Ras Puts the Brake on Doxorubicin-mediated Cell Death inp53-expressing Cells*□S

Received for publication, October 5, 2010, and in revised form, December 3, 2010 Published, JBC Papers in Press, December 14, 2010, DOI 10.1074/jbc.M110.191916

Sunil K. Manna1,2, Charitha Gangadharan1, Damodar Edupalli, Nune Raviprakash, Thota Navneetha,Sidharth Mahali, and Maikho ThohFrom the Laboratory of Immunology, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad 500 001, India

Doxorubicin is one of the most effective molecules used inthe treatment of various tumors. Contradictory reports oftenopen windows to understand the role of p53 tumor suppressorin doxorubicin-mediated cell death. In this report, we provideevidences that doxorubicin induced more cell death in p53-negative tumor cells. Several cells, having p53 basal expres-sion, showed increase in p53 DNA binding upon doxorubicintreatment. Doxorubicin induced cell death in p53-positivecells through expression of p53-dependent genes and activa-tion of caspases and caspase-mediated cleavage of cellular pro-teins. Surprisingly, in p53-negative cells, doxorubicin-medi-ated cell death was more aggressive (faster and intense).Doxorubicin increased the amount of Fas ligand (FasL) by en-hancing activator protein (AP) 1 DNA binding in both p53-positive and p53-negative cells, but the basal expression of Faswas higher in p53-negative cells. Anti-FasL antibody consider-ably protected doxorubicin-mediated cell death in both typesof cells. Activation of caspases was faster in p53-negative cellsupon doxorubicin treatment. In contrast, the basal expressionof Ras oncoprotein was higher in p53-positive cells, whichmight increase the basal expression of Fas in these cells. Over-expression of Ras decreased the amount of Fas in p53-negativecells, thereby decreasing doxorubicin-mediated aggressive celldeath. Overall, this study will help to understand the muchstudied chemotherapeutic drug, doxorubicin-mediated cellsignaling cascade, that leads to cell death in p53-positive and-negative cells. High basal expression of Fas might be an im-portant determinant in doxorubicin-mediated cell death inp53-negative cells.

Doxorubicin, an anthracycline compound, is one of themost effective chemotherapeutic agents available to treatbreast cancer and leukemia patients. The drug is able to in-duce regression of metastatic breast cancer (1, 2). Several re-ports suggest that it generates oxidative stress that leads toDNA damage and culminates in cell death through mitoticcatastrophe (3–5). DNA damage activates p53, which in turn

activates several genes such as p21, p17, etc., causing cell cyclearrest followed by apoptosis. Several tumor cells includingbreast cancer cells have a mutation in p53. Still, these cells areequally potent for cell death against several apoptosis-pro-moting agents. The oncogenic mutation in Ras family genesincluding H-, K-, and N-ras is often observed in several hu-man cancers (5). Ras family proteins are predominantly in-volved in cell cycle progression. It has been reported that on-cogenic Ras induces senescence and apoptosis through p53activation (6). In hepatocellular carcinoma, restoration of p53rapidly regressed H-ras (7). It is also reported that p53 cancoexist with K-ras in human cancer tissues and cells (8). In amouse model, the K-ras can progress tumor despite the intactp53 (9). Oncogenic K-ras has shown to repress p53 functionby stabilizing the Snail (10).Sustained elevation of calcium in cells keeps high calcineu-

rin activity. The family of transcription factors of nuclear fac-tor of activated T-cells (NF-AT)3 is the target substrate forcalcineurin (11). Upon stimulation of calcineurin, several resi-dues at the regulatory domain of NF-AT are dephosphory-lated, and this leads to nuclear translocation of NF-AT andactivation of target genes such as AP-1,MEF2, FasL, andGATA (11–13). As doxorubicin increases reactive oxygen spe-cies production, it promotes NF-�B activation via activationof I�B kinase complex. Aberrantly active NF-�B complexescan contribute to tumorigenesis by regulating genes that notonly promote the growth but also survival of cancer cells (14,15) and also induce resistance against doxorubicin (16).Doxorubicin has been shown to induce cell death via a non-classical pathway involving a biphasic induction of NF-�B,which in turn expresses interleukin-8 (IL-8), and this IL-8induces cell death through a sequential process: increase inintracellular Ca2� release, calcineurin activation, dephosphor-ylation of NF-AT, nuclear translocation of NF-AT, NF-AT-dependent FasL expression, FasL-mediated caspase activa-tion, and induction of cell death (17). FasL expression againdepends upon the transcriptional activation of AP-1 throughactivation of c-Jun N-terminal kinase (JNK) (18, 19). Ex-pressed FasL acts through its specific receptor Fas and acti-vates caspases, the cysteine proteolytic enzymes, which arereported to be the executioners of apoptosis. Oleandrin, acardiac glycoside, has shown to induce cell death potently inseveral human cell types (11, 20, 21).

* This work was supported by a grant from the Department of Science andTechnology (DST), Government of India, and a core grant from the Cen-tre for DNA Fingerprinting and Diagnostics (CDFD). This work was alsosupported by Council for Scientific and Industrial Research (CSIR), Gov-ernment of India fellowships (to C. G., S. M., and M. T.).

□S The on-line version of this article (available at http://www.jbc.org) con-tains supplemental Figs. 1– 4.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed. Tel.: 011-91-40-24749412;

Fax: 011-91-40-24749448; E-mail: manna@cdfd.org.in.

3 The abbreviations used are: NF-AT, nuclear factor of activated T-cells; AP-1,activator proteins 1; FasL, Fas ligand; MTT, 3-(4,5-dimethyl-2-thiozolyl)-2,5-diphenyl-2H-tetrazolium bromide; PARP, poly (ADP-ribose) polymer-ase; NE, nuclear extracts; Ab, antibody; pNA, para-nitroaniline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 9, pp. 7339 –7347, March 4, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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In this report, we have found that doxorubicin-mediatedcell death is slow and less potent in p53-positive cells. Breasttumor cell line MCF-7 has basal expression of p53, whereasother breast cells such as SKBr3 and MDA-MB-231 have mu-tated p53 (22, 23). Cells, such as U-937 (monocytic cells),THP1 (monocytic macrophages), and HeLa (epithelial cells)have either mutated or no p53 expression (24, 25). HCT116cells are knocked out of p53 by homologous recombinationand designated as (HCT116 (p53�/�)), and non-transfectedcells (HCT116 (Wild)) are used for this study. We have pro-vided the evidences for the first time that p53-positive cellshave high basal K-ras, but low Fas expression, which mightdictate p53-positive cells for slow and less potent cell deathmediated by doxorubicin than p53 mutant or null cells, al-though both types of cells showed equal expression of FasLupon treatment of doxorubicin. This study will help under-stand how p53, although it is a well known tumor suppressor,puts the brake via the Fas-Ras pathway on doxorubicin-medi-ated cell death, which might help target the specific moleculesto regulate sensitization of p53-positive cells for aggressivecell death.

EXPERIMENTAL PROCEDURES

Materials—Doxorubicin, EDTA, EGTA, PMSF, propidiumiodide, Ac-DVED-pNA, Ac-ITED-pNA, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT), gly-cine, oleandrin, and anti-tubulin antibody were obtained fromSigma. DMEM and fetal bovine serum (FBS) were obtainedfrom Invitrogen. DAPI and goat anti-rabbit IgG conjugatedwith Alexa Fluor 594 were purchased fromMolecular Probes.Antibodies against p53, p21, PARP, JNK1, Fas, FasL, K-ras,IL-8, goat anti-rabbit IgG conjugated with HRP, and gel shiftoligonucleotides for AP-1, NF-AT, and p53 were obtainedfrom Santa Cruz Biotechnology (Santa Cruz, CA). Antibodiesagainst phospho-JNK and SP-600125 (JNK inhibitor) wereobtained from Calbiochem. The plasmid constructs for FasL-luciferase and NF-AT-luciferase were a kind gift from Prof.B. B. Aggarwal of the University of Texas, M.D. AndersonCancer Center (Houston, TX). The K-ras wild type and mu-tant constructs were obtained from the Laboratory of CancerBiology, Centre for DNA Fingerprinting and Diagnostics(CDFD), Hyderabad, India.Cell Lines—MCF-7, SKB3, MDA-MB-231, U-937, HeLa,

and THP1 cells were obtained from American Type CultureCollection (Manassas, VA). HCT116 cells and p53 knock-outHCT116 cells were a kind gift from the Prof. B. Vogelsteinlaboratory (Johns Hopkins Oncology Center, Baltimore, MD).NF-�B, AP-1, NF-AT, and p53 DNA Binding Assay—DNA

binding activity of NF-�B, AP-1, NF-AT, and p53 was deter-mined by EMSA (26). Briefly, after different treatments, cells,were used to prepare cytoplasmic extracts and nuclear ex-tracts (NE). NE proteins (8 �g) were incubated with 32P end-labeled double-stranded NF-�B oligonucleotide of HIV-LTR,5�-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCA-GGGAGGCGTGG-3�, for 30 min at 37 °C and run on 6.6%native PAGE. Similarly, AP-1, NF-AT, and p53 were assayedusing specific double-stranded labeled oligonucleotides. Visu-

alization of radioactive bands was done in a FluorescentImage Analyzer FLA-3000 (Fuji).NF-AT- or FasL-dependent Luciferase Gene Transcription

Assay—The expression of NF-AT- or FasL-dependent lucifer-ase reporter gene was carried out as described previously (11).Cells were transiently transfected with SuperFect transfectionreagent containing 0.5 �g of each reporter plasmid containingan NF-AT or a FasL binding site cloned upstream of luciferase(designated as NF-AT-luciferase or FasL-luciferase) and GFPconstructs. After 3 h of transfection, cells were washed andcultured for 12 h. GFP-positive cells were counted. Transfec-tion efficiency was observed as 37–40% in different transfec-tion conditions. After various treatments, the cells were ex-tracted with lysis buffer (part of the luciferase assay kit fromPromega), and the extracts were incubated with the fireflyluciferin (substrate from Promega). Light emission was moni-tored with a luminometer, and values were calculated as -foldof activation over vector-transfected value.Cytotoxicity Assay—The cytotoxicity was measured by

MTT assay (15). Briefly, cells (1 � 104 cells/well of a 96-wellplate) were treated with different agents for the indicated con-centrations and times, and thereafter, 25 �l of MTT solution(5 mg/ml in PBS) was added. After a 2-h incubation, 100 �l ofextraction buffer (20% SDS in 50% dimethylformamide) wasadded. After an overnight incubation at 37 °C, absorbance wasread at 570 nm with the extraction buffer as blank.Determination of Nuclear Fragmentation—U-937, MCF-7,

THP1, and HeLa cells were treated with doxorubicin. Cellswere then harvested and fixed in 80% methanol, stained withpropidium iodide, and viewed under a fluorescence micro-scope (20).Determination of DNA Fragmentation—MCF-7 and HeLa

cells were treated with doxorubicin and then harvested, andDNA was extracted following the method described earlier(20). Extracted DNA (2.0 �g) was analyzed by electrophoresison a 2% agarose gel. DNA fragments were visualized withethidium bromide under UV light.LIVE/DEAD Assay—The cytotoxicity was determined by

the LIVE/DEAD assay (27) (Molecular Probes, Eugene, OR).Briefly, after different treatments, 1 � 105 cells were stainedwith the LIVE/DEAD cell assay reagent. Red (as dead) andblue (as live) cells were analyzed under a fluorescence micro-scope (Labophot-2, Nikon, Tokyo, Japan).Immunocytochemistry—The amount of Ras was examined

by the immunocytochemical method as described (11).Briefly, cells were cultured on chamber slides, washed afterdifferent treatments, air-dried, fixed with 3.5% formalde-hyde, and permeabilized with 0.5% Triton X-100. Slideswere blocked by 5% goat serum and incubated with anti-K-ras Ab for 8 h followed by incubation with goat anti-rabbitIgG-Alexa Fluor 594 for 1 h. Slides were mounted withmounting medium with DAPI and analyzed under a fluo-rescence microscope.Caspase 3 and 8 Activities Assay—To evaluate caspase 3

and 8 activities, cell lysates were prepared after their respec-tive treatments. 50 �g of the cell lysate proteins was incubatedwith 200 �M caspase 3 substrate (Ac-DVED-pNA) or caspase8 substrate (Ac-ITED-pNA) in 100 �l of reaction buffer (1%

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Nonidet P-40, 20 �M Tris-HCl, pH 7.5, 137 mM NaCl, and10% glycerol) and incubated for 2 h at 37 °C. The release ofchromophore pNA (para-nitroaniline) was monitored spec-trophotometrically at 405 nm (11).In Vitro JNK Assay—The JNK was assayed by a method de-

scribed previously (28). Briefly, JNK complex from whole-cellextract (300 �g) was precipitated with anti-JNK1 antibody (1�g) followed by incubation with protein A/G-Sepharosebeads (Pierce). After a 2-h incubation, the beads were washedwith lysis buffer and assayed for activity of JNK using 5 �g ofGST-Jun (amino acids 1–100) substrate protein.Western Blot—PARP, p21, p53, Fas, FasL, Ras, phospho-

JNK, JNK1, and tubulin were detected by Western blot tech-nique using specific antibodies followed by detection bychemiluminescence (Amersham Biosciences).Reverse Transcription (RT)-PCR—Total RNA was isolated

using the standard TRIzol method, and 1 �g of total RNAwasreverse-transcribed into cDNA by the One-Step Access RT-PCRkit (Promega, Madison,WI) followed by amplification usinggene-specific primers for FasL, caspase 8, and actin. PCRwasperformed, and amplified products were separated by agarose gelelectrophoresis (2%) and visualized by ethidium bromide stain-ing. The primer sequence and product size are as follows: FasL,344 bp (forward) 5�-GGATTGGGCCTGGGGATGTTTCA-3�,(reverse) 5�-TTGTGGCTCAGGGGCAGGTTGTTG-3�;caspase 8, 380 bp (forward) 5�-TTATTCAGGCTTGTCAGG-3�,(reverse) 5�-GCACCATCAATCAGAAGGGAAG-3�; actin, 616bp (forward) 5�-CCAACCGTGAAAAGATGACC-3�, (reverse)5�-GCAGTAATCTCCTTCTGCATCC-3�.Statistical Analysis—Results were expressed as mean �

S.D. of three independent experiments. Statistical analyses of

the samples were done by the Student’s t test wherever appli-cable. The p � 0.05 was considered to be significant.

RESULTS

In this study, the effects of doxorubicin in p53-positive and-negative cells were examined. Doxorubicin was used as a so-lution in dimethyl sulfoxide (DMSO) at 10 mM concentration.Further dilution was carried out in cell culture medium. Theconcentrations and duration of exposure of the chemicalsemployed in these studies had no effect on cytolysis as de-tected by lactate dehydrogenase assay from culture superna-tant of treated cells (data not shown).Doxorubicin Increases Cell Death More Potently in SKBr3,

U-937, THP1, and HeLa Cells than MCF-7 Cells—Differentcells were treated with 1 �M doxorubicin for different times,and an MTT assay was done to detect cell death. Doxorubicinincreased cell death almost 15–25% at any time of treatmentin U-937, THP1, HeLa, or MDA-MB-231 cells when com-pared with MCF-7 cells. At 72 h of doxorubicin treatment,cell death was observed at 88% (p � 0.005), 85% (p � 0.005),83% (p � 0.01), 87% (p � 0.005), and 63% (p � 0.01) forU-937, THP1, HeLa, MDA-MB-231, and MCF-7 cells, respec-tively (Fig. 1A). Almost 20% more cell death was observed inSKBr3 cells than in MCF-7 cells at any concentration of doxo-rubicin when treated for 72 h (supplemental Fig. 1A). Nuclearfragmentation data as detected by propidium iodide-stainedcell nuclei supported the similar cell death in these cells (Fig.1B). Doxorubicin induced cell death at 72% (p � 0.01), 77%(p � 0.005), 58% (p � 0.005), 79% (p � 0.01), and 76% (p �0.005) in U-937, THP1, MCF-7, HeLa, and SKBr3 cells, re-spectively, at 72 h of treatment as detected by the LIVE/

FIGURE 1. Effect of doxorubicin on induction of cell death in U-937, HeLa, THP1, SKBr3, and MCF-7 cells. A, THP1, U-937, HeLa, MDA-MB-231, andMCF-7 cells were treated with 1 �M doxorubicin for different times, and inhibition of cell viability was calculated from the MTT assay. B, U-937, THP1, MCF-7,HeLa, and SKBr3 cells were treated with 1 �M doxorubicin for 72 h. Cells were fixed, stained with propidium iodide, and viewed under a fluorescence micro-scope. U-937, THP1, HeLa, and MCF-7 cells were treated with doxorubicin (1 �M) for 72 h. Cells were incubated with LIVE/DEAD assay solution for 30 min.Cells were viewed under a fluorescence microscope. C, the number of apoptotic cells (red color) and live cells (green color) was counted, and the percentageof apoptotic cells is indicated in the figure. MCF-7 and HeLa cells were treated with doxorubicin (1 and 2.5 �M) for 24 h. Cells were harvested, and DNA wasprepared and run in a 1.5% agarose gel. D, the gel was stained with ethidium bromide and viewed in Gel Doc.

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DEAD assay (Fig. 1C). At 24 h of doxorubicin treatment, al-most no DNA fragmentation was observed in MCF-7 cells,but HeLa cells showed sufficient DNA fragmentation (Fig.1D). Partial PARP cleavage was observed at 48 and 72 h inMCF-7 cells upon doxorubicin treatment, although in THP1cells, complete PARP cleavage was observed at the same timeof treatment (supplemental Fig. 1B). Phase contrast micro-scope views for the doxorubicin-treated cells also suggestedmore cell death in HeLa cells when compared with MCF-7cells (supplemental Fig. 1C). All these data suggest that doxo-rubicin-mediated cell death is faster and more potent inU-937, THP1, SKBr3, MDA-MB-231, and HeLa cells thanMCF-7 cells.Doxorubicin Increases Cell Death in More Potently in p53-

negative Cells—Doxorubicin enhanced p53 DNA binding withincreasing concentrations in MCF-7 cells, but not in SKBr3cells (Fig. 2A). The p53 DNA binding was increased with in-creasing time of doxorubicin treatment in MCF-7 cells, butno significant p53 DNA binding was observed in U-937 cells(supplemental Fig. 2A). We have shown that doxorubicin in-duces p53 in MCF-7 cells, but not in U-937 or SKBr3 cells.MCF-7 cells have wild type p53, and SKBr3, U-937, THP1, orHeLa cells either are null for p53 or have mutated p53 (21,22). To detect the role of p53 in differential cell death medi-ated by doxorubicin, we used HCT116 cells, having wild typep53 (HCT116 (Wild)), and p53 negative HCT116 cells inwhich p53 was knocked out by homologous recombination(HCT116 (p53�/�)). The high basal p53 DNA binding activitywas marginally increased in HCT116 (Wild) cells with theincreasing time of doxorubicin treatment, whereas HCT116(p53�/�) cells did not show any basal p53 DNA binding anddoxorubicin had no effect in altering the p53 DNA binding(Fig. 2B, upper panel). Doxorubicin showed biphasic NF-�B

DNA binding in both p53-positive and p53-negative HCT116cells (Fig. 2B, second upper panel). The amounts of p53 andp21 increased with increasing time of doxorubicin treatmentin HCT116 (Wild) cells, but not in HCT116 (p53�/�) cells(Fig. 2B, lower panels). These data suggest that HCT116(p53�/�) cells do not have p53 expression and that HCT116(Wild) cells have basal expression of p53. Doxorubicin in-duces p53 and p53-dependent p21 gene in p53-positive cells.Doxorubicin induces biphasic activation of NF-�B. HCT116(p53�/�) cells showed almost 20–25% more cell death thanHCT116 (Wild) cells at any point of doxorubicin treatment(Fig. 2C). Doxorubicin treatment also showed almost 20–25%less cell death in HCT116 (Wild) cells than HCT116 (p53�/�)cells at any concentration (supplemental Fig. 2B).Doxorubicin Increases AP-1 DNA Binding, JNK Activation,

FasL Expression, and Caspase Activation in Both p53-positiveand p53-negative Cells—To understand the differential po-tency of doxorubicin-mediated cell death in p53-positive and-negative cells, we have measured the amount of FasL, whichis transcriptionally regulated by AP-1 (18, 19), upon treat-ment of doxorubicin. Doxorubicin increased AP-1 DNA bind-ing with increasing time of treatment as shown from the nu-clear extracts by gel shift assay in both HCT116 (Wild) andHCT116 (p53�/�) cells (Fig. 3A, lower panel). It showed bi-phasic activation of NF-�B DNA binding in both types of cells(Fig. 3A, upper panel), which is supported by our previousobservation (17). Doxorubicin almost equally induced JNKactivation in both types of cells as shown by detecting theamount of phospho-JNK by Western blot (supplemental Fig.3A) or by in vitro JNK assay using GST-Jun as substrate pro-tein (Fig. 3B). The amount of FasL expression was almostequal in both of these cells upon doxorubicin treatment asshown by RT-PCR (Fig. 3C1), Western blot (Fig. 3C2), or

FIGURE 2. Effect of doxorubicin on induction of p53 and cell death in p53-positive and -negative cells. A, MCF-7 and SKBr3 cells were treated with dif-ferent concentrations of doxorubicin for 24 h, and nuclear extracts were assayed for p53 DNA binding by gel shift assay. HCT116 cells (HCT116 (Wild)) andp53 knock-out cells (HCT116 (p53�/�)) were treated with 1 �M doxorubicin for different times. B, upper panels, NE were prepared, and 8 �g of NE proteinsper sample was used to detect p53 and NF-�B by gel shift assay. Lower panels, whole-cell extracts were prepared, and 100 �g of proteins was used to detectp53 and p21 by Western blot. Blots were reprobed for tubulin. HCT116 p53�/� and wild type cells were treated with 1 �M doxorubicin for different times,and cell viability was detected by MTT assay. C, inhibition of cell viability was indicated in the figure.

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FasL-dependent luciferase gene expression (supplemental Fig.3B). The amount of caspase 8 expression was almost equal inboth types of cells upon doxorubicin treatment as shown byRT-PCR data (supplemental Fig. 3C), whereas activities ofcaspase 8 (Fig. 3D1) and caspase 3 (Fig. 3D2) were muchhigher in than HCT116 (p53�/�) cells than HCT116 (Wild)cells upon doxorubicin treatment. The cleavage of PARP wasmore intense and faster in HCT116 (p53�/�) cells thanHCT116 (Wild) cells (Fig. 3E). These data suggest that al-though the AP-1-dependent FasL expression is equal in bothp53-positive and p53-negative cells, the activity of caspases isdifferent in these cells.Inhibition of FasL Protects Doxorubicin-mediated Aggressive

Cell Death in p53-negative Cells—To detect the role of FasLin aggressive cell death mediated by doxorubicin in p53-nega-tive cells, both HCT116 (Wild) and HCT116 (p53�/�) cellswere treated with different concentrations of SP-600125 (JNKinhibitor) and then stimulated with doxorubicin for 24 h.AP-1 DNA binding activity was assayed from nuclear extracts.

As shown in Fig. 4, A1 and A2, doxorubicin-mediated AP-1DNA binding decreased gradually with the increasing concen-trations of SP-600125. The SP-600125 did not alter p53 DNAbinding in MCF-7 or HCT116 (Wild) cells (supplemental Fig.4A). Doxorubicin induced 67% (p � 0.005) and 84% (p �0.01) cell death in HCT116 (Wild) and HCT116 (p53�/�)cells, respectively. In SP-600125-pretreated cells, doxorubicininduced cell death 29 and 40% in HCT116 (Wild) andHCT116 (p53�/�) cells, respectively (Fig. 4B1). This resultsuggests that almost 40% cell death is protected by JNK inhib-itor. In anti-FasL Ab-pretreated cells, doxorubicin induced 28and 29% cell death in HCT116 (p53�/�) and HCT116 (Wild)cells, respectively (Fig. 4B2). These data suggest that additive(almost 20%) cell death in HCT116 (p53�/�) cells is com-pletely protected by anti-FasL Ab. As JNK inhibitor alonecannot block the additive cell death but anti-FasL Ab blocks itcompletely, we have looked at the possible other pathway forFasL expression, via IL-8 (17), in these cells. Both HCT116(p53�/�) and HCT116 (Wild), when preincubated with anti-

FIGURE 3. Effect of doxorubicin on induction of NF-�B, AP-1, p53, JNK, FasL, caspases, and PARP cleavage in p53-positive and -negative cells.A, HCT116 (p53�/�) and wild type cells were treated with 1 �M doxorubicin for different times. NE were prepared, and 8 �g of NE proteins per sample wasused to detect NF-�B and AP-1 by gel shift assay. HCT116 (p53�/�) and wild type cells were treated with 1 �M doxorubicin for different times, and whole-cell extracts were prepared. 300 �g of whole-cell extracts was incubated with anti-JNK Ab (200 ng) for 2 h and then pulled down by protein A/G-Sepharosebeads and assayed for JNK using 2 �g of GST-Jun as substrate protein and [�-32P]ATP. The reaction mixture was boiled with SDS-PAGE Laemmli buffer andrun in a 9% SDS-PAGE. B, the gel was dried and exposed to a phospho-screen, and the radioactive bands were detected as activity of JNK. MCF-7 and U-937cells were treated with 1 �M doxorubicin for different times, and total RNA was extracted. C1, the amount of FasL was measured from total RNA by RT-PCR.M, Marker lane for DNA ladder. C2, HCT116 (p53�/�) and wild type cells were treated with 1 �M doxorubicin for different times, whole-cell extracts wereprepared, and the amount of FasL was detected from 100 �g of proteins by Western blot. HCT116 wild type and p53�/� cells were treated with 1 �M doxo-rubicin for different times and harvested in lysis buffer. D1 and D2, cellular lysates were incubated with caspase 8 substrate (Ac-ITED-pNA) (D1) or caspase 3substrate (Ac-DVED-pNA) (D2), and absorbance was recorded at 405 nm. Results are indicated as the percentage of caspase activities, considering 1-fold foruntreated cells. Oleandrin (OL) (100 ng/ml) was used to treat the cells as a positive control. E, HCT116 (p53�/�) and wild type cells were treated with 1 �M

doxorubicin for different times, and PARP was measured from 100 �g of whole-cell extracts.

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FasL or -IL-8 Ab and SP-600125 followed by treatment withdoxorubicin, demonstrated that anti-IL-8 Ab- and SP-600125-preincubated cells showed complete inhibition, butanti-FasL Ab- and SP-600125-preincubated cells showed par-tial inhibition of AP-1 DNA binding (supplemental Fig. 4B).

Anti-IL-8 Ab or SP-600125 partially but in combination com-pletely inhibited FasL expression in both HCT116 (p53�/�)and HCT116 (Wild) cells as shown by the amount of FasLdetected by Western blot (Fig. 4C1) and FasL-dependent lu-ciferase activity (Fig. 4C2). These data suggest that expression

FIGURE 4. Effect of JNK inhibitor, anti-FasL Ab, or anti-IL-8 Ab on doxorubicin-mediated cell death in p53-positive and -negative cells. A1 and A2,HCT116 (p53�/�) and wild type cells were treated with different concentrations of JNK inhibitor (SP-600125) for 2 h and then stimulated with 1 �M doxoru-bicin for 24 h. After these treatments, nuclear extracts were prepared and assayed for AP-1 by gel shift assay. B1 and B2, HCT116 (p53�/�) and wild type cellswere treated with 10 �M SP-600125 (B1) or 1 �g/ml anti-FasL antibody (B2) for 2 h and then treated with 1 �M doxorubicin for 72 h in triplicate. Cell viabilitywas detected by MTT assay, and inhibition of cell viability was indicated in the figure considering untreated cell viability as 100%. C1 and D1, HCT116(p53�/�) and wild type cells were pretreated with SP-600125, anti-IL-8 Ab, or both in combination for 2 h followed by treatment with doxorubicin (1 �M) for24 h, the amount of FasL was detected from whole-cell extracts by Western blot (C1), and NF-AT DNA binding activity was measured from nuclear extract bygel shift assay (D1). HCT116 (p53�/�) and wild type cells were transfected with Qiagen SuperFect reagent for 3 h with plasmids for FasL or NF-AT promoterDNA that had been linked to luciferase (FasL-luciferase or NF-AT-luciferase) and GFP. After washing, cells were cultured for 12 h. The GFP-positive cells werecounted, and transfection efficiency was calculated. C2 and D2, cells were pretreated with SP-600125, anti-IL-8 Ab, or both in combination for 2 h followedby doxorubicin (1 �M) for 24 h, and luciferase activity was measured and indicated as -fold of activation for FasL-dependent genes (C2) or NF-AT-dependentgenes (D2). E1 and E2, HCT116 (p53�/�) and wild type cells were treated without or with 10 �M SP-600125 and 1 �g/ml anti-FasL Ab or -IL-8 Ab for 2 h andthen stimulated with 1 �M doxorubicin for 72 h. Cell viability was detected by MTT assay, and inhibition of cell viability was indicated in the figure, consider-ing untreated cell viability as 100%.

p53-negative Cells Are More Sensitive to Doxorubicin

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of FasL is dependent upon IL-8-mediated signaling and AP-1-dependent transcription. As IL-8 expresses FasL via NF-AT(17), the DNA binding activity of NF-AT was completely in-hibited by anti-IL-8 Ab, but not by SP-600125, as shown bygel shift assay (Fig. 4D1) or NF-AT-dependent luciferase ac-tivity assay (Fig. 4D2). These data further suggest that bothIL-8-mediated expression and AP-1-dependent FasL expres-sion interplay in doxorubicin-mediated cell death. Doxorubi-cin induced 63% (p � 0.005) and 87% (p � 0.01) cell death inHCT116 (Wild) and HCT116 (p53�/�) cells, respectively. InSP-600125- or anti-IL-8 Ab-preincubated cells, doxorubicininduced cell death 29 and 40% in HCT116 (Wild) andHCT116 (p53�/�) cells, respectively. In a combination of SP-600125 and anti-IL-8 Ab, only 22% cell death was observed inboth HCT116 (Wild) (Fig. 4E1) and HCT116 (p53�/�) (Fig.4E2) cells. The amount of cleaved PARP also showed the sim-

ilar extent of cell death as shown in the MTT assay (supple-mental Fig. 4C). These data suggested that by inhibiting FasL,both p53-positive and p53-negative cells show a similar extentof cell death mediated by doxorubicin.HCT116 (Wild) Cells Show High Basal Expression of K-ras,

but Low Basal Expression of Fas—As FasL is expressed in bothp53-positive and p53-negative cells upon doxorubicin treat-ment, the amount of Fas was measured to understand the dif-ferential cell death. A high basal amount of Fas was observedin HCT116 (p53�/�) cells, and doxorubicin did not alter thisbasal amount of Fas (Fig. 5A). The high basal amount of K-ras, but not H-ras, was observed in HCT116 (Wild) cells, asshown by Western blot (Fig. 5B1) and immunofluorescence(Fig. 5B2). Doxorubicin treatment did not alter this basal ex-pression of K-ras. HCT116 (p53�/�) cells, transfected withvector, K-ras (wild), and K-ras (mutant) constructs were

FIGURE 5. Effect of doxorubicin in expression of K-ras and Fas on p53-positive and -negative cells. A and B1, HCT116 (p53�/�) and wild type cells weretreated with 1 �M doxorubicin for different times, and whole-cell extracts were used to detect Fas (A) and H-ras and K-ras (B1) by Western blot analysis. Blotswere reprobed for tubulin. B2, both HCT116 (p53�/�) and wild type cells were treated with 1 �M doxorubicin for 24 h, and then cells were used to detectK-ras by anti-K-ras Ab conjugated with Alexa Fluor 594 by immunofluorescence microscope. HCT116 p53 (p53�/�) cells were transfected with vector, K-ras(wild), or K-ras (mutant), and GFP constructs were transfected with Qiagen SuperFect reagent for 3 h. After transfection, cells were washed and cultured for12 h. GFP-positive cells were counted under a fluorescence microscope, and transfection efficiency was calculated. C, cells were treated with doxorubicin (1�M) for 24 h, and then the amounts of K-ras and Fas were measured by Western blot. Transfected cells, HCT116 (p53�/�), and HCT116 (Wild) cells weretreated with doxorubicin (1 �M) for 12 and 24 h, and cell viability was assayed by MTT assay. D, results are indicated as inhibition of cell viability in percent-age. HCT116 (p53�/�) and wild type cells were incubated with 1 �g/ml anti-Fas Ab for 2 h and then treated with doxorubicin (1 �M) for 72 h. Cell viabilitywas assayed by MTT assay. E, results are indicated as inhibition of cell viability in percentage. HCT116 (p53�/�) cells were transfected with vector, K-ras(wild), or K-ras (mutant) for 3 h, and GFP constructs were transfected with Qiagen SuperFect reagent for 3 h. After transfection, cells were washed and cul-tured for 12 h. Cells were incubated with 1 �g/ml anti-Fas Ab for 2 h and then treated with doxorubicin (1 �M) for 72 h. F, cell viability was assayed by theMTT assay and indicated as inhibition of cell viability in percentage.

p53-negative Cells Are More Sensitive to Doxorubicin

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treated with doxorubicin, and the amount of Fas and K-raswas measured. In K-ras (wild)-transfected cells, the amount ofK-ras was observed (Fig. 5C, upper panel). The basal amountof Fas, which was detected in vector or K-Ras (mutant) cells,was almost completely inhibited in K-ras (wild)-transfectedcells (Fig. 5C, lower panel). These data suggest that K-ras in-terplays in the basal amount of Fas, which is shown in p53-negative cells. K-ras-transfected HCT116 (p53�/�) cellsshowed a similar cell death as shown in HCT116 (Wild) cells,as shown by MTT assay (Fig. 5D). Anti-Fas Ab-pretreatedcells showed 38% (p � 0.001) cell death in HCT116 (Wild)cells, but 75% (p � 0.005) cell death was observed in HCT116(p53�/�) cells (Fig. 5E). Doxorubicin further potentiated celldeath in both types of cells. In HCT116 (p53�/�) cells, trans-fected with K-ras showed similar cell death by anti-Fas Aband doxorubicin as shown in HCT116 (Wild) cells. HCT116(p53�/�) cells, transfected with vector of mutant K-ras,showed almost 75% cell death by anti-Fas Ab alone and al-most 80% cell death by doxorubicin treatment. Co-incubationof anti-Fas Ab and doxorubicin showed almost 95% (p � 0.01)cell death in HCT116 (p53�/�) cells, transfected with vectoror mutant K-ras (Fig. 5F). These data suggest that K-ras regu-lates the expression of Fas, which helps in differential celldeath mediated by doxorubicin and anti-Fas Ab in p53-posi-tive and -negative cells.

DISCUSSION

Doxorubicin is a widely used chemotherapeutic drug fortreating breast cancer and many other tumors. Doxorubicintreatment often causes DNA damage and oxidative stress thatleads to change in mitochondrial membrane potential fol-lowed by cell death (3, 4). DNA damage often leads to activa-tion of p53, which is a well known tumor suppressor. In manytumor cells, the p53 is non-functional either by deletion or bymutation of it. Doxorubicin is a very potent chemotherapeuticdrug against breast tumor. MCF-7 cells have wild type p53that might be a target of doxorubicin, although p53-mutatedbreast tumor cells such as SKBr3 or MDA-MB-231 are alsotargeted by doxorubicin more aggressively for cell death.However, the role of p53 in tumor biology is very confusing.Chemotherapeutic drugs are effective even in the absence ofp53. We have found that in p53-positive cells, doxorubicin-mediated cell death is slower and less potent than in p53down-regulated cells, which is supported by a number of evi-dences such as conversion of MTT dye, nuclear and DNAfragmentation, LIVE/DEAD assay, caspase activation, andcaspase-dependent cellular protein fragmentation. In p53-positive cells, activation of p53 and its dependent genes is ob-served, which always induces cell cycle arrest and therebyapoptosis. Although in p53-positive cells, apoptosis is ob-served, when compared with p53-negative cells, the apoptosisis 20–25% less at any time of doxorubicin treatment. It is sur-prising that p53-negative cells lack several p53-mediatedgenes that arrest cell cycle; still, doxorubicin-mediated celldeath is much efficient. It is obvious that the unansweredquestion is that p53-mediated cell death might be proceedingthrough cell cycle arrest, but in p53-negative cells, the cell

death may be independent of cell cycle arrest, and this needsto be studied further.The p53 transcription factor activates several genes that

usually help in cell cycle arrest. Some of the genes expressedby p53 are prosurvival genes such as Cox2, DDR2, EGF, etc.(29, 30). It has been reported that myosin VI, a p53-depen-dent gene that is involved in the endocytosis pathway, inhibitsDNA damage in a p53-dependent manner (31). The highamount of p53 is also regulated by MDM2 (murine doublemutant 2) through proteasomal degradation (32). However,all these reports suggest the down-regulation of p53. Al-though we did not observe the decrease in p53 activation,which was shown either by DNA binding activity or by p53-dependent gene expression, MDM2 helps proteasome-medi-ated degradation of p53. As we did not observe the decreasein the amount of p53 as shown by Western blot, the role ofMDM2 in repression of doxorubicin-mediated cell deathshould be ruled out. It is important to look for the moleculesthat interact with p53 for its repression.Doxorubicin increased activities of caspases. Recruitment

of proteins containing death domain is a prerequisite for acti-vation of caspases. Recruitment of these proteins is often pre-ceded by interaction of death ligands such as FasL, TNF, andTNF-related apoptosis-inducing ligand (TRAIL) with theirrespective receptors. Several studies have shown that com-monly used chemotherapeutic drugs can stimulate the ex-pression of NF-�B-dependent inflammatory cytokines, suchas TNF, IL-1, FGF, G-CSF, VEGF, and IL-6 (33, 34). Activa-tion of NF-�B and AP-1 is redox-sensitive (35, 36). Doxorubi-cin is known to increase reactive oxygen species by reactingwith cellular iron (37), and this may cause the activation ofNF-�B and AP-1. Doxorubicin treatment increased DNAbinding to AP-1 and the amount of FasL irrespective of theamount of p53. FasL expression is AP-1-dependent. Surpris-ingly, the inhibition of AP-1 DNA binding did not block com-plete inhibition of FasL expression by doxorubicin treatment.Doxorubicin treatment was found to enhance a biphasic acti-vation of NF-�B, further supporting our previous observation(17). It has been reported that doxorubicin-mediated firstphase activation of NF-�B leads to expression of FasL througha sequential process: NF-�B-dependent IL-8 expression, IL-8-mediated increase in intracellular free Ca2�, Ca2�-dependentcalcineurin activation, calcineurin-mediated dephosphoryla-tion of NF-AT, nuclear translocation of NF-AT, and NF-AT-dependent FasL expression (17). Suppression of FasL expres-sion mediated by NF-�B (via IL-8) and AP-1 inhibits celldeath almost 60% in p53-positive cells and 75% in p53-nega-tive cells. Doxorubicin-mediated additive cell death (20–25%)in p53-negative cells is completely inhibited by suppression ofFasL. These data suggest that FasL-mediated cell death inter-plays in aggressive cell death in p53-negative cells induced bydoxorubicin. Although FasL expression is observed in bothp53-positive and p53-negative cells, the FasL-mediated re-sponse is still the determining factor for aggressive cell deathin p53-negative cells. FasL interacts with its specific cell sur-face receptor Fas/CD95 to induce cell death via caspase acti-vation. To our surprise, higher basal expression of Fas is ob-served in p53-negative cells when compared with p53-positive

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cells. The p53-negative cells showed more cell death uponexposure to anti-Fas Ab. These data further support that theexpression of Fas in these cells is higher resulting in pro-nounced cell death, which is further potentiated by doxorubi-cin treatment. Mutant p53 has been shown to suppress Fasexpression (38), which may not be true in the case of HCT116cells where we are not detecting the basal expression of Fas.We have also noticed that p53-positive cells have a high basalexpression of K-ras. Ras family proteins are usually an activa-tor of cell proliferation, but we are getting the opposite effectin K-ras, which is suppressing p53-mediated cell death indoxorubicin-treated cells. Basal K-ras expression is shown tobe absent in colon cancer cells (9), which is supporting outobservation that HCT116 cells have no basal expression ofK-ras, whereas p53 overexpressed HCT116 cells show highbasal expression of K-ras, which further supports that Ras isone of the p53-dependent genes (39). It has been reportedthat K-ras suppresses p53 through stabilization of Snail pro-tein, which directly binds with p53, via expression of ataxiatelangiectasia-mutated and Rad3-related proteins (40). Howp53 regulates K-ras expression needs to be studied further.The Ras family of oncogenes, especially H-ras, has beenshown to down-regulate Fas via the PI3 kinase pathway andthus execute an antiapoptotic effect (36). It has been reportedthat sustained activation of the Ras/Raf/MAPK cascade hasbeen observed in activation of p53 (41). The possible role ofbasal expressed K-ras might be decreasing the amount of Fasin p53-positive cells.Considering the usefulness of doxorubicin in treating dif-

ferent tumors, especially breast cancer, the detailed mecha-nism of action would help to design the chemotherapeuticdrugs for effective therapy especially for combination therapyand drug-resistance. We have detected the role of p53 indoxorubicin-mediated cell death in different tumor cells. Howp53 interplays in doxorubicin-mediated cell death involvingFasL expression might help to detect the role of these mole-cules in doxorubicin drug resistance as p53 exerts its regu-lated cell death effect in p53-positive cells. Thus, this studywould be useful to understand the role of p53 to regulate celldeath where K-ras puts a brake and down-regulates Fas expres-sion, thereby slowing down apoptosis in p53-positive cells.

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p53-negative Cells Are More Sensitive to Doxorubicin

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VOLUME 286 (2011) PAGES 7339 –7347DOI 10.1074/jbc.A110.191916

Ras puts the brake on doxorubicin-mediated cell death in p53-expressing cells.Sunil K. Manna, Charitha Gangadharan, Damodar Edupalli, Nune Raviprakash, Thota Navneetha, Sidharth Mahali, and Maikho Thoh

PAGE 7341:

Line 7 from the bottom should read as follows. Nuclear fragmentation data as detected by propidium iodide-stained cell nuclei supported thesimilar cell death in these cells (Fig. 1B). Doxorubicin-induced cell death 68% (p� 0.01), 76% (p� 0.005), 56% (p� 0.005), 80% (p� 0.01), and 72%(p � 0.005) in U-937, THP1, MCF-7, HeLa, and SKBr3 cells, respectively, at 72 h of treatment as detected by LIVE/DEAD assay (Fig. 1C).

FIGURE 1. Effect of doxorubicin on induction of cell death in U-937, HeLa, THP1, SKBr3, and MCF-7 cells. THP1, U-937, HeLa, MDA-MB-231, and MCF-7 cellswere treated with 1 �M doxorubicin for different times, and inhibition of cell viability was calculated from MTT assay (A). U-937, THP1, MCF-7, and HeLa cellswere treated with 1 �M doxorubicin for 72 h. Cells were fixed, stained with propidium iodide, and viewed under fluorescence microscope (B). U-937, THP1, HeLaMCF-7, and SKBr3 cells were treated with doxorubicin (1 �M) for 72 h. Cells were incubated with LIVE/DEAD assay solution for 30 min. Cells were viewed undera fluorescence microscope. The number of apoptotic cells (red) and live cells (green) was counted, and the percentage of apoptotic cells is indicated in the figure(C). MCF-7 and HeLa cells were treated with doxorubicin (1 and 2.5 �M) for 24 h. Cells were harvested, and DNA was prepared and run in 1.5% agarose gel. Gelwas stained with ethidium bromide and viewed in Gel Doc (D).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 15, p. 12157, April 6, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

APRIL 6, 2012 • VOLUME 287 • NUMBER 15 JOURNAL OF BIOLOGICAL CHEMISTRY 12157

ADDITIONS AND CORRECTIONS

Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.

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Navneetha, Sidharth Mahali and Maikho ThohSunil K. Manna, Charitha Gangadharan, Damodar Edupalli, Nune Raviprakash, ThotaRas Puts the Brake on Doxorubicin-mediated Cell Death in p53-expressing Cells

doi: 10.1074/jbc.M110.191916 originally published online December 14, 20102011, 286:7339-7347.J. Biol. Chem. 

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VOLUME 286 (2011) PAGES 7339 –7347DOI 10.1074/jbc.A110.191916

Ras puts the brake on doxorubicin-mediated cell death in p53-expressing cells.Sunil K. Manna, Charitha Gangadharan, Damodar Edupalli, Nune Raviprakash, Thota Navneetha, Sidharth Mahali, and Maikho Thoh

PAGE 7341:

Line 7 from the bottom should read as follows. Nuclear fragmentation data as detected by propidium iodide-stained cell nuclei supported thesimilar cell death in these cells (Fig. 1B). Doxorubicin-induced cell death 68% (p� 0.01), 76% (p� 0.005), 56% (p� 0.005), 80% (p� 0.01), and 72%(p � 0.005) in U-937, THP1, MCF-7, HeLa, and SKBr3 cells, respectively, at 72 h of treatment as detected by LIVE/DEAD assay (Fig. 1C).

FIGURE 1. Effect of doxorubicin on induction of cell death in U-937, HeLa, THP1, SKBr3, and MCF-7 cells. THP1, U-937, HeLa, MDA-MB-231, and MCF-7 cellswere treated with 1 �M doxorubicin for different times, and inhibition of cell viability was calculated from MTT assay (A). U-937, THP1, MCF-7, and HeLa cellswere treated with 1 �M doxorubicin for 72 h. Cells were fixed, stained with propidium iodide, and viewed under fluorescence microscope (B). U-937, THP1, HeLaMCF-7, and SKBr3 cells were treated with doxorubicin (1 �M) for 72 h. Cells were incubated with LIVE/DEAD assay solution for 30 min. Cells were viewed undera fluorescence microscope. The number of apoptotic cells (red) and live cells (green) was counted, and the percentage of apoptotic cells is indicated in the figure(C). MCF-7 and HeLa cells were treated with doxorubicin (1 and 2.5 �M) for 24 h. Cells were harvested, and DNA was prepared and run in 1.5% agarose gel. Gelwas stained with ethidium bromide and viewed in Gel Doc (D).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 15, p. 12157, April 6, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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VOLUME 286 (2011) PAGES 7339 –7347DOI 10.1074/jbc.A113.191916

Ras puts the brake on doxorubicin-mediated cell deathin p53-expressing cells.Sunil K. Manna, Charitha Gangadharan, Damodar Edupalli, Nune Raviprakash,Thota Navneetha, Sidharth Mahali, and Maikho Thoh

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 12, p. 8562, March 22, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

8562 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 12 • MARCH 22, 2013

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