in MDM2-overexpressing tumor cells. Clin Cancer Res 1999;5:2242–50. 27. Mu Z, Hachem P, Hensley H, et al. Antisense MDM2 enhances the responseof androgen insensitive human prostate cancer cells to androgen deprivationin vitro and in vivo . Prostate 2008;68:599–609. 28. Kang KH, Kim WH, Choi KH. p21 promotes ceramide-induced apoptosisand antagonizes the antideath effect of Bcl-2 in human hepatocarcinoma cells.Exp Cell Res 1999;253:403 –12. 29. Heinlein CA, Chang C. Androgen receptor in prostate cancer. Endocr Rev2004;25:276–308. 30. Bhuiyan MM, Li Y, Banerjee S, et al. Down-regulation of androgenreceptor by 3,3¶-diindolylmethane contributes to inhibition of cellproliferation and induction of apoptosis in both hormone-sensitive LNCaPand insensitive C4–2B prostate cancer cells. Cancer Res 2006;66:10064 –72. 31. Cronauer MV, Schulz WA, Burchardt T, Ackermann R, Burchardt M.Inhibition of p53 function diminishes androgen receptor-mediated signaling inprostate cancer cell lines. Oncogene 2004;23:3541 –9. 32. Powers JT, Hong S, Mayhew CN, Rogers PM, Knudsen ES, Johnson DG.E2F1 uses the ATM signaling pathway to induce p53 and Chk2 phosphorylationand apoptosis. Mol Cancer Res 2004;2:203 –14. 33. Fueyo J, Gomez-Manzano C, Yung WK, et al. Overexpression of E2F-1 inglioma triggers apoptosis and suppresses tumor growth in vitro and in vivo . NatMed 1998;4:685 –90. 34. Kowalik TF, DeGregori J, Leone G, Jakoi L, Nevins JR. E2F1-specificinduction of apoptosis and p53 accumulation, which is blocked by Mdm2. CellGrowth Differ 1998;9:113–8. 35. Sun Y. p53 and its downstream proteins as molecular targets of cancer. MolCarcinog 2006;45:409–15. 36. Kowalik TF, DeGregori J, Schwarz JK, Nevins JR. E2F1 overexpression inquiescent fibroblasts leads to induction of cellular DNA synthesis and apoptosis.J Virol 1995;69:2491– 500. 37. Wu X, Levine AJ. p53 and E2F-1 cooperate to mediate apoptosis. Proc NatlAcad Sci U S A 1994;91:3602 –6. 38. Shono T, Tofilon PJ, Schaefer TS, Parikh D, Liu TJ, Lang FF. Apoptosisinduced by adenovirus-mediated p53 gene transfer in human glioma correlateswith site-specific phosphorylation. Cancer Res 2002;62:1069 –76. 39. She QB, Bode AM, Ma WY, Chen NY, Dong Z. Resveratrol-inducedactivation of p53 and apoptosis is mediated by extracellular-signal-regulatedprotein kinases and p38 kinase. Cancer Res 2001;61:1604– 10. 40. Hunt KK, Deng J, Liu TJ, et al. Adenovirus-mediated overexpressionof the transcription factor E2F-1 induces apoptosis in human breast andovarian carcinoma cell lines and does not require p53. Cancer Res 1997;57:4722–6. 41. Ross JS, Sheehan CE, Dolen EM, Kallakury BV. Morphologic and molecularprognostic markers in prostate cancer. Adv Anat Pathol 2002;9:115–28. 42. Tamboli P, Amin MB, Xu HJ, Linden MD. Immunohistochemical expressionof retinoblastoma and p53 tumor suppressor genes in prostatic intraepithelialneoplasia: comparison with prostatic adenocarcinoma and benign prostate. ModPathol 1998;11:247–52. 43. Stackhouse GB, Sesterhenn IA, Bauer JJ, et al. p53 and bcl-2 immunohis-tochemistry in pretreatment prostate needle biopsies to predict recurrence ofprostate cancer after radical prostatectomy. J Urol 1999;162:2040–5. 44. Takayama H, Shin M, Nonomura N, Okuyama A, Aozasa K. p53 mutationsin prostatic intraepithelial neoplasia and concurrent carcinoma: analysis of lasercapture microdissected specimens from non-transition and transition zones. Jpn JCancer Res 2000;91:941–7. 45. Ittmann M, Wieczorek R, Heller P, Dave A, Provet J, Krolewski J.Alterations in the p53 and MDM-2 genes are infrequent in clinically localized,stage B prostate adenocarcinomas. Am J Pathol 1994;145:287 –93. 46. Osman I, Drobnjak M, Fazzari M, Ferrara J, Scher HI, Cordon-Cardo C.Inactivation of the p53 pathway in prostate cancer: impact on tumor progression.Clin Cancer Res 1999;5:2082–8. 47. Leite K, Franco M, Strougi M, et al. Abnormal expression of MDM2 inprostate carcinoma. Mod Pathol 2001;14:428–36. 48. Macleod KF, Sherry N, Hannon G, et al. p53-dependent and independentexpression of p21 during cell growth, differentiation, and DNA damage. GenesDev 1995;9:935 –44. 49. Blagosklonny MV, Prabhu NS, El-Deiry WS. Defects in p21WAF1/CIP1,Rb, and c-myc signaling in phorbol ester-resistant cancer cells. Cancer Res 1997;57:320–5. 50. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases.Genes Dev 1995;9:1149 –63. 51. el-Deiry WS, Tokino T, Velculescu VE, et al. WAF1, a potential mediator ofp53 tumor suppression. Cell 1993;75:817–25. 52. Sherr CJ. Mammalian G1 cyclins. Cell 1993;73:1059 –65. 53. Waga S, Hannon GJ, Beach D, Stillman B. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature1994;369:574 –8. 54. Xiong Y, Zhang H, Beach D. D type cyclins associate with multipleprotein kinases and the DNA replication and repair factor PCNA. Cell 1992;71:505 –14. 55. Sheikh MS, Li XS, Chen JC, Shao ZM, Ordonez JV, Fontana JA.Mechanisms of regulation of WAF1/Cip1 gene expression in human breastcarcinoma: role of p53-dependent and independent signal transduction pathways.Oncogene 1994;9:3407–15. 56. Sheikh MS, Garcia M, Zhan Q, Liu Y, Fornace AJ, Jr. Cell cycle-independentregulation of p21Waf1/Cip1 and retinoblastoma protein during okadaic acid-induced apoptosis is coupled with induction of Bax protein in human breastcarcinoma cells. Cell Growth Differ 1996;7:1599–607. 57. Matsushita H, Morishita R, Kida I, et al. Inhibition of growth of humanAS-MDM2 and Radiation Enhances E2F1-Induced Apoptosis Mol Cancer Res 2008;6(11). November 20081753 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from vascular smooth muscle cells by overexpression of p21 gene through induction ofapoptosis. Hypertension 1998;31:493–8. 58. Agus DB, Cordon-Cardo C, Fox W, et al. Prostate cancer cell cycleregulators: response to androgen withdrawal and development of androgenindependence. J Natl Cancer Inst 1999;91:1869–76. 59. Navone NM, Troncoso P, Pisters LL, et al. p53 protein accumulation andgene mutation in the progression of human prostate carcinoma. J Natl Cancer Inst1993;85:1657 –69. 60. Aprikian AG, Sarkis AS, Fair WR, Zhang ZF, Fuks Z, Cordon-Cardo C.Immunohistochemical determination of p53 protein nuclear accumulation inprostatic adenocarcinoma. J Urol 1994;151:1276–80. 61. Burchardt M, Burchardt T, Shabsigh A, et al. Reduction of wild type p53function confers a hormone resistant phenotype on LNCaP prostate cancer cells.Prostate 2001;48:225–30. 62. Zhou JR, Yu L, Zerbini LF, Libermann TA, Blackburn GL. Progression toandrogen-independent LNCaP human prostate tumors: cellular and molecularalterations. Int J Cancer 2004;110:800–6. 63. Shenk JL, Fisher CJ, Chen SY, Zhou XF, Tillman K, Shemshedini L. p53represses androgen-induced transactivation of prostate-specific antigen bydisrupting hAR aminoto carboxyl-terminal interaction. J Biol Chem 2001;276:38472–9.64. Nesslinger NJ, Shi XB, deVere White RW. Androgen-independent growth ofLNCaP prostate cancer cells is mediated by gain-of-function mutant p53. CancerRes 2003;63:2228 –33. 65. Nantermet PV, Xu J, Yu Y, et al. Identification of genetic pathways activatedby the androgen receptor during the induction of proliferation in the ventralprostate gland. J Biol Chem 2004;279:1310 –22. 66. Davis JN, Wojno KJ, Daignault S, et al. Elevated E2F1 inhibits transcriptionof the androgen receptor in metastatic hormone-resistant prostate cancer. CancerRes 2006;66:11897–906. 67. Ebelt H, Hufnagel N, Neuhaus P, et al. Divergent siblings: E2F2 and E2F4but not E2F1 and E2F3 induce DNA synthesis in cardiomyocytes withoutactivation of apoptosis. Circ Res 2005;96:509–17. 68. Mu Z, Hachem P, Pollack A. Antisense Bcl-2 sensitizes prostate cancer cellsto radiation. Prostate 2005;65:331–40. 69. Alcock RA, Dey S, Chendil D, et al. Farnesyltransferase inhibitor(L-744,832) restores TGF-h type II receptor expression and enhances radiationsensitivity in K-ras mutant pancreatic cancer cell line MIA PaCa-2. Oncogene2002;21:7883–90. 70. Dey S, Spring PM, Arnold S, et al. Low-dose fractionated radiationpotentiates the effects of Paclitaxel in wild-type and mutant p53 head and necktumor cell lines. Clin Cancer Res 2003;9:1557 –65.Udayakumar et al. Mol Cancer Res 2008;6(11). November 20081754 on April 19, 2017. © 2008 American Association for Cancer Research.mcr.aacrjournals.orgDownloaded from Correction: Article on AS-MDM2 and Radiation EnhancesE2F1-Induced Apoptosis In the article by Udayakumar and colleagues on AS-MDM2, beginning on page 1742of the November 2008 issue ofMolecular Cancer Research , there was an error in the title.The correct title appears here.Antisense MDM2 Enhances E2F1-Induced Apoptosis and the Combination SensitizesAndrogen-Sensitive and Androgen-Insensitive Prostate Cancer Cells to Radiation. Udayakumar TS, Hachem P, Ahmed MM, Agrawal S, Pollack A. Antisense MDM2enhances E2F1-induced apoptosis and the combination sensitizes androgen-sensitive andandrogen-insensitive prostate cancer cells to radiation. Mol Cancer Res 2008;6:1742–54. Copyright D 2008 American Association for Cancer Research.doi:10.1158/1541-7786.MCR-6-12-COR Mol Cancer Res 2008;6(12). December 20081957 2008;6:1742-1754.Mol Cancer ResThirupandiyur S. Udayakumar, Paul Hachem, Mansoor M. Ahmed, et al.Androgen-Independent Prostate Cancer Cells to RadiationCombination Sensitizes Androgen-Dependent andAntisense MDM2 Enhances E2F1-Induced Apoptosis and the Updated versionhttp://mcr.aacrjournals.org/content/6/11/1742Access the most recent version of this article at: Cited articleshttp://mcr.aacrjournals.org/content/6/11/1742.full.html#ref-list-1This article cites 70 articles, 37 of which you can access for free at: Citing articles/content/6/11/1742.full.html#related-urlsThis article has been cited by 2 HighWire-hosted articles. 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