The CD24 Protein Inducible Expression System Is an IdealTool to Explore the Potential of CD24 as an Oncogene and aTarget for Immunotherapy in Vitro and in Vivo*

Received for publication, July 29, 2011, and in revised form, September 29, 2011 Published, JBC Papers in Press, October 5, 2011, DOI 10.1074/jbc.M111.286534

Shiran Shapira‡§1, Dina Kazanov‡, Samuel Weisblatt‡§, Alex Starr¶�, Nadir Arber‡§2, and Sarah Kraus‡§

From the ‡Integrated Cancer Prevention Center and ¶Lung and Allergy Institute, Tel Aviv Sourasky Medical Center, Tel Aviv 64239and the �Department of Molecular Genetics and Biochemistry and §Sackler Faculty of Medicine, Tel Aviv University,Tel Aviv 69978, Israel

Background: It is critical to tightly regulate gene expression to study its biological role.Results:A tetracycline-dependent CD24 expression system was successfully implemented. An efficient study of its potential asan oncogene and a target for immunotherapy was performed.Conclusion:This is a valuable tool to studyCD24 pathogenesis and novel treatment options for CD24-expressingmalignancies.Significance: The CD24 inducible expression system allows accurate analysis of its role and function.

CD24 is a cell surface, heavily glycosylated glycosylphosphati-dylinositol-anchored mucin-like protein that is overexpressedin various human malignancies. To accurately analyze CD24function and dissect its biological role in a defined genetic back-ground, it is critical to tightly regulate its expression and be ableto turn it on/off in a restricted environment and at a specifictime.The tetracycline-induced expression system ismost prom-ising as it exhibits such regulation, lack of pleiotropic effects,and high and rapid induction levels. To evaluate the oncogenicand immunotherapeutic potential of CD24 by applying theTet-On system, the human CD24 gene was cloned down-stream to two tetracycline operator sequences, resulting inpCDNA4/TO-CD24, which was then transfected into tetracy-cline (Tet) repressor-expressing cells (293T-REx), allowing tighton/off regulation, thereby resulting in a very low background orleaky CD24 expression. Selected clones were chosen for furtherstudies and characterized in vitro and in vivo, and several treat-mentmodalitieswere examined. In addition, the role ofCD24 inpromoting cell proliferation and tumor growthwas studied. Thetetracycline-dependent system was successfully implemented.Tetracycline treatment induced CD24 expression in a dose- andtime-dependent fashion, which was abrogated following treat-ment with anti-CD24monoclonal antibodies (mAbs). CD24-in-duced expression led to an increased proliferation rate that wasinhibited bymAb treatment. In vivo, significantly larger tumorswere developed in tetracycline-fedmice. The CD24 Tet-On sys-tem is a good model to unravel the role and underlying CD24pathogenesis in vivo. This valuable tool allows the successfulstudy of novel treatment options, whose effectiveness depends

on the CD24 expression level. This set of experiments supportsCD24 oncogenic properties.

CD24 is a small, heavily glycosylated mucin-like cell sur-face protein anchored to the membrane via glycosyl phos-phatidylinositol (1). CD24, first attributed as a differentia-tion marker for B cells, is involved in lymphocyte maturation(2, 3), regulates the proliferation of neuronal precursors (4),and was shown to be involved in the regulation of homeo-static cell renewal (5). CD24 is known to be overexpressed invarious human malignancies, both solid and hematological(6, 7), and is usually tied with a more aggressive course of thedisease (1, 8, 9).In our previous studies, we had shown the importance of

CD24 in themultistep process of colorectal cancer carcinogen-esis, already at the early stage of adenoma (10). Stringently con-trolled conditional expression systems are crucial for the func-tional characterization of genes. The development of anexpression system with the ability to control and regulate thelevel and duration of expression is useful for elucidating thefunctions of CD24 and its impact in the pathogenesis process.The tight regulation of CD24 expression was achieved by thewidely accepted inducible Tet-On system. The tetracycline-controlled transcription activation system has been alreadysuccessfully employed in a variety of eukaryotic cells, includingmammals (11, 12), plants (13), and yeast (14) cells, and at theorganism level, in mice (15, 16), plants (17), and Drosophila(18).The CD24 Tet-On system allows a precise study to evaluate

the role of CD24 and its function, partly because it is a definedsystem with minimal noise and background. Each experimentwas performed on the same cell population; therefore, there areno unknown differences between the control and the experi-mental groups that can enhance the heterogeneity of theresults. Therefore, this model system may also serve to effec-tively evaluate the effectiveness of new immunotherapy optionsagainst CD24-expressing cells.

* This work was supported in part by the Cooperation Program in CancerResearch of the Deutsches Krebsforschungszentrum (DFKZ) and the IsraelMinistry of Science and Technology (MOST).

1 This work was performed in partial fulfillment of the requirements for aPh. D. degree at the Sackler Faculty of Medicine, Tel Aviv University, TelAviv, Israel.

2 To whom correspondence should be addressed: 3/3rd Fl., Arison MedicalTower, Tel Aviv Sourasky Medical Center, Integrated Cancer PreventionCenter, Tel Aviv Medical Center, 6 Weizmann St., Tel Aviv 64239, Israel. Tel.:972-3-6974968; Fax: 972-3-6974867; E-mail: nadir@tasmc.health.gov.il ornarber@post.tau.ac.il.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 47, pp. 40548 –40555, November 25, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

40548 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 47 • NOVEMBER 25, 2011

by guest on April 11, 2020

http://ww

w.jbc.org/

Dow

nloaded from

EXPERIMENTAL PROCEDURES

Materials

All reagents were purchased from Sigma (Rehovot, Israel),unless otherwise stated. Secondary horseradish peroxidase-conjugated antibodies were from Jackson ImmunoResearchLaboratories Inc. (West Grove, PA). EZ-ECL detection kit andcell culture supplements were from Beit-Haemek, Israel.

Methods

Establishment of CD24-expressing Cells

Plasmid Construction—Initially, a DNA fragment coding fora full-length human CD24 fragment was amplified by PCRusing the plasmid pCMV-SPORT6-CD24 as a template usingprimers Kozak-HindIII-CD24-F (5�-CTGGAAGCTTGCC-ACCATGGATGGGCAGAGCAATGGTGGC-3�) and XbaI-CD24-R (5�-TCATCTAGAGTATTAAGAGTAGAGATGC-AGAAG-3�). The PCR product was digested by HindIII andXbaI and inserted into the pcDNA4/TO (pcDNA4 tetracyclineoperator) plasmid, downstream to two tetracycline operatorsequences, TetO2, which was cleaved with the same enzymes.The resulting plasmid was named pcDNA4/TO-CD24.The T-RExTM System—The T-RExTM system is a tetracy-

cline-regulated mammalian expression system (19, 20).pcDNA4/TO-CD24 was transfected into 293T-RExTM stablecells expressing the tetracycline repressor from thepcDNA6/TR vector (Invitrogen), using the calcium phosphatetransfection method. 48 h after transfection, the cellswere seeded intoDMEMmedium supplementedwith 10% fetalbovine serum (FBS), containing the selectable marker Zeocin(InvivoGen, 100 �g/ml). Several clones were isolated andcharacterized.

CD24 Binding Assay

Evaluation of CD24 induction was done by specific bindingof anti-CD24 mAb using flow cytometry. Approximately 1 �106 293T-RExTM stable transfected cells were used in eachexperiment. After trypsinization, the cells were washed inFACSbuffer (10%FBS, 0.01% sodiumazide in ice-cold PBS) andfixedwith 2% formaldehyde (in PBS) for 15min. Then, 100�l of10 �g/ml anti-CD24 mAb were added for 30 min at room tem-perature. Following washes, FITC-labeled goat anti-mouseantibodies diluted 1:100 in FACS buffer were added for 30 minat room temperature and protected from light. Detection ofbound antibodies was performed on a FACSCalibur (BD Bio-sciences), and results were analyzed with the CellQuest pro-gram (BD Biosciences).

Plating Efficiency

293T-RExTM stable transfected cells (1000 or 3000 cells/well) were seeded in 10-cm plates with or without 1 �g/mltetracycline in DMEM supplemented with 2.5% FBS. After 10days, attached cells were fixed with 4% formaldehyde in PBSand stained with crystal violet. Colonies larger than 2 mmwerecounted.

Proliferation Assay

Two different 293T-REx-CD24 clones were examined.30,000 cells were seeded in 12-well plates in complete medium

containing 5% FBS. On the next day, the serum was reduced to2.5% with or without 1 �g/ml tetracycline. Every 3 days, cellswere collected and counted from three wells to assess thegrowth rate.

Preparation of ZZ-PE38 Fusion Proteins

The armed anti-CD24mAb is a novel antibody-toxin immu-noconjugate where the targeting moiety is an anti-CD24SWA11 mAb, whereas the toxic moiety is a truncated form ofthe Pseudomonas exotoxin (PE)3 (Shapira et al. (21)). Theexpression and purification of the wild-type (WT) PE,ZZ-PE38, and the fusion proteins, SWA11/IgG-ZZ-PE38,were performed as described by Shapira et al. (21) Briefly, thepET22b-ZZ-PE38 plasmid (22), which carries an in-framefusion of ZZ to PE38, was designed for the expression of solubleZZ-PE38 fusion protein in the Escherichia coli periplasm.The Fc-binding protein ZZ is a duplication of mutated Bdomain of Staphylococcus aureus protein A, which is quiteeffective at binding the Fc domain of mouse IgG2a immuno-globulins (22, 23). The conjugation of SWA11 and normal IgG(control) antibodies to ZZ-PE38 fusion protein was performedas follows. Antibodies, diluted in PBS, were mixed withZZ-PE38 in PBS (3-fold molar excess of ZZ-PE38 over IgG) for16 h at 4 °C. Separation of excess ZZ-PE38 from the IgG-ZZ-PE38 complex was performed by applying the sample onto a25-ml Superdex 200 size exclusion column (GE Healthcare) asrecommended by the manufacturer. The resulting immuno-complexes were SWA11-ZZ-PE38 and IgG-ZZ-PE38.

Viability Assay

293T-RExTM stable transfected cells were seeded in 96-wellplates (2 � 104 cells/well) in complete medium. On the nextday, ZZ-PE38, WT PE, SWA11, SWA11-ZZ-PE38, and IgG-ZZ-PE38 were added to the cells in quadruplicates at variousconcentrations. 48 h later, the medium was replaced by immu-nocomplex-free medium (100 �l/well) containing 1 mg/ml3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) reagent and incubated for 2–4 h. MTT-formazan crys-tals were dissolved by the addition of acid isopropyl alcohol(0.04 M HCl in isopropyl alcohol). Absorbance at 570 nm and areference wavelength of 690 nm were recorded on an auto-matedmicroplate reader. IC50 was determined as the immuno-toxin concentration that caused 50% death when comparedwith untreated cells.

Xenograft Model for Measuring in Vivo Tumor Development

Athymic nude mice (Harlan Laboratories, Rehovot, Israel)were housed in sterile cages and handled with aseptic precau-tions. Themicewere fed ad libitum. To test the tumor-promot-ing potential of CD24, exponentially growing 293T-REx-CD24cells were harvested and resuspended at a final concentration of5 � 106 or 10 � 106 cells per 0.2 ml of PBS per injection. Thecells were injected subcutaneously at two sites on the flanks ofthe mice. The mice were weighed, and tumor growth was mea-sured twice weekly with a caliper; volume was calculated as

3 The abbreviations used are: PE, Pseudomonas exotoxin; Tet, tetracycline;MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Ideal Model System for the Inducible Expression of CD24

NOVEMBER 25, 2011 • VOLUME 286 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 40549

by guest on April 11, 2020

http://ww

w.jbc.org/

Dow

nloaded from

4⁄3� � a � b2. At the end of the experiment, the mice weresacrificed after anesthesia by cervical dislocation, and thetumors were excised. The study was approved by the Institu-tional Committee for AnimalWelfare at the Tel Aviv SouraskyMedical Center.

RESULTS

Establishment of an Inducible CD24 Tet-On ExpressionSystem—Stable clones were established, and their CD24 genetetracycline inducibility was evaluated. Two representativeclones (clones 13 and 15) with high inducible expression werechosen for further studies. Tetracycline increased significantlythe expression of CD24, as can be seen inWestern blot (Fig. 1).These results were confirmed using flow cytometry for mem-brane CD24 levels (data not shown). In the absence of tetracy-cline, the clones expressed only low basal level of CD24, indi-cating that CD24 expression is tightly regulated withoutleakiness.

CD24Expression Level Is Regulated byTetracycline in aDose-and Time-dependent Manner—293T-REx-CD24 cells weretreated for 24 h with different concentrations of tetracycline (0,0.01, 0.05, 0.1, and 1�g/ml). CD24 expression level was propor-tional to the tetracycline concentration as can be seen byWest-ern blot (Fig. 2A) and FACS analysis (Fig. 2B).The CD24 expression level was increased in a time-depen-

dentmanner following exposure to 1�g/ml tetracycline for 0, 4,6, 16, 24, 30, or 48 h. Its expression level increased from a base-line to amaximal level as shown by time lapse, as analyzed usingTINA20 software (Fig. 2C).Increased Expression of CD24 Increases Colony Formation

and Cell Proliferation—Fig. 3A shows representative experi-ments that demonstrate the increase in growth rate followinginduction of CD24 expression (squares). Fig. 3B demonstratesqualitatively, by crystal violet staining, the different growthrates between induced and uninduced cells. Increased prolifer-ation rate is correlated to the tetracycline concentration (Fig.

FIGURE 1. Representative clones screened for CD24 expression under regulation of tetracycline. The cells were exposed to 1 �g/ml tetracycline for 48 h.20 �g from each sample were subjected to Western blot analysis for CD24. The membrane was reprobed with anti-tubulin antibody to assess the uniformityof sample loading.

FIGURE 2. Tetracycline regulates CD24 expression levels in a dose- and time-dependent manner. The cells were treated with 0, 0.01, 0.05, 0.1, and 1 �g/mltetracycline for 24 h. A, cell extracts were subjected to Western blotting, and CD24 levels were analyzed with anti-CD24 SWA11 mAb. The membrane wasreprobed with anti �-tubulin to verify that protein was uniformly loaded across the gel. B, membrane expression levels of CD24 were examined by FACSanalysis. C, the cells were treated with 1 �g/ml tetracycline for 0, 4, 6, 16, 24, 30, and 48 h. CD24 expression levels were examined by Western blot analysis.Protein expression levels are presented as optical density (arbitrary units) per unit area (OD/mm2).

Ideal Model System for the Inducible Expression of CD24

40550 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 47 • NOVEMBER 25, 2011

by guest on April 11, 2020

http://ww

w.jbc.org/

Dow

nloaded from

3C). In addition, the plating efficiency was 3-fold higher whenCD24 expression was triggered (Fig. 3D).Susceptibility of CD24-expressing Cells to Anti-CD24-tar-

geted Therapy, Using Naked and Armed Antibodies—CD24-induced expression levels were examined after treatment withanti-CD24 mAb. The expression levels decreased followingantibody treatment, as can be demonstrated by the Westernblot (Fig. 4A) and FACS analysis (Fig. 4B).In addition, the cells were grown in the absence or presence

of various concentrations of anti-CD24 mAb for 48 and 96 h.The growth of the induced cells was inhibited in a dose-depen-dent manner (Fig. 4C), whereas no significant effect was dem-onstrated in the uninduced cells (data not shown).We evaluated the cytotoxicity of the armed anti-CD24,

SWA11-ZZ-PE38 immunotoxin, when compared with theWTPE toxin and with a nonspecific immunotoxin, IgG-ZZ-PE38.According to the cell killing experiments, CD24-inducedexpression induced sensitivity to the SWA11-ZZ-PE38 immu-notoxin treatment (Fig. 5A) with an IC50 value of 20 ng/ml.Conversely, no sensitivity was observed toward the control

nonspecific immunotoxin (Fig. 5B), IgG-ZZ-PE38, nor to theindividual components of the immunoconjugate (Fig. 5D). Ascan be seen in Fig. 6, the therapeutic window between theinduced and uninduced cells was opened only in the cells thatwere treated with the SWA11-ZZ-PE38, emphasizing the spec-ificity of this targeted tool and its dependence on CD24expression.As expected, the WT PE, which has its own cell binding

domain, is able to kill the cells regardless of CD24 expression(Fig. 5C). This is demonstrated by the similar IC50 values of theinduced and uninduced cells (10 ng/ml for both) and resulted inno therapeutic window. However, the IC50 values of SWA11-ZZ-PE38 in inducible CD24-expressing cells (20 ng/ml) weresimilar to those of theWTPE toxin (10 ng/ml), emphasizing thepotency of the immunocomplex against CD24-expressingtumor cells.CD24 Promotes Tumor Growth in Vivo—Two experiments

were performed. In the first one, the cells were grown with orwithout 1�g/ml tetracycline 1week prior to injection into nudemice. Eachmousewas injected subcutaneouslywith two clones;

FIGURE 3. Cell proliferation and colony formation are increased following induction of CD24 expression. A, 30,000 cells from two different clones wereseeded in 12-well plates in complete medium. The next day, the medium was replaced, and serum was reduced to 2.5%. Cells were counted every 3 days. Errorbars indicate S.D. B, different amounts of cells were seeded in 12-well plates in 2.5% FBS-supplemented medium, with or without 1 �g/ml tetracycline. After 7days of incubation, cells were fixed and stained with crystal violet. C, 30,000 cells were seeded in 12-well plates in medium supplemented with 2.5% FBS, anddifferent concentrations of tetracycline were added. Each time point represents the number of cells that were counted. D, 1000 or 3000 293T-REx-CD24 cellswere seeded in 10-cm plates in 2.5% FBS-supplemented medium with or without 1 �g/ml tetracycline and grown to form colonies. After 10 days of incubation,cells were fixed and stained with crystal violet, and colonies were counted.

Ideal Model System for the Inducible Expression of CD24

NOVEMBER 25, 2011 • VOLUME 286 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 40551

by guest on April 11, 2020

http://ww

w.jbc.org/

Dow

nloaded from

clone 13 was injected in the right flank and clone 15 wasinjected in the left flank, to rule out a clone-specific effect. Intotal, there were four groups as described in Table 1.Mice from group 3 developed tumors in both flanks (Fig. 6A),

whereas no tumors were developed in group 4 (Fig. 6B). Group1, which was injected with a smaller amount of cells, also devel-oped tumors but after a longer period of time (�1month). Still,the uninduced mice did not develop any tumors.Tumors from both groups were removed, and CD24 expres-

sion was analyzed by Western blotting. All the tumorsexpressed high levels of CD24, similar to those obtained withinduced cells in vitro (data not shown).

The second experiment included two groups with multiplesubjects (six mice in each group). All the mice were injectedsubcutaneously with 10� 106 cells. In this experiment, the cellswere grown with or without 1 �g/ml tetracycline 48 h prior tothe injection. Fig. 6C shows that CD24 expression indeed pro-motes tumor formation and development, as demonstrated inthe previous experiment. As expected, 293T-RExTM cells alsodeveloped tumors, but at a slower growth rate.Before cell implantation, samples from the growing cultures

were analyzed by Western blot to confirm the induction ofCD24 expression (Fig. 6D). In addition, 1month after cell injec-

tion (at the end of the experiment), tumors were removed, andCD24 expression levels were examined (Fig. 6E). Expressionwas induced by tetracycline in the tumor and was increasedwhen compared with the control mice that received onlysucrose. There was a close association between CD24 expres-sion level and tumor volume.

DISCUSSION

A proof of concept of the Tet-On inducible expression sys-tem is reported herein. This system can serve as an importantresearch tool to unravel and clarify the functional roles of theCD24 gene and its importance in the multistep carcinogenesisprocess. Indeed, it has been confirmed that CD24 is a potentialoncogene.Usually, to study these goals, human cancer cell lines

expressing a specific gene are comparedwith a different cell linethat does not express the gene. Obviously, there aremanymoreknown and unknown differences between these cell lines thatcan enhance the diversity of the outcome analysis due tounknown factors embedded in the cells, and their effect and/orimpact cannot be discarded. Therefore, it is difficult to isolatethe effect of a specific gene.

FIGURE 4. Anti-CD24 mAb decreases CD24 inducible expression and inhibits cell proliferation. A, 2 � 105 cells were seeded in 12-well plates in thepresence or absence of 0.01 or 0.05 �g/ml tetracycline for 24 h. Then, the cells were washed, the medium was replaced, and different concentrations (5, 25, and50 �g/ml) of SWA11 mAb were added for 48 h. 20 �g of total cell lysates were loaded on 10% SDS-PAGE followed by CD24 immunoblotting. The membranewas then reprobed with anti-�-tubulin. B, the cells were grown in the presence of 0.05 �g/ml tetracycline for 24 h. Then, they were washed with PBS, andmedium containing 50 �g/ml chimeric anti-CD24 mAb was added for another 48 h. Approximately 1 � 106 cells were then used for FACS analysis withanti-CD24 murine mAb. The arrow shows the curve which represents the antibody-treated cells, and the right-most curve represents the untreated cells. C, 3 �104 cells were seeded in 12-well plates in the absence or presence of 1 �g/ml tetracycline in medium supplemented with 2.5% FBS and were treated with 50,100, and 200 �g/ml anti-CD24 mAb. The cells were counted after 48 and 96 h in a Coulter counter device. con, control.

Ideal Model System for the Inducible Expression of CD24

40552 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 47 • NOVEMBER 25, 2011

by guest on April 11, 2020

http://ww

w.jbc.org/

Dow

nloaded from

It is apparent that a stable cell line that overexpresses a spe-cific gene, such as the CD24 gene demonstrated in this study,can be compared with the one that contains only the emptyvector. Representative clones can be chosen for further studies,but one should keep in mind that different clones are, in fact,different cell populations with variations between the stableclone and the empty vector control. In addition, constitutiveand forcefully high expression of the protein may become aburden for the cells, leading to a change in their behavior. Theinability to control the level of expression is another significantdisadvantage of these constitutive expression systems. Theinducible expression systemmay overcome these obstacles andwould serve as an ideal tool for unraveling the downstreameffectors of the gene and shedding light on the role of the gene,its function, and its potential as a therapeutic target.Regulation of gene expression in a temporal and spatial man-

ner provides a useful tool for the study of mammalian genefunction particularly during oncogenesis. The Tet regulatorysystems are currently the most widely used models for condi-tional gene expression. Maximal expression levels in these sys-tems are very high and comparable with the maximal levelsreachable from a constitutive and strongmammalian promotersuch as CMV (24). Indeed, the level of CD24 protein followingexposure to tetracycline was comparable with the level of theprotein after transient expression (data not shown).The current study demonstrates the effect of increased

expression of CD24 in 293T-RExTM cells, where its expressionis regulated by tetracycline. It was confirmed that inducibleexpression of CD24 resulted in a more malignant phenotype.The inducible cells proliferated faster and demonstrated anincreased saturation density and plating efficiency. Most

importantly, they weremore tumorigenic in vivowhen injectedsubcutaneously to nude mice. These results are compatiblewith numerous publications linking CD24 with rapid cancercell growth (25–27).This system is very sensitive, and the control of CD24 can be

achieved in a time- and dose-dependent manner. These char-acteristics of the system are very important as they can carefullydissect the oncogenic potential of CD24. The very low back-ground ofCD24when the cells were not exposed to tetracyclineconfirms the non-leakiness of this inducible system.The potential for targeting CD24 in cancer therapy seems

promising asCD24 is overexpressed inmany human cancers (9,28). Using this system, we were able to study new treatmentmodalities for CD24-expressing cancer cells where the only dif-ference between the treated and untreated cultures is the levelof CD24 expression. We were able to compare the effects ofseveral existing mAbs targeting CD24, e.g. SWA11 (29), as wellas novel immunoconjugates that were produced in our labora-tory (Shapira et al. (21)). Down-regulation of CD24 expressionusing mAbs resulted in decreased expression level and growthinhibition of the induced cells in vitro. In addition, specificdelivery of more potent treatment modalities, such as theSWA11-ZZPE38 immunotoxin, effectively killed the cellsdepending on CD24 expression levels. In contrast, the WTPseudomonas exotoxin, which possesses its own cell bindingdomain, enters into mammalian cells via a receptor-mediatedendocytic pathway through binding to themultiligand receptorfound in most cultured cells (30). Therefore, it kills the cellsregardless of CD24 expression.Above all, the system proved itself in vivo. HEK293 cells are

tumorigenic, but it was clearly shown that up-regulation of

FIGURE 5. Cytotoxicity of SWA11-ZZ-PE38 immunotoxin correlates with CD24 expression. A–D, 2 � 104 cells were seeded in 96-well plates in the presenceor absence of 1 �g/ml tetracycline. The following day, varying concentrations of SWA11-ZZ-PE38 immunotoxin (A), IgG-ZZ-PE38 control immunocomplex (B),WT PE toxin (C), and the individual components of the complex (SWA11 and ZZ-PE38) at the highest concentration equivalent to that of the immunotoxin, wereadded to quadruplicate samples for 48 h. The relative number of viable cells when compared with untreated cells was determined using an enzymatic MTTassay. The IC50 values were determined as the concentration needed to kill 50% of the cells and are indicated by broken lines. The bidirectional arrows indicatethe therapeutic window of the immunocomplex treatment. The data shown are from a representative experiment. Error bars indicate S.D.

Ideal Model System for the Inducible Expression of CD24

NOVEMBER 25, 2011 • VOLUME 286 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 40553

by guest on April 11, 2020

http://ww

w.jbc.org/

Dow

nloaded from

CD24 expression increases cell tumorigenicity in vivo. Nudemice that were implanted with the cells and received theinducer in their drinking water developed impressive tumors incontrast to the uninduced mice. Not only was tumor incidencewas significantly lower in the control group, but the rate oftumor growth was significantly slower as well, resulting insmaller tumor volume. These results provide strong evidencefor the potential oncogenicity of CD24.In summary, the Tet-controlled CD24 expression system has

a tight and dose-dependent inducibility for tetracycline. This

further suggests that CD24 is a tumor-promoting agent thatmarkedly enhances tumor development. This system is apotential tool to study the functional role of CD24 and evaluatenovel therapeutic modalities against it.

Acknowledgment—We are in debt to Professor Rolf Stahel (Universityof Zurich, Switzerland) for kindly providing the SWA11 hybridomacells used in this study.

REFERENCES1. Kristiansen, G., Sammar, M., and Altevogt, P. (2004) J. Mol. Histol. 35,

255–2622. Taguchi, T., Kiyokawa, N., Mimori, K., Suzuki, T., Sekino, T., Nakajima,

H., Saito, M., Katagiri, Y. U., Matsuo, N., Matsuo, Y., Karasuyama, H., andFujimoto, J. (2003) J. Immunol. 170, 252–260

3. Li, O., Zheng, P., and Liu, Y. (2004) J. Exp. Med. 200, 1083–10894. Nieoullon, V., Belvindrah, R., Rougon, G., and Chazal, G. (2005)Mol. Cell.

Neurosci. 28, 462–4745. Nieoullon, V., Belvindrah, R., Rougon, G., and Chazal, G. (2007) Cell Tis-

sue Res. 329, 457–4676. Suzuki, T., Kiyokawa, N., Taguchi, T., Sekino, T., Katagiri, Y. U., and

FIGURE 6. Evaluation of CD24 potential to promote tumor formation in nude mice. A, in the first experiment, tumor size was measured with a caliper, andthe volume was calculated. B, uninduced mice. C, in the second experiment, tumor size was measured with a caliper, and the volume was calculated. Error barsindicate S.D. D and E, the induced and uninduced cells (clones 13 and 15) were analyzed for CD24 expression levels before the cells were implanted into themice (D) and after becoming a tumor in the living organism (E). CD24 was detected with SWA11 mAb, and the membrane was then reprobed with anti-tubulinantibody.

TABLE 1Study design of in vivo experiment 1

Number ofmice

Number of cellsinjected intoeach flank

Induceda oruninducedb

Group 1 3 5 �106 InducedGroup 2 3 5 �106 UninducedGroup 3 3 10 �106 InducedGroup 4 3 10 �106 Uninduced

a Mice administered with 1 mg/ml tetracycline � 3% sucrose are “induced” mice.b Mice administered with only 3% sucrose are “uninduced” mice.

Ideal Model System for the Inducible Expression of CD24

40554 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 47 • NOVEMBER 25, 2011

by guest on April 11, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Fujimoto, J. (2001) J. Immunol. 166, 5567–55777. Chappel,M. S., Hough,M. R.,Mittel, A., Takei, F., Kay, R., andHumphries,

R. K. (1996) J. Exp. Med. 184, 1639–16498. Lim, S. C., and Oh, S. H. (2005) Pathol. Res. Pract. 201, 479–4869. Sagiv, E., and Arber, N. (2008) Expert Rev. Gastroenterol. Hepatol. 2,

125–13310. Sagiv, E.,Memeo, L., Karin, A., Kazanov, D., Jacob-Hirsch, J.,Mansukhani,

M., Rechavi, G., Hibshoosh, H., and Arber, N. (2006) Gastroenterology131, 630–639

11. Petry, I. B., Fieber, E., Schmidt, M., Gehrmann, M., Gebhard, S., Hermes,M., Schormann, W., Selinski, S., Freis, E., Schwender, H., Brulport, M.,Ickstadt, K., Rahnenführer, J., Maccoux, L.,West, J., Kölbl, H., Schuler,M.,and Hengstler, J. G. (2010) Clin. Cancer Res. 16, 451–460

12. Resnitzky, D., Gossen,M., Bujard, H., and Reed, S. I. (1994)Mol. Cell. Biol.14, 1669–1679

13. Weinmann, P., Gossen, M., Hillen, W., Bujard, H., and Gatz, C. (1994)Plant J. 5, 559–569

14. Park, Y. N., and Morschhäuser, J. (2005) Eukaryot. Cell 4, 1328–134215. Aiba, A., and Nakao, H. (2007) Neurosci. Res. 58, 113–11716. Kistner, A., Gossen, M., Zimmermann, F., Jerecic, J., Ullmer, C., Lübbert,

H., and Bujard, H. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 10933–1093817. Matzke, A., van der Winden. J., and Matzke M. A. (2003) Plant Mol Biol

Rep. 21, 9–1918. Stebbins, M. J., Urlinger, S., Byrne, G., Bello, B., Hillen, W., and Yin, J. C.

(2001) Proc. Natl. Acad. Sci. U.S.A. 98, 10775–1078019. Hillen, W., and Berens, C. (1994) Annu. Rev. Microbiol. 48, 345–36920. Yao, F., Svensjö, T., Winkler, T., Lu, M., Eriksson, C., and Eriksson, E.

(1998) Hum. Gene Ther. 9, 1939–195021. Shapira, S., Shapira, A., Starr, A., Kazanov, D., Kraus, S., Benhar, I., and

Arber, N. (2011) Gastroenterology 140, 935–94622. Mazor, Y., Barnea, I., Keydar, I., and Benhar, I. (2007) J. Immunol.Methods

321, 41–5923. Nizard, P., Chenal, A., Beaumelle, B., Fourcade, A., and Gillet, D. (2001)

Protein Eng. 14, 439–44624. Yin, D. X., Zhu, L., and Schimke, R. T. (1996)Anal. Biochem. 235, 195–20125. Baumann, P., Cremers, N., Kroese, F., Orend, G., Chiquet-Ehrismann, R.,

Uede, T., Yagita, H., and Sleeman, J. P. (2005) Cancer Res. 65,10783–10793

26. Kim,H. J., Kim, J. B., Lee, K.M., Shin, I., Han,W., Ko, E., Bae, J. Y., andNoh,D. Y. (2007) Cancer Lett. 258, 98–108

27. Wang, W., Wang, X., Peng, L., Deng, Q., Liang, Y., Qing, H., and Jiang, B.(2010) Cancer Sci. 101, 112–119

28. Sagiv, E., Kazanov, D., and Arber, N. (2006) Biomed. Pharmacother. 60,280–284

29. Sagiv, E., Starr, A., Rozovski, U., Khosravi, R., Altevogt, P., Wang, T., andArber, N. (2008) Cancer Res. 68, 2803–2812

30. Mucci, D., Forristal, J., Strickland, D., Morris, R., Fitzgerald, D., and Sael-inger, C. B. (1995) Infect. Immun. 63, 2912–2918

Ideal Model System for the Inducible Expression of CD24

NOVEMBER 25, 2011 • VOLUME 286 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 40555

by guest on April 11, 2020

http://ww

w.jbc.org/

Dow

nloaded from

KrausShiran Shapira, Dina Kazanov, Samuel Weisblatt, Alex Starr, Nadir Arber and Sarah

in Vivo and in VitroPotential of CD24 as an Oncogene and a Target for Immunotherapy

The CD24 Protein Inducible Expression System Is an Ideal Tool to Explore the

doi: 10.1074/jbc.M111.286534 originally published online October 5, 20112011, 286:40548-40555.J. Biol. Chem. 

  10.1074/jbc.M111.286534Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/286/47/40548.full.html#ref-list-1

This article cites 30 references, 12 of which can be accessed free at

by guest on April 11, 2020

http://ww

w.jbc.org/

Dow

nloaded from