IL-2-granzyme A chimeric protein overcomes multidrug resistance (MDR) through a caspase...

IL-2-granzyme A chimeric protein overcomes multidrugresistance (MDR) through a caspase 3-independentapoptotic pathway

Inna Grodzovski1, Michal Lichtenstein1, Hanan Galski2 and Haya Lorberboum-Galski1

1 Laboratory of Molecular Immunobiology, Department of Biochemistry and Molecular Biology, Faculty of Medicine, Hebrew University, Jerusalem, Israel2 Department of Hematology and BMT, Chaim Sheba Medical Center, Tel Hashomer, Israel

One of the main problems of conventional anticancer therapy is multidrug resistance (MDR), whereby cells acquire resistance

to structurally and functionally unrelated drugs following chemotherapeutic treatment. One of the main causes of MDR is

overexpression of the P-glycoprotein transporter. In addition to extruding the chemotherapeutic drugs, it also inhibits

apoptosis through the inhibition of caspases. To overcome MDR, we constructed a novel chimeric protein, interleukin (IL)-2

granzyme A (IGA), using IL-2 as a targeting moiety and granzyme A as a killing moiety, fused at the cDNA level. IL-2 binds to

the high-affinity IL-2 receptor that is expressed in an array of abnormal cells, including malignant cells. Granzyme A is known

to cause caspase 3-independent cell death. We show here that the IGA chimeric protein enters the target sensitive and MDR

cancer cells overexpressing IL-2 receptor and induces caspase 3-independent cell death. Specifically, after its entry, IGA

causes a decrease in the mitochondrial potential, triggers translocation of nm23-H1, a granzyme A-dependent DNase, from

the cytoplasm to the nucleus, where it causes single-strand DNA nicks, thus causing cell death. Moreover, IGA is able to

overcome MDR and kill cells resistant to chemotherapeutic drugs. We believe that overcoming MDR with targeted molecules

such as IGA chimeric protein that causes caspase-independent apoptotic cell death could be applied to many other resistant

types of tumors using the appropriate targeting moiety. Thus, this novel class of targeted molecules could open up new vistas

in the fight against human cancer.

Today many anticancer drugs are being developed to succeedwhere the conventional methods of treatment such as chemo-therapy, surgery and radiotherapy have failed. One of themain struggles of conventional therapy is against multidrugresistance (MDR), whereby cells acquire resistance to a vari-ety of structurally and functionally unrelated drugs followingchemotherapeutic treatment.1,2

MDR is a phenomenon, which results from various rea-sons. The most-characterized cause of MDR is the overex-pression of a 170-kDa membrane glycoprotein known as P-glycoprotein (Pgp). It belongs to a super family of ATP-bind-ing cassette transporters. These transporters, through anATP-dependent mechanism, actively efflux a wide variety ofchemotherapeutic drugs used to treat cancer out of the cells.Thus, they lower the drug concentration inside the cells, ren-dering them resistant.1

Most chemotherapeutic drugs trigger cell death via apo-ptosis. On induction of apoptosis, a sequence of well-organ-ized events lead to the activation of caspases, especially cas-pase 3, which are activated by proteolytic cleavage.3,4 In itsclassical form, apoptosis can occur only when caspases, inparticular caspase 3, are activated.

In addition, there is another type of cell death that hasthe morphological features of apoptosis; however, in thispathway there is no caspase activation or caspase substrateinvolvement. Although mitochondrial damage occurs andreactive oxygen species accumulate, there is no release ofcytochrome C or mitochondrial apoptotic mediators.5–8 TheDNA damage is characterized by the generation of single-stranded DNA nicks9–11 rather than the oligonucleosomalDNA typical of classical caspase-dependent apoptosis.5,7

It has been shown that in MDR cells, Pgp has the abilityto confer resistance to apoptosis induced by a wide range ofcaspase-dependent apoptotic stimuli.12 Pgp inhibits caspaseactivation, specifically that of caspase 3, even when its efflux-ing ability is damaged.13 Therefore, Pgp regulates cell deathnot only by effluxing the chemotherapeutic agents out of cellsbut also by inhibiting caspase activation, thus interfering withits mechanism of action in addition to lowering the cellulardrug concentration.14 However, it has been demonstratedthat Pgp does not protect against death induced by a cas-pase-independent mechanism.12,14–17 We decided to takeadvantage of this feature and to utilize a human protein,

Key words: IL-2-granzyme A, MDR, Pgp, targeting, caspase 3, nm23-

H1, Dox

DOI: 10.1002/ijc.25527

History: Received 7 Jan 2010; Accepted 10 Jun 2010; Online 22 Jun

2010

Correspondence to: Haya Lorberboum-Galski, Department of

Biochemistry and Molecular Biology, Faculty of Medicine, Hebrew

University, Jerusalem 91120, Israel, Tel.: [972-2-6757465], Fax:

þ[972-2-6415848], E-mail: [email protected]

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Int. J. Cancer: 128, 1966–1980 (2011) VC 2010 UICC

International Journal of Cancer

IJC

granzyme A, which specifically triggers caspase-independentcell death to kill MDR cells.

Granzyme A is a serine protease stored in the cytoplasmicgranules of killer lymphocytes. When these lymphocyte cellsencounter their target, an immunological synapse is formed.The granules migrate toward the synapse, and their contents,including various granzymes, are released and delivered intothe target cell.5,8,18 Following Granzyme A’s entry into thecell, it causes mitochondrial damage, reactive oxygen specieselevation and subsequent translocation of the endoplasmicreticulum-associated multiprotein complex, SET, from thecytoplasm to the nucleus.19 In the nucleus, among its manyactions, granzyme A cleaves SET protein, thus releasing gran-zyme A-activated DNase, nm23-H1, which initiates DNAdamage.11,19 In this way, granzyme A’s delivery to the cellinduces caspase-independent cell death.

To target and deliver granzyme A into MDR cells, wefused this enzyme at the cDNA level to a targeting moiety,thereby creating a new chimeric protein. We chose interleu-kin (IL)-2 as the targeting moiety. IL-2 recognizes the highaffinity IL-2 receptor (IL-2R), composed of three subunits,alpha, beta and gamma (the latter two are common to severalIL family receptors), which are mainly expressed on activatedT and B cells. Less than 5% of unstimulated human periph-eral blood cells express the alpha subunit of IL-2R.

However, high IL-2R alpha expression was demonstratedon an array of abnormal cells including the malignant cells inpatients with adult T-cell leukemia, cutaneous T-cell lym-phoma, anaplastic large-cell lymphoma, hairy cell B-cell leuke-mia and the Reed Sternberg and associated polyclonal T cellsin Hodgkin’s disease and acute and chronic granulocytic leuke-mia cells.20–22 Such abnormalities of IL-2R alpha expressionhave also been demonstrated in autoimmune diseases.21–23

Furthermore, elevations of IL-2 R alpha expression have beendetected in the serum of patients during organ allograft rejec-tion and from those with graft-versus-host disease.22,24

This major difference in IL-2R alpha expression betweennormal and abnormal cells in disease provides the basis forthe development of targeted T-cell therapies. In cancer ther-apy, many IL-2-based chimeric proteins have been developed,with the most well known being an FDA-approved chimericprotein termed Ontak. This molecule is composed of the IL-2molecule fused to a modified form of diphtheria toxin25,26

and has been approved for the treatment of cutaneous T-celllymphoma. Our group has also constructed several IL-2-based human chimeric proteins, such as IL-2-Bax27 and IL-2-caspase-3, which seem to be promising targeting therapeuticmolecules in the fight against immunological conditions suchas inflammatory bowel disease and experimental autoimmuneencephalomyelitis.28,29

The new chimeric protein, IL-2-granzyme A (IGA),designed to kill MDR cells via a caspase-independent celldeath, was expressed and partially purified. Our results showthat IGA chimeric protein enters and kills efficiently and spe-cifically its target cells, both sensitive and MDR cells, express-

ing the high affinity IL-2R, whereas having no effect on non-target cells. Moreover, IGA chimeric protein kills target cellsvia a caspase 3-independent mechanism. Following its entryinto the target cells, IGA chimera causes a decrease in themitochondrial potential. Later on, IGA causes nm23-H1DNase translocation from the cytoplasm to the nucleuswhere the DNase induces single-strand DNA nicks, thusleading to alternative cell death.

Material and MethodsCell lines

The drug-sensitive Lm1 mouse lymphoma cell line and itsdrug-resistant cell variant Lm1-MDR, as well as Ls and Ls-MDR cells, were developed in one of our laboratories. Lm1-MDR is a subline of Lm1 cells transduced with a retroviralvector encoding the human MDR1 gene.30 Ls-MDR cells areLs cells (lymphoma cells) transduced with a retroviral vectorencoding the human MDR gene. These cell lines were testedand authenticated according to the AACR cell line policy.B16 mouse melanoma cell line, D122 mouse Lewis lung car-cinoma cell line, Lm1 and Lm1-MDR were cultured inDulbecco’s modified Eagle medium (Biological industries,Beit-Haemek, Israel). D122 was additionally supplementedwith 10% nonessential amino acids and 10% sodium pyru-vate. L1210 mouse lymphocytic leukemia cells (ATCC) weremaintained in RPMI 1640 (Biological Industries). All mediawere supplemented with 10% heat-inactivated fetal calf serum(HyClone, Logan, UT), except Ls-MDR, which was main-tained in DMEM supplemented with heat-inactivated fetalhorse serum (Biological Industries), 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin (Biological Indus-tries). Colchicine (Sigma-Aldrich, St. Louis, MO) was addi-tionally supplemented to the growth medium at 700 ng/mlfor Lm1-MDR and 400 ng/ml for Ls-MDR. All cells wereincubated in humidified atmosphere with 5% CO2 at 37�C.All cultures were tested for mycoplasma contamination withEZ-PCR mycoplasma kit (Biological Industries) according tothe manufacturer’s instructions and found to be negative.

Construction, expression and partial purification of the

chimeric protein

The cDNA for human Granzyme A was obtained by reversetranscriptase polymerase chain reaction (PCR) using RNAisolated from fresh phytohemagglutinin-ctivated human lym-phocytes. Total RNA was isolated and reverse transcribedinto the first strand cDNA, using a reverse transcription kit(ABgene, United Kingdom). The granzyme A fragment wasgenerated by PCR using the above cDNA and a pair of pri-mers covering the coding sequence of the active enzyme, 50-CCCAAGCTTTCATTATTGGAGGA AAT-30 (forward), 50-CCATCGATTTAAACTGC TCCCTT-30 (reverse). The PCRproduct was inserted into pAY1 expression vector20 down-stream to human IL2 sequence using HindIII and ClaIrestriction enzymes. Next, the IGA sequence was cut outfrom the pAY1 and subcloned into pET28b expression vector

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Grodzovski et al. 1967


using EcoRI and NdeI enzymes resulting in pIGA plasmid.The clone was confirmed by sequencing analysis.

Escherichia coli BL21 CodonPlus (kDE3) competent cellswere transformed with the pIGA plasmid. The cells weregrown at 37�C in saline broth medium containing kanamycin(50 lg/ml), tetracycline (12.5 lg/ml) and chloramphenicol(34 lg/ml). IGA expression was induced at an OD600 of 0.6–0.8 with 1 mM isopropyl-D-thiogalactoside (1 mmol/l; Biolab,Jerusalem, Israel) for 2 hr. The cells were harvested by cen-trifugation at 450g for 15 min. The pellet of expressing cellswas suspended in 20 mM Tris-HCl pH 7.9, 1 mM EDTAcontaining 0.2 mg/ml lysozyme (Sigma-Aldrich), sonicatedand centrifuged at 35,000g for 30 min at 4�C. The pelletobtained was denatured for 1–4 hr in buffer containing 6 Murea, 0.1 M Tris-HCl pH 8.8, 1 mM EDTA, 50 mM NaCland 10 mM DTT. The supernatant was cleared by centrifuga-tion at 35,000g for 15 min and diluted 1:50, to approximately0.1 lg/ll in refolding buffer (20 mM Tris-HCl pH 8.8, 1 mMEDTA, 0.25 M NaCl and 10 mM DTT). The chimeric pro-tein was left to refold for 48 hr at 4�C. The refolded chimericprotein fraction was dialyzed against PBS at 4�C, aliquotedand stored at �20�C.

Western blot analysis

Various samples were loaded on 12–15% (wt/vol) sodium do-decyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)gels. The proteins were transferred onto Immobilon-P trans-fer membrane (Millipore, Billerica, MA). Western blot analy-sis was performed using anti-human Granzyme A (1:10,000;Serotec, Oxford, United Kingdom), anti-caspase 3 and anti-active caspase 3 subunit, (1:500; Cell Signaling Technology,Danvers, MA), anti-nm23-H1 (1:700; Santa Cruz Biotechnol-ogy, Santa Cruz, CA) and antitubulin (1:10,000; Serotec)antibodies. Antibodies were detected using the EZ-ECL kit(Biological Industries) according to the manufacturer’sinstructions.

IGA in vitro enzymatic activity

The enzymatic activity of the IGA chimera was tested usinga fluorogenic substrate, Z-GPR-AMC (BIOMOL, PlymouthMeeting, PA). The substrate stock (10 mM) was prepared inDMSO and diluted to 0.5 mM in 20 mM Tris-HCl, 150 mMNaCl, pH 7.9. Approximately 2.6 lg of the IGA chimera (ina total volume of 20 ll) were added to 80 ll of the substratesolution and incubated at 37�C for 4 hr. Fluorescence inten-sity was measured using a Fluostar fluorometer (BMG Lab-technologies GmbH, Germany) at an excitation wavelengthof 390 nm and emission wavelength of 460 nm.

FACS analysis

To verify the amount of IL-2R alpha chain expressed on thevarious cell lines surface (Lm1, Lm1-MDR, L1210, Ls.T1 Ls-MDR, B16 and D122), the cells (0.5–1 � 106 cells in total)were washed twice in PBS, centrifuged at 300g for 5 min,resuspended in 50 ll of the FACS buffer (3% FCS, 0.02%

Azide and PBS) and incubated for 30 min at 4�C with anti-mouse CD25-PE-conjugated (Serotec) or anti-human CD25(a kind gift from T. Waldmann, NIH), at 1:10 and 1:50,respectively. The cells were washed twice with FACS buffer,resuspended in 50 ll of the same buffer, and the human celllines were incubated with goat anti-mouse FITC-conjugatedsecondary antibody (Jackson ImmunoResearch Laboratories,Bar Harbor, ME) for 20 min at 4�C. The cells were washedtwice with FACS buffer, resuspended in 0.5 ml PBS and ana-lyzed by FACScan (Becton Dickinson Immunocytometry Sys-tems, San Jose, CA) using the CellQuest program.

For cell cycle analysis, Lm1 and Lm1-MDR cells (1 � 106

cells/5 ml medium) were incubated with PBS, IGA (12 lg/ml) or 0.1 lM doxorubicin (Dox). MDR cells were grown ina cholchicin-free medium for at least 3 days before and dur-ing the experiment to increase the efficiency of the pump in-hibitor verapamil. This treatment was performed to preventPgp from pumping propidium iodide (PI) out of the cells.Following the treatment, cells were washed twice with coldPBS, centrifuged at 4,000g for 5 min and resuspended in hy-potonic fluorochrome solution (50 lg/ml PI in 0.1% sodiumcitrate and 0.1% Triton X-100). Cells were incubated at 4�Cin the dark overnight and then analyzed by FACS for DNAcontent as a function of cell number, using FACScan (BectonDickinson) and the CellQuest program.

Cell viability assay

Cells were seeded in 96-well plates (104 cells per 100 ll well;Nunc, Rochester, NY) and treated for various time periods.Cell viability was assessed using the CellTiter-BlueTM kit(Promega, Madison, WI) according to manufacturer’sinstructions.

Internalization

Lm1 cells (5 � 106 cells/ml) were incubated with the IGAchimera for various time periods. Following incubation, thecells were washed four times with PBS and lysed for 10 minwith P300 lysis buffer (20 mM NaH2PO4, 250 mM NaCl, 30mM Na4P2O710H2O, 0.1% NP-40 and 5 mM EDTA) andcentrifuged (15,000g, 10 min.). Samples of the supernatantwith equal amounts of protein were loaded on a SDS-PAGEgel, and the internalized protein was detected using the anti-granzyme A antibody.

Immunodepletion

IGA chimeric protein (8 lg) was incubated with 20 lg anti-IL-2 antibodies overnight at 4�C with rotation. The followingday, protein-G PLUS-Agarose beads (Santa Cruz Biotechnol-ogy) were added to the sample and incubated for additional4 hours at 4�C with rotation. The beads were pelleted by cen-trifugation at 1,000g for 5 min at 4�C, and the supernatantwas dialyzed in Midi GeBAflex-tube (Geba, Kfar-Hanagid,Israel) against PBS. The dialyzed fraction was analyzed byWestern blot using anti-granzyme A antibodies and by cellviability assays.

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1968 IL-2-granzyme A kills MDR cancer cells


Caspase-3 activity assay

Various treatments were added to 106 Lm1 or Lm1-MDRcells seeded in 3 ml medium. The cells were collected afterparticular time periods, washed with PBS, incubated at�80�C for 30 min and then all the liquid removed. Onepacked cell volume of Dounce buffer (10 mM Tris-HCl pH7.5, 0.5 mM MgCl2 and 1 mM PMSF) was added to thedefrosted samples, which were incubated on ice for 30 minwith pipetation every 10 min. Samples were then centrifugedat 425g for 10 min. Protein concentrations were measured byBradford reagent (Bio-Rad, Hercules, CA). Lysates of equalprotein amounts were used for the assay. Caspase 3 activitywas measured using the Apo-ONETM homogeneous caspase3 assay kit (Promega) according to manufacturer’sinstructions.

Additionally, to detect caspase 3 within treated cell lysates,3 � 106 Lm1 and Lm1-MDR cells in 5 ml medium wereincubated with different treatments for various time periods.Cell lysates were extracted, and equal protein amounts wereloaded, run on SDS-PAGE and immunoblotted with anti-cas-pase 3 antibody and anti-active caspase 3 antibody.

Mitochondrial membrane potential measurements

Lm1 (0.5 � 106 cells/3 ml) were seeded and incubated withvarious treatments for certain periods of time. Lm1-MDRcells (0.5 � 106 cells/3 ml) were preincubated for 30 min at37�C with the 20 lM of the Pgp pump inhibitor, verapamil(Sigma-Aldrich) in a colchicine-free medium to prevent Pgpfrom pumping Jc-1 out of the MDR cells. All following pro-cedures were carried out under the same conditions, i.e.,Lm1-MDR cells were incubated with verapamil and withoutcolchicine. After the incubation, the cells were harvested,incubated with 10 lM Jc-1 (Biotium, Hayward, CA) for 20min, washed with PBS and seeded in a 96-well plate. Theanalysis was performed using a Fluostar flurometer (BMGLabtechnologies).

Jc-1 is a lipophilic fluorescent cation that incorporatesinto the mitochondrial membrane, where it can form aggre-gates or monomers, according to the physiological membranepotential of the mitochondria. The aggregates exhibit redfluorescence, detected at 520 nm. When the mitochondrialpotential is damaged, Jc-1 monomers appear and exhibitgreen fluorescence detected at 590 nm. Thus, the ratio ofred/green represents the state of the mitochondria. Approxi-mately, 20 lM CCCP was used as a positive control.

Confocal microscopy

Lm1 and Lm1-MDR cells (4 � 105 cell/ml) were seeded on18-mm glass slides covered with poly-D-lysine hydrobromide(Sigma-Aldrich). The cells were incubated with IGA chimericprotein (12 lg/ml) or PBS. A colchicine-free medium wasused for both cell lines. Following the incubation period,Lm1-MDR cells were first incubated at 37�C for 30 min with20 lM verapamil. The same medium was used when cells

were incubated with 200 nM MitoTracker CMXRos (Invitro-gen, Carlsbad, CA) for an additional 30-min incubation pe-riod at 37�C. Following treatment, the slides were washedwith PBS, fixed with 3.7% formaldehyde in PBS, washedagain and then analyzed with an Olympus confocal laserscanning system Flu View FV300 equipped with an UPlan-SApo 60 � 3.5 oil lens at a wavelength of 488 nm.

To detect apoptotic morphology of Lm1 and Lm1-MDRcells (105 cell/ml) were seeded on 18-mm glass slides. Thecells were incubated with IGA chimeric protein (12 lg/ml)or PBS for various time periods and then analyzed by differ-ential interference contrast microscopy according to Nomar-ski using Olympus confocal laser scanning system He-NeFV300 lens equipped with UPlanSApo 60 � 1.35� oil lens4.5� zoom at an excitation of 633 nm.

Subcellular fractionation

Approximately, 4 � 106 Lm1 or Lm1-MDR cells in 5 ml me-dium were treated with the IGA chimera (12 lg/ml) for vari-ous time periods, then collected and washed three times withcold PBS. After brief centrifugation (500g, 5 min, 4�C), thecells were resuspended in one packed cell volume (100 llbuffer per million cells) of buffer A (10 mM Hepes, pH 7.9,1.5 mM MgCl2, 10 mM KCl, 0.5 mM PMSF and 0.5 mMDTT) and allowed to swell on ice for 15 min. The cells werethen lysed by rapidly pushing them through a 29-narrowgauge hypodermic needle 30 times. The cell homogenate wascentrifuged at 2,000g for 10 min to produce a crude nuclearpellet and a cytoplasmic supernatant. The nuclear pellet wasfurther purified by resuspension in one packed cell volume ofbuffer B (20 mM Hepes pH 7.9, 25% glycerol, 0.42 M NaCl,1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mMDTT) followed by stirring for 30 min at 4�C. The lysate wasthen centrifuged at 20,000g for 90 min. The supernatantcontained the nuclear proteins. Samples of equal proteinamounts of the cytoplasmic and nuclear subfractions wererun on SDS-PAGE, transferred and immunoblotted withanti-nm23-H1 to assess the quality of the fractionation andwith anti-tubulin as a marker of the cytoplasmic fraction.

Detection of DNA fragmentation

Lm1 or Lm1-MDR (106 cells/5 ml) cells were washed withPBS after incubation with the IGA chimeric protein (12 lg/ml), PBS or Dox (0.2 lM final concentration; Sigma-Aldrich)for various time periods. DNA was extracted using ProteinaseK (200 lg/ml final concentration; Roche Diagnostics Corpo-ration, Indianapolis, IN) and the phenol-chloroform extrac-tion procedure31; 1 lg of each sample was loaded on a 1%agarose gel and analyzed by electrophoresis and ethidiumbromide (EtBr) staining.

Klenow incorporation assay

Equal amounts of DNA were used for the Klenow labelingprocedure. Each reaction (20 ll) included 1 lg DNA, 10%Klenow buffer, 3.5 U Klenow fragment (Takara, Shiga,

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Japan), 2 lCi 32P-dCTP (IZ-Top, Budapest, Hungary) and12.5 mM unlabeled dNTPS (without dCTPs). The sampleswere incubated at 55�C for 1 hr and loaded on ProberTM col-umns (Talron Biotech, Rehovot, Israel) to get rid of unincor-porated labeled nucleotides. The samples were then loadedand run on 4% polyacrylamide denaturing gel and analyzed.

ResultsIGA cloning, expression and partial purification

IL2-based apoptosis-inducing chimeric proteins have alreadybeen shown to specifically target cells expressing the high af-finity IL-2R and to promote cell specific apoptotic death bothin vitro27,32 and in vivo.28,33 However, to target and kill MDRcells, we constructed a new IL-2-based chimeric protein, withgranzyme A, which triggers caspase-independent cell death,as its killing moiety (Fig. 1a).

The construct consists of the human IL-2 sequence(encoding aa 2–131), followed downstream by the humangranzyme A’s active sequence (encoding aa 29–251). Thesequence encoding IGA was inserted into a pET28b expres-sion vector under the control of the T7 promoter (Fig. 1a).

The vector was transformed into E. coli strain BL21 codþ,and the IGA chimeric protein was expressed. SDS-PAGE(Fig. 1b) and Western blot (Fig. 1c) using anti-granzyme Aantibodies confirmed the expression of the chimeric protein(42 kDa) and revealed that the chimera accumulated mainlyin the insoluble subcellular fraction of the bacterial expressingcells (Figs. 1b and 1c). The partially purified and enriched in-soluble preparations were used in the following experimentsafter denaturation and refolding.

IGA exhibits in vitro enzymatic activity

The functionality of the IGA chimeric protein was tested bymeasuring its enzymatic ability to cleave the synthetic sub-strate of granzyme A, Z-GPR-AMC, using an in vitro cell-free assay. IGA exhibits in vitro enzymatic activity, and itsability to cleave the substrate was highly specific, being 5-foldhigher than a control sample without any protein (PBS) or anonrelevant chimeric protein, IL-2-PE, which does not haveany granzyme A enzymatic activity (Fig. 1d).

IGA chimeric protein: activity and specificity

on various cell lines

First, FACS analysis was performed to verify and quantifythe expression levels of the alpha subunit of the IL-2 high-af-finity receptor (IL-2R) on the surface of the various cellstested for their response to IGA chimeric protein. The celllines expressed variable levels of the receptor subunit, rangingfrom those that expressed high levels of the alpha chain (83%on L1210 cells and 63% on Lm-1, 64% on Ls.T1 and 69% onLm1-MDR cells; Fig. 2a) to those that express relatively lowlevels of the receptor subunit (30% on Ls-MDR cells;Fig. 2a). D122 and B16 had no detectable expression of theIL-2R alpha subunit (Fig. 2a). The graphical presentation ofIL-2a subunit expression is summarized in Figure 2b.

Next, L1210, Lm-1, Lm1-MDR, Ls.T1 and Ls-MDR cellswere incubated with the chimera, and cell death was meas-ured after 72 hr. IGA effectively inhibited the growth of alltarget cells expressing IL-2R, eventually leading to cell death(Fig. 2c). However, the response of the cells varied, with thechimera killing, 80% of Lm-1 and Lm1-MDR cells, 75% ofL1210 cells, 65% of Ls.T1 cells and 40% of Ls-MDR cells. Itshould be pointed out that the chimera efficiently killed boththe sensitive cells—Lm1, Ls.T1 and L1210—and the resistantcells—Lm1-MDR and Ls-MDR (Fig. 2c). Control, nontarget

Figure 1. Schematic representation of the IGA chimeric protein and

its characterization. (a) Schematic presentation of IGA chimeric

protein. The construct consists of the human IL-2 (aa 2–131),

followed downstream by the sequence of the human active

granzyme A (aa 29–251). Arrows indicate the IGA chimeric protein.

(b) SDS-PAGE and (c) Western blot analysis using anti-granzyme A

antibody of bacterial subfractions expressing the IGA chimeric

protein: 1, whole cell extract; 2, soluble fraction; 3, insoluble

fraction (for both b and c). (d) IGA’s enzymatic activity. The

enzymatic activity was measured as described in Methods, using

Z-GPR-AMC as a substrate. IL-2-PE chimeric protein, which lacks

granzyme A enzymatic activity, was used as a negative control. The

values presented are mean 6 SE of at least three separate assays,

each carried out in triplicate.

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Figure 2. The effect of IGA on cell growth, specificity of its activity, and its internalization into the target cells. (a) FACS analysis measuring

IL-2-receptor alpha chain expression levels. Various cell lines were incubated with anti-CD25-antibodies and submitted to FACS analysis as

described in Methods. M2 represents the positive cell population, expressing the IL-2 receptor alpha subunit. (b) Graphical presentation of

IL-2 receptor alpha subunit expression. Each column represents a mean 6 SE of at least three independent experiments. (c) Cell viability

assay. Different cell lines were incubated with IGA for 72 hr at 37�C, with two concentrations of IGA chimeric protein: 6 lg/ml (gray columns)

or 12 lg/ml (white columns), as described in Methods. Each column represents a mean 6 SE of 3–6 independent experiments. (d) Lm1 cells

(5 � 106 cells/ml) were incubated with IGA (40 lg/ml) for various time periods at 37�C or with IGA in the presence of 0.2% Azide at 4�C

(for 1 hr). Cell lysates were prepared and run on SDS-PAGE gels. The IGA chimera was detected within the lysates by Western blot analysis

using anti-granzyme A antibody. The IGA chimera itself was used as a positive control. Anti-tubulin was used as a control for protein loading.

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cell lines, lacking IL-2R alpha chain expression (D122 andB16 cells) were not affected by treatment with the IGA chi-mera. In addition, the IGA chimera killed its target cells in adose-dependent manner (Fig. 2c). Thus, IGA showed a highspecificity for cells expressing the high affinity IL-2R, whetherthey were sensitive or resistant. Comparing the response ofthe various cells with the level of IL-2R expression suggeststhat cells expressing lower levels of the receptor were lesssensitive to the activity of IGA, whereas cells expressing highlevels of IL-2R had greater susceptibility to the activity of thechimera (Figs. 2b and 2c). These results suggest that sensitiv-ity to the chimera’s activity is correlated with IL-2R expres-sion levels.

IGA chimeric protein enters Lm1 target cells

We tested the ability of the IGA chimera to enter its targetcells. By using Western blot analysis on lysates of Lm1 cellstreated with IGA, we found that the chimera enters cells asearly as 5 min after the beginning of the treatment (Fig. 2d).After 1 hr treatment, the amount of the chimera increasedwithin the target cells. Treatment of the Lm1 cells with thechimera in the presence of sodium azide prevents the inter-nalization of the chimera into the cells, and no IGA chimericprotein is detected within the cell lysate (Fig. 2d). Theseresults emphasize the specificity of the internalization of theIGA into its target cells (Fig. 2d).

Specificity of IGA chimeric protein activity

To confirm the specificity of the IGA chimeric protein activ-ity, it was incubated with anti-IL-2 antibodies following by theaddition of protein-G-beads to precipitate the IGA from thefraction. Following dialysis against PBS, the depletion of IGAwas confirmed by Western blot analysis using anti-granzymeA antibodies (Fig. 3a). The activity of the depleted fractionwas analyzed by cell viability assay using Lm1 cells and com-pared it with the activity of the un-depleted fraction whileusing the same protein concentration (Fig. 3a). As seen in Fig-ure 3a, IGA was completely depleted from IGA protein frac-tion. This depleted fraction had no effect on Lm1 cells whereasthe un-depleted fraction caused 80% death of Lm1 cells (Fig.3b). This result supports the fact that the killing effect of theIGA protein preparation is due to the IGA chimeric proteinitself and not due to any other factor in the fraction.

The effect of IGA on sensitive and on MDR cell lines

As the IGA chimeric protein was able to efficiently kill MDRcells, we compared the ability of IGA and a routine chemo-therapeutic drug, Dox, to kill MDR cells. The chimera orDox were incubated with sensitive, Lm1 and resistant, Lm1-MDR cell lines at various concentrations for various timeperiods. Cell death caused by IGA was concentration andtime dependent for both cell types (Fig. 4). Lm1-MDR cellswere more susceptible to the killing by the chimera after 48hr in all concentrations of the chimeric protein, comparedwith the parental Lm1 cells (Fig. 4a). At the highest protein

concentration the chimera (12 lg/ml) killed about 45% ofLm1-MDR cells and about 30% of Lm1 cells. Dox had verylittle effect on the resistant Lm1-MDR cells compared withthe sensitive Lm1 cells after 48 hr (Fig. 4a).

After 72 hr of treatment, the chimera successfully killed theresistant and the sensitive cells at about the same efficiency(Fig. 4b). It should be pointed out that at the two lower con-centrations of the IGA chimera, cell death in MDR cells wasmuch greater than that seen in sensitive Lm1 cells. However,at the higher concentration of IGA about the same percentageof cell death was reached (Fig. 4b). In contrast, although Doxkilled the sensitive Lm1 cells to a similar extent as did IGA, itfailed to kill the resistant Lm1-MDR cells (Fig. 4).

We tested the ability of the chimeric protein to kill targetcells after shorter incubation periods (Fig. 4c). After 6 and 24hr of incubation, the chimeric protein did not have any effecton viability of the target cells (Fig. 4c).

Figure 3. Specificity of IGA’s activity. (a) IGA was depleted using

anti-IL2 antibodies followed by protein G beads, as described in

Methods. Identical protein concentrations of the depleted and un-

depleted samples were loaded onto SDS-PAGE. The blot was

incubated with anti-granzyme A antibodies. (b) The depleted and

un-depleted samples were incubated for 72 hr with Lm-1 cells, and

viability assays were performed as described in Methods.

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IGA causes typical apoptotic morphology in sensitive and

MDR cell lines

To gain some insight regarding the mechanism of action ofIGA within target cells, we first analyzed the morphology ofthe cells treated with IGA. As mentioned before, both classi-cal and nonclassical apoptosis manifest a typical morphology,which includes: condensation of chromatin, shrinkage of thecytoplasm, blebbing of the plasma membrane and formationof apoptotic bodies.

As seen in Figure 5, in Lm1 cells, a small population withapoptotic morphology starts to appear following 48 hr oftreatment with the chimera. At that time point, a largerpopulation of morphologically apoptotic cells is seen in Lm1-MDR cells. After 72 hr of incubation with the chimera, inboth cell lines, the majority of cells manifest a typical apopto-tic morphology, blebbing of cell membranes, which indicatesof final stages of apoptosis (Fig. 5). These results support theeffect of IGA chimera on cell viability of Lm1 and Lm1-MDR cells (Figs. 4a and 4b).

IGA chimeric protein’s entry triggers mitochondrial

damage in Lm1 and Lm1-MDR cells

We next analyzed the effect of IGA on the mitochondrialpotential of treated cells using Jc-1 and MitoTracker reddyes. Lm1 and Lm1-MDR cells were incubated with IGA (12lg/ml) or without any treatment (PBS) for various time peri-ods. The cells were then harvested and stained with eitherMitoTracker Red or Jc-1. MitoTracker stains mitochondria inlive cells, and its accumulation is dependent on the mem-brane potential.

A decrease in the MitoTracker intensity was observed inLm1 cells already 3 hr after the start of treatment (Figs. 6aand 6b), with even a more pronounced decrease after 12 hrof incubation with the chimeric protein (Figs. 6a–6c). Therewas a similar decrease in the mitochondrial potential inLm1-MDR cells (Figs. 6d–6f).

A similar tendency was observed when samples were ana-lyzed with Jc-1 dye. After 3 hr of IGA chimera treatment, a30% decrease in mitochondrial potential was observed in

Figure 4. The effect of IGA chimeric protein on sensitive and MDR cells is concentration and time dependent. Lm1 (black columns) and

Lm1-MDR cells (white columns) were incubated with different concentrations of IGA chimera or with 0.2 lM Dox for 48 hr (a) or 72 hr (b).

(c) Lm1 (black columns) and Lm1-MDR cells (white columns) were incubated with 12 lg/ml IGA chimera for various time periods. Both cell

lines were incubated with 0.2 lM Dox for 72 hr. After the indicated incubation period, a cell viability assay was performed (as described in

Methods). Each column represents a mean 6 SE of 2–4 independent experiments.

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both Lm1 and Lm1-MDR cells (Fig. 6g). This decrease wasalso apparent 12 and 18 hr after treatment. An uncoupler,CCCP, was used as a positive control, to monitor thedecrease in mitochondrial potential. This decrease in mito-chondrial potential suggests that the mitochondria may havea certain role in the onset of events triggered by the IGA chi-meric protein.

IGA induced death that is Caspase-3 independent

Granzyme A is known to trigger caspase-independent celldeath. Therefore, we tested whether caspase 3 is involved indeath caused by the IGA chimera. We first investigated cas-pase 3 activation by Western blot analysis (Fig. 7a). We incu-bated Lm-1 parental cells and their MDR counterpart, Lm1-MDR cells, with the chimera, Dox, because it is a chemother-apeutic drug causing conventional and caspase 3-dependentcell death, or PBS (no treatment). Cell lysates were preparedand analyzed by Western blots using anticaspase 3 or specificanti-activated caspase 3 antibodies. There was no evidence ofcaspase-3 activation in any of the lysates from cells treated

with the IGA chimera (Fig. 7a). However, caspase-3 was acti-vated in Lm1 sensitive cells after 48 hr of Dox treatment, asevident by the appearance of its 17-kDa subunit (Fig. 7a). InLm1-MDR cells treated with Dox, caspase-3 remained inac-tive, at its full length, as expected.

Next, we examined caspase 3 activation using an in vitrocaspase 3 enzymatic activity assay. Caspase 3 enzymatic activ-ity was measured in cell lysates 24 and 48 hr after treatment.There was no evidence of caspase 3 activation followingtreatment with IGA, either in Lm1 cells or in Lm1-MDR atboth 24 and 48 hr post-treatment. In contrast, there was asubstantial three-fold increase in caspase-3 enzymatic activitywhen Lm1 cells were treated with Dox for 48 hr (Fig. 7b). Asexpected, there was no caspase 3 activation in the MDR cellsfollowing Dox treatment.

To ensure that the molecules used in these assays werebiologically functional, their ability to cause cell death wastested in parallel (Fig. 7c). Cells were incubated for 24 and 48hr with the IGA chimera or Dox, and then, cell death wasmeasured. The chimera killed almost 40% of Lm1 cells and50% of Lm1-MDR cells after 48 hr treatment (there was nodetectable killing after 24 hr, results not shown). Dox killedmore than 50% of the Lm1 cells and less than 20% of theLm1-MDR cells, confirming that both the IGA chimera andDox are active molecules that cause apoptotic death by differ-ent pathways.

IGA induces DNA damage undetectable by PI staining

To further define the nature of cell death caused by IGA,Lm1 and Lm1-MDR cells were treated with the chimera andstained with PI. There was almost no effect on the cell cycleof cells treated with IGA for various time periods in both theLm1 (Fig. 8a) and the Lm1-MDR cell lines (Fig. 8c) com-pared with the control samples. No effect was also observedafter 48 hr treatment with IGA (results not shown). In con-trast, Dox had a profound effect on Lm1 cells; most of thecell population appeared to be arrested at the G2 phase (Fig.8b). This is a characteristic arrest following Dox treatment.34

Dox had no apparent effect on Lm1-MDR cells (Fig. 8d).These results show that the DNA damage caused by IGA chi-meric protein is different from the DNA damage seen inclassical apoptosis, a finding that is in accordance with whathas been reported about granzyme A.5,7–10,19,35 The type ofDNA damage was further investigated, as described below.

IGA chimeric protein triggers nm23-H1 translocation

to the nucleus

To further analyze the cell death mechanism and DNA dam-age caused by the IGA chimera, we examined the role ofnm23-H1 DNase. It was previously reported that nm23-H1 isan endoplasmic reticulum-bound granzyme A-activatedDNase that translocates from the cytoplasm to the nucleus inresponse to granzyme A treatment.10,11,19

Lm1 and Lm1-MDR cells were incubated with IGA, Doxor without any treatment (PBS) for 24 and 48 hr, and we

Figure 5. Apoptotic morphology of target cells treated with IGA

chimeric protein. Lm-1 and Lm-1-MDR cells (105 cells/ml) were

incubated with IGA (12 lg/ml) for 48 and 72 hr. The cells were

visualized by differential interface contrast microscopy according to

Nomarski. Magnification: �60 � 4.5.

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followed up the fate of nm23-H1 within the treated cells. Todo so, subfractionation was performed on cell lysates, thusobtaining the nuclear and cytoplasmic subfractions, andnm23-H1 was identified by Western blot analysis using anti-nm23-H1 antibodies and anti-tubulin as a cytoplasmicmarker (Fig. 9a). After 24 hr of IGA treatment, there was noobvious change in nm23-H1 localization in Lm1 cell lysates(data not shown); however, after 48 hr, nm23-H1 wasdetected in the nucleus of the Lm1-treated cells compared

with control untreated cells (PBS, Fig. 9a), thus indicatingthe translocation of nm23-H1 from the cytoplasm to the nu-cleus. After Dox treatment, no nm23-H1 was detected in thenucleus. In Lm1-MDR cells, nm23-H1 DNase was detectablewithin the nucleus even earlier, at 24 hr posttreatment (Fig.9b). These results are in accordance with the physiologicalrole of granzyme A whereby it causes translocation of theDNase, nm23-H1, to the nucleus. The early translocation ofnm23-H1 into the nucleus of MDR cells is in correlation

Figure 6. IGA reduces the mitochondrial potential of Lm1 and Lm1-MDR cells. Following treatment with IGA chimeric protein, Lm1 and Lm1-

MDR cells were incubated with MitoTracker CMXros as described in Methods. Lm1 cells (a–c), Lm1-MDR (d–f); a, d: PBS; b, e: 6 hr; c, f: 12

hr. Magnification: �60 � 3.5. (g) Lm1 (black colums) and Lm1-MDR cells (white columns) (106 cells/5 ml) treated with 12 lg/ml IGA were

incubated with Jc-1 as described in Methods. 590/520 is the ratio between Jc-1 aggregates and Jc-1 monomeric state. The uncoupler,

CCCP, was used as positive control. Each column represents a mean 6 SE of at least three different experiments.

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with the early effect of IGA chimeric protein on these cells.Significant death was detected in MDR cells already 48 hrpost-IGA treatment, much earlier than in the sensitive Lm1cells (see Fig. 4a).

IGA induces single-stranded and not double-stranded DNA

breaks in MDR cells

To assess the type of DNA damage caused by the chimera,DNA was extracted from cells treated with IGA or Dox andanalyzed. DNA double-strand nicks were analyzed by electro-phoresis of the DNA on agarose gel stained with EtBr. Nonicks were seen in control (PBS) DNA samples or DNA sam-ples of cells treated with IGA, both in Lm1 and Lm1-MDRcells (Fig. 10a). However, after treatment of Lm1 cells withDox, DNA degradation was clearly observed (Fig. 10a). Therewas also no degradation following Dox treatment of resistantLm1-MDR cells, as expected (Fig. 10a).

Next, DNA samples from cells after various treatmentswere analyzed by the Klenow incorporation assay and visual-

ized on denaturing PAGE. A major ‘‘smear’’ graduallyappeared in Lm1-MDR cells 24 hr after IGA chimera treat-ment, indicating single-strand DNA nicking (Fig. 10b). Thesmear became even more apparent 48 hr after chimeric pro-tein treatment. Dox did not affect the Lm1-MDR cells.

DNA samples of Lm1 cells treated with IGA showedincreasing DNA digestion from 6 until 24 hr post-treatment,resulting in a major smear 48 hr posttreatment; however, itsintensity was less than that seen in the Lm1-MDR cells. Amajor smear appeared also in Lm1 cells following Dox treat-ment (Fig. 10b). In contrast, the smear seen after 48 hr of IGAtreatment in MDR cells shows no such pattern. However, IGAtreatment of Lm1 cells resulted in some similar oligonucleoso-mal fragmentation, suggesting a possible involvement of othertypes of DNA nucleases in addition to nm23-H1, as reportedpreviously.36 This experiment demonstrates an alternativeDNA digestion pattern caused by the IGA chimeric proteinthat influenced mainly MDR cells 48 hr posttreatment and toa lesser extent the sensitive parental Lm1 cells.

Figure 7. IGA activates a caspase 3-independent cell death. (a) 3 � 106 cells/5 ml Lm1 (1) and Lm1-MDR (2) were incubated with the IGA

chimeric protein (12 lg/ml) or with Dox (0.2 lM) for the indicated time periods. Whole cell lysates were extracted as described in Methods

and analyzed by Western blot analysis using anti-caspase 3, anti-activated caspase 3 subunit and anti-tubulin as a control. (b) Caspase-3

in vitro activity was measured using lysates of Lm-1 cells (gray columns, 24 hr incubation; black columns, 48 hr incubation) and Lm-1-MDR

cells (white columns, 48 hr incubation) treated with the IGA (12 lg/ml) or Dox (0.2 lM). The lysates were incubated with the caspase-3

synthetic substrate, as described in Methods, and the fluorescence intensity was measured at excitation wavelengths of 490 nm and 520

nm. The graph indicates fold increases of caspase-3 activity from that measured in the PBS samples. The experiment represents the mean

6 SD of 3 separate experiments. (c) Cell death of Lm1 and Lm1-MDR cells incubated for 48 hr with the IGA chimera or Dox was measured

parallel to the caspase 3 activity assays.

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DiscussionMDR is a major obstacle in the successful treatment of can-cer. One of the main causes of MDR is the overexpression ofthe Pgp transporter. In addition to its ability to efflux chemo-therapy drugs, Pgp also interferes with the action of chemo-therapy drugs through inhibition of the apoptotic process, in

particular by inhibiting caspases, the executioners of the apo-ptotic cascade.

We decided to tackle this obstacle by designing a novelchimeric protein termed IGA. The chimera is composed ofIL-2 as a targeting moiety and granzyme A as a killing moi-ety. In this study, we demonstrated that the semipurified IGA

Figure 8. IGA has no effect on the cell cycle of target cells. Lm1 and Lm1-MDR cells (106 cells/5ml) were incubated with 12 lg/ml IGA or

0.2 lM Dox for the indicated time periods. Following incubation the cells were harvested, counted and processed for cell cycle analysis as

described in Methods. (a) Lm1 cells treated with the chimera for 6–24 hr. (b) Lm1 cells treated with Dox for 24 hr. (c) Lm1-MDR cells

treated with the chimera. (d) Lm1-MDR cells treated with Dox.

Figure 9. IGA triggers the translocation of nm23-H1 from the cytosol to the nucleus. Western blot analysis of cytosolic and nuclear

subfractions extracted from Lm1 (a) and Lm1-MDR (b) cells (3 � 106 cells/5 ml) incubated with PBS, IGA chimera (12 lg/ml) or Dox

(0.2 lM) for 24 and 48 hr. The subcellular fractionation procedure was preformed as described in Methods. The blot was incubated with

anti-nm23-H1 antibody and with anti-tubulin, as a cytoplasmic marker. Cyt, cytoplasmic subfraction; nuc, nuclear subfraction.

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chimeric protein is able to enter cells, which overexpress thehigh-affinity IL-2R and kill them in a specific and dose-dependent manner through a similar mechanism to that bywhich granzyme A itself kills cells (Fig. 2); by an alternative,caspase 3-independent cell death (Fig. 7).9–11,19,37 IGA chi-meric protein entry causes mitochondrial damage (Fig. 6),which may serve as a cue triggering subsequent events, aswas reported by Lieberman and coworkers.6 As we did notsee any changes in the cell cycle (Fig. 8), we concluded that,as in the case of granzyme A, the pattern of DNA damagecaused by IGA chimera is different from the DNA damageobserved in classical apoptotic cell death. Indeed, we showed

that, after the treatment with the IGA chimeric protein, gran-zyme A-activated DNase, nm23-H1, translocates from thecytoplasm to the nucleus (Fig. 9). Moreover, IGA chimericprotein causes single-strand cuts, characteristic of the cutsmade by nm23-H1 (Fig. 10). The intracellular events wereaccompanied by emergence of morphological changes charac-teristic of apoptosis (Fig. 5).

In addition, we compared the effect of the IGA chimerawith that of the chemotherapeutic drug, Dox, on MDR andparental, sensitive cell lines. The chimera efficiently kills bothsensitive and MDR cells (Fig. 4), as opposed to Dox, whichkills only the sensitive parental cells and has no effect on theMDR cells (Fig. 4). Furthermore, the killing of the MDR cellsis more extensive at 48 hr of treatment than the killing of thesensitive parental cell line (Figs. 4a and 4b). In conclusion,MDR cells were affected by IGA chimeric protein treatmentearlier than Lm1 cells, although the same level of death wasreached after 72 hr of treatment (Fig. 4).

Granzyme A was chosen as the killing moiety in our chi-meric protein mainly because of its ability to cause caspase-independent cell death. To confirm that the death induced bythe chimera was caspase independent, we compared the acti-vation of caspase 3 following treatment by the IGA chimerato that following Dox treatment. Caspase 3 is the master cas-pase in the apoptotic cascade. It is activated both after an ex-trinsic stimulus and in response to mitochondrial damage.38

Hence, its activation is crucial for the execution of the classicapoptotic death pathway. We demonstrated by Western blotanalysis and in vitro caspase 3 enzymatic assay that caspase 3was not activated in cells treated with IGA chimeric protein,neither in parental Lm1 nor in the resistant Lm1-MDR cells,although the chimera killed both cell lines (Figs. 7a and 7b).These observations are in contrast to the activity of the che-motherapeutic drug, Dox, which activated caspase 3 in theparental cancer cell line Lm1, but had no effect on the MDRcell line, Lm1-MDR (Fig. 7).39 Thus, IGA chimera causedcaspase-independent cell death as opposed to Dox, whichkills cells via classic apoptotic cell death, through caspase-3activation.

Granzyme A was used as the killing moiety of IGA chi-meric protein also because of the optimum pH for its activ-ity. It cleaves substrates at approximately pH 7.5 and above.40

This feature is useful when trying to specifically kill MDRcells, in which the pH is abnormally high and interferes withnormally occurring cellular events.41 Furthermore, an ele-vated intracellular pH in MDR cells compared with thehealthy and parental cancer cells has been suggested to beassociated with Pgp overexpression or its effect on otherpumps involved in pH balance, such as the NHE1pump.12,16,42 Some studies have linked the alkalosis that char-acterizes the MDR cells with the inhibition of caspase activa-tion, which usually occurs in acidic conditions.12,14–17

Another reason for choosing granzyme A to be deliveredand kill MDR cells is the fact that the MDR phenomenacannot, most probably, be solved solely by inhibiting the

Figure 10. IGA treatment causes single-strand DNA breaks. (a) DNA

samples (1 lg total, each sample) extracted from Lm1 and

Lm1-MDR cells treated with the IGA chimera (12 lg/ml) and Dox

(0.2 lM) for various time periods were run on an agarose gel and

stained with EtBr. (b) Autoradiograph of DNA samples from Lm1 or

Lm1-MDR cells treated with the chimera or Dox for various time

periods and labeled with P32. The samples were labeled using a

Klenow incorporation assay and were run on a denaturing PAGE for

analysis. *A brighter image to visualize the digestion pattern.

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pump itself. MDR cells have many abnormalities, includingdecreased ceramide levels,43 elevated pH and an expanded ve-sicular compartment.41 Specifically, it is known that granzymeA targets the cell repair mechanism by cleaving a SET com-plex member, Ape1, which is involved in base excision repairand maintenance of the function of key transcription factorssensitive to inhibition by oxidation.35,44 In addition, gran-zyme A cleaves a number of proteins including SET, nucleo-some assembly protein, HMG-2, DNA bending protein andhistone H1, thus disrupting their activities. This leads to theopening of the chromatin, consequently making the DNAaccessible to various nucleases and specifically to the nm23-H1 nuclease.5,7–9,11,19,35,45 Thus, it seems logical to kill MDRcells by specifically delivering granzyme A into these cells, amaster protein that triggers caspase-independent cell death,thus bypassing the many defense mechanisms of the MDRcells.

Another downside for targeting Pgp itself is the problemof nonspecific toxicity.46 In normal tissues, Pgp has a crucialrole. It is involved in protection of the host against cytotox-ins, either by accelerating their excretion or by preventingtheir uptake. It is expressed in tissues such as gastrointestinaltract, blood-brain barrier, testis, uterus and others.2,46 Inhibi-ting Pgp means exposing these organs to various toxins, sincethe inhibition will occur nonselectively on the surface of ev-ery cell that expresses the Pgp pump. In this case, the IGAchimeric protein appears a promising candidate as it deliversgranzyme A only to cells expressing high-affinity IL-2R,namely, activated lymphocytes. Although activated T and Bcells are crucial for normal ongoing immune responses, tem-porary elimination of theses cells will cause minor damage asthey are constantly being renewed.

In conclusion, by triggering granzyme A-dependent celldeath, we are not just targeting a specific component, thePgp pump, but the whole cell repair and chromatin integrity

system, thus increasing the chances of killing the MDR cells.The use of IL-2 as a targeting moiety is not new. It has beenproven successful and is used as part of many chimeric pro-teins, including the chimeric protein already approved by theFDA, Ontak.47 In our laboratory, IL-2-caspase 3 and IL-2-Bax were constructed and used to specifically target and killcancer cells via apoptosis.27,28 As opposed to Ontak, whichhas a bacterial toxin as its killing moiety and, thus, causesside effects and a strong immune response, our constructscontain only human-based protein moieties, and thereforethese proteins have an advantage because they will notinduce an immune response.20,48

Targeting hematological malignancies in general and re-sistant types in particular is especially appropriate for severalreasons. First, these tumor cells often contain antigens thatare not present on stem cells. Second, targeted chimeric pro-teins such as IGA immediately encounter the cells in thebloodstream allowing less agent to be given than would beneeded for the treatment, for example, of poorly vascularizedsolid tumors. Moreover, lymph node deposits are known tobe more vascular than many solid tumors. Finally, patientshaving these types of tumors are known to have a suppressedhumoral immune response, which would completely avoidthe production of antibodies in case a minimal immuneresponse against the chimeric protein was to occur.

IGA chimeric protein was designed to target and killMDR lymphocyte cells. However, the use of human intracel-lular proteins that induce caspase-independent apoptotic celldeath, such as granzyme A, as novel killing moieties ofdesigned targeted chimeric proteins, could also be applied tomany other resistant types of tumors using the appropriatetargeting moiety. Thus, overcoming MDR with targetedmolecules such as IGA chimeric protein that induces cas-pase-independent apoptotic cell death should open up newvistas in the fight against human cancer.

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