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Virus Research 136 (2008) 75–80

Contents lists available at ScienceDirect

Virus Research

journa l homepage: www.e lsev ier .com/ locate /v i rusres

ecombinant Newcastle disease virus expressing human interleukin-2erves as a potential candidate for tumor therapy

Volky, 10080, He

diseaics teavethe hJurkampers alreposethe a

Heng Zhaoa,∗, Markus Jankeb, Philippe Fournierb,a Department of Food Science and Nutritional Engineering, China Agricultural Universitb Division of Cell Immunology, German Cancer Research Center, Im Neuenheimer Feld 2

a r t i c l e i n f o

Article history:Received 15 February 2008Received in revised form 14 April 2008Accepted 22 April 2008Available online 5 June 2008

Keywords:Recombinant NDVIL-2Anti-tumor

a b s t r a c t

A recombinant Newcastleby applying reverse genet2 for cancer therapy. We htumor cell lines, includingcell line HT29, and humanup till 16 days, at body teexpression level of IL-2 wainfection. As NDV was proour data strongly support

1. Introduction

Newcastle disease virus (NDV) is a member of the genusAvulavirus in the family Paramyxoviridae within the order Monone-

avirales (Mayo, 2002). The 15-kb NDV genome encodes 6 proteins:ucleocapsid protein (NP), phosphoprotein (P), matrix proteinM), fusion protein (F), hemagglutinin-neuraminidase (HN) andarge polymerase protein (L) (Krishnamurty and Samal, 1998;hillips et al., 1998). The genome of NDV contains a series ofandemly linked genes in the order 3′-NP-P-M-F-HN-L-5′ sepa-ated by non-transcribed intergenic (IG) sequences. These junctionegions consist of three elements, which are known as gene-endGE), IG, and gene-start (GS) sequences (Emerson, 1982; Collins etl., 1996).

NDV was proposed as a very promising therapeutic agent amonghe group of oncolytic viruses. It has a long history as an oncolyticgent that can selectively replicate in and destroy tumor cells,nd stimulate immune system. It has pleiotropic immune stimu-atory properties, introduces T cell co-stimulatory activity, causes

acrophage activation (Cassel and Garrett, 1965; Csatary, 1971;iyahira et al., 1998; Schirrmacher and Haas, 1998). Clinical stud-

es using NDV vaccine for tumor therapy have been performed withromising results during the past decades. Patients suffering fromdvanced cancers resistant to conventional anti-cancer treatment

∗ Corresponding author. Tel.: +86 10 62737457; fax: +86 10 62736479.E-mail address: hengzhao2000@gmail.com (H. Zhao).

168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2008.04.020

er Schirrmacherb

083 Beijing, PR Chinaidelberg, Germany

se virus (NDV) containing human interleukin-2 (IL-2) gene was generatedchnique and further evaluated for its suitability to express and deliver IL-further analyzed the ability of rNDV/IL2 to express IL-2 in several humanuman breast carcinoma cell line MCF-7, the human colon-adenocarcinomat cell line. IL-2 expressed by tumor cells infected with rNDV/IL-2 was stableature, and with biological activity. Expression kinetics indicated that theady high at 24 h after infection and reached the highest level at 48 h after

d as a very promising oncolytic agent in a new age of therapeutic viruses,pplication of recombinant NDV/IL-2 virus as an anti-tumor agent.

© 2008 Elsevier B.V. All rights reserved.

received the attenuated NDV vaccine by frequently repeated highdose inhalations, ingestions, injections or enemas. NDV 73T straininduces IFN� and TNF� release from peripheral blood mononuclearcells of healthy individuals (Cassel et al., 1983; Csatary et al., 1999,2004; Liebrich et al., 1991; Reichard et al., 1992; Schirrmacher etal., 1999; Zorn et al., 1994). Furthermore, NDV has a natural avianhost range, and is non-pathogenic to mammals including humans.

The present work involves NDV as a potential therapeuticagent for cancer treatment. Our designed approach focuses onthe cytokine in the tumor microenvironment to enhance the hostimmune response against cancer by expressing human interleukin-2 (IL-2) in the oncolytic NDV. IL-2 has been demonstrated as apowerful drug for treating tumor, which can selectively stimulatehuman T cells (Maas et al., 1993). The mature IL-2 consists of 133amino acids, with a molecular weight of 15.4 kDa. CD4+ and CD8+

T lymphocytes are the major source of IL-2, it can stimulate prolif-eration, cytolytic activity, and cytokine secretion of T lymphocytesand natural killer cells (Gaffen et al., 1998; Bukreyev et al., 2000). Ithas been reported that 52 cancer patients in Germany were immu-nized with NDV vaccine adding IL-2, followed up experiment onpancreatic carcinoma stage G3 revealed that 22% were alive formore than 5 years free of tumor, 33% survived for 24, 21 and 18months, while 45% survived for 9–10 months (Schirrmacher andHaas, 1998).

NDV rescue from cloned cDNA by reverse genetics has made itpossible to manipulate the NDV genome to express heterologousgenes. We have done previously the insertion of the secreted alka-line phosphatase (SEAP) gene into four different positions in the

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76 H. Zhao et al. / Virus R

NDV genome (Zhao and Peeters, 2003). Non-virulent NDV La Sotawas used to express the IL-2 gene as it is harmless to human, andprevious experiment in our lab showed that this strain is a verypromising oncolytic agent. In the present study, we have inserted ahuman IL-2 gene into the NDV genome at the position between theHN and L genes. We evaluated further this recombinant NDV/IL-2as a potential viral candidate for cancer therapy in the aspects ofIL-2 expression, replication and virus yield. We have also demon-strated IL-2 protein expressed by NDV/IL-2 has biological functionand remains stable at body temperature. Apoptosis analysis alsoconfirmed that it is suitable to express and deliver IL-2 gene forcancer therapy.

2. Materials and methods

2.1. Cell lines and culture

Primary chicken embryo fibroblasts (CEF) cells were grown inGlasgow modification of Eagle medium-Eagle’s minimal essentialmedium (GMEM-EMEM), containing 5% fetal calf serum (FCS), and2% antibiotic mix at 37 ◦C in a 5% CO2 incubator. All tumor cellswere obtained from the Tumorbank DKFZ (Heidelberg, Germany).The human Jurkat cell line was grown in RPMI-1640 mediumsupplemented with 10% inactivated fetal calf serum (FCS), 2 mMl-glutamine, 10 mM HEPES, 100 U ml/1 penicillin and 100 mg ml/1streptomycin. The human breast carcinoma cell line MCF-7 and thehuman colon-adenocarcinoma cell line HT29 were maintained inDulbecco’s modified Eagle’s medium containing 10% inactivatedFCS, 2 mM l-glutamine, 10 mM HEPES, 100 U ml/1 penicillin and100 mg ml/1 streptomycin. The non-tumorigenic but immortalizedhuman breast epithelial cell line MCF-10A was cultured in Dul-becco’s modified Eagle’s medium with 5% horse serum, 10 mMHEPES, 100 U ml/l penicillin, 100 mg ml/l streptomycin, 500 ng ml/lhydrocortisone, 0.01 mg ml/l insulin, 20 ng/ml epidermal growthfactor and 100 ng ml choleratoxin. The IL-2-dependent CTLL cellline was purchased from ATCC (Rockville.), and cultured in Iscovesmedium, 100 U/ml penicillin, 100 mg/ml streptomycin, 10 mMHEPES, 10% FCS, 0.05 mM �-mercaptoethanol, 25 �g/ml IL-2. Allreagents for cell culture were purchased from Gibco Life Technolo-gies. All cell lines were incubated at 37C in a humidified atmosphereof 5% CO2.

2.2. Construction of full-length NDV cDNAs containing thehuman IL-2 gene

The IL-2 gene was cloned into pGEM-T vector (Promega) with theExpandTM Long Template PCR System kit (Roche). Two oligos weredesigned (5′-gcACTAGTacaggatgcaactcctgtcttgc-3′, positive sense;5′-g aCGTACGattaagtcagtgttgagatgatgc-3′, negative sense (SpeI siteand BsiWI site in upper case, IL-2 specific sequences in lower case),and the resulting plasmid was named pGEMT/IL-2. Sequencing wasperformed to confirm that no mutation occurred.

The IL-2 gene was inserted into the HN-L junction of NDV at theSpeI and BsiWI sites in the same manner as the secreted alkalinephosphate gene described previously (Zhao and Peeters, 2003). Inthe end, an additional transcriptional unit including the NDV genestart, gene end signals and IL-2 open reading frame were insertedinto the full length NDV genome. The integrity of the entire foreignsequence was confirmed by sequencing. The resulting plasmid wasnamed pNDV/IL-2.

2.3. Rescue of the recombinant virus NDV/IL-2

The rescue procedure for obtaining the recombinant NDV/IL-2virus has been as previously described. In brief, CEF cells grown in

ch 136 (2008) 75–80

six-well plates were infected with fowlpox-T7 virus at a multiplic-ity of infection (MOI) of 0.5. Then cells were transfected with a totalamount of 2 �g DNA mixture containing three expression plasmidsencoding the NP, P, and L proteins and a fourth plasmid pNDV/IL-2.Immuno peroxidase monolayer assay (IPMA) was applied to detectthe presence of F protein at the surface of virus-infected cells in themonolayer. NDV infected cells were colored and observed underthe light microscope. Supernatants and cell monolayers with pos-itive IPMA staining were harvested and passed through a 0.2-�mpore-size filter. A volume of 100 �l was inoculated into the allan-toic cavity of 10-day-old embryonated SPF chicken eggs to amplifythe recovered recombinant virus. Five days after inoculation, theallantoic fluids were harvested, and the presence of virus was deter-mined by a rapid plate hemagglutination (HA) test using chickenerythrocytes (Mukherjees et al., 2000). A positive hemagglutina-tion reaction indicated that virus was present in the allantoic fluidof inoculated eggs.

2.4. Viruses and viral RNA isolation

The NDV strain La Sota and the recombinant NDV/IL-2 weregrown in 9–11-days old embryonated specific pathogen free (SPF)eggs. NDV La Sota is commonly applied as a live attenuated vac-cine in poultry industry. The fowlpox recombinant virus (FPV-T7)expressing T7 RNA polymerase was grown on primary chickenembryo liver cells. Viral RNA was recovered from allantoic fluidusing a High Pure Viral RNA isolation kit (Roche). Viral cDNA wasprepared using Superscript reverse transcriptase (Gibco BRL, LifeTechnologies, Scotland).

2.5. ELISA

The expression levels of IL-2 gene in the supernatant of trans-fected monolayers and in the allantoic fluid of chicken eggs weremeasured by means of enzyme immuno assay as described byOptEIATM Human IL-2 kit (Pharmingen, California). The OD450 of thesamples was determined and plotted against a standard curve. Thestandard curve was generated by serially diluting the stock enzymeIL-2 in dilution buffer supplied by the kit.

2.6. Determination of virus growth

Virus growth was determined both in cell culture and embry-onated chicken eggs. CEF cells in 24-well plates were infected with

recombinant virus at 37 ◦C in EMEM supplemented with 5% fetalcalf serum (FCS), 2% antibiotics in a 5% CO2 atmosphere. Cells withsupernatants were frozen at the time indicated i.e. 0, 12, 24, 36, 48,72 h post-infection. Titrations were performed in duplicate. 100 �lof 10-fold serial dilutions in PBS were added 5 × 104 cells/well, theconcentration of virus was determined by end-point titration onCEF cells and was expressed as mean log10 50% tissue culture infec-tive dose (TCID50) per ml. 200 �l of the recombinant virus dilutioncontaining 104 TCID50 per ml was inoculated into the allantoic cav-ity of 10-day-old embryonated chicken eggs. At the time indicated,the allantoic fluids of inoculated eggs were harvested and clari-fied by centrifugation for 10 min at 1000 × g. Supernatants werepooled, frozen and thawed and used for titration in tissue culture,as described above.

2.7. Expression of IL-2 by tumor cells infected with NDV/IL-2

Tumor cell lines MCF-7, HT29, Jurkat, and the immortalizedepithelial cell line MCF-10 were infected with 100 HU NDV/IL-2or NDV as a control for an hour at 37 ◦C. After 5 min centrifu-gation at 4000 rpm to remove the cell debris, the supernatants

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Fig. 1. Construction of the NDV/IL-2 bearing the IL-2 gene. Diagram showing theIL-2 gene has been inserted into the NDV genome at the position between the HNand L genes. The ORF of the IL-2 gene was PCR amplified with SpeI and BsiWI taggedprimers, digested and introduced into the newly created SpeI and BsiWI site of theintergenic region.

3.2. Growth characteristics of the recombinant virus

To compare the growth characteristics of the recombinantviruses, the virus titers in the supernatant were determined byplating 10-fold dilutions on CEF cells at 0, 24, 36, 48 and 72 h post-infection. Allantioic fluid was harvested at different time points andinfectious titers were determined in duplicate. As shown in Fig. 2A,both the kinetics and magnitude of replication of the two viruseswere very similar; while the production of NDV/IL-2 was slightlydelayed, but not distinguishable from that of the parental virus at all

H. Zhao et al. / Virus R

were analyzed by IL-2 specific ELISA. Furthermore, time course ofexpression kinetic of IL-2 in response to an infection was inves-tigated. MCF-7 cells were infected with 100 HU NDV/IL-2 for anhour at 37 ◦C, washed before being slipped into four cell culturalflasks. After 24, 48, 72 and 96 h, the supernatants were harvested,centrifuged at 4000 rpm for 5 min and analyzed in IL-2-specificELISA.

2.8. Apoptosis assays

Apoptosis was measured by Nicoletti staining (Nicoletti et al.,1991). 5 × 105 cells were washed in PBS, incubated with 250 �lNicoletti solution (0.05 mg/ml propidium iodide, 0.1% sodium cit-rate, 0.1% Triton X-100 in PBS) for 48 h at 4 ◦C in the dark and thenanalyzed by flow cytometry. Cells were incubated with mouse IgGanti-NDV-F mAb (produced in our lab) for 20 min on ice in thedark. After twice washing, cells were incubated with PE-labeledgoat-anti-mouse IgG secondary antibody (Dianova, Hamburg, Ger-many). After washing twice with flow cytometry buffer, adjusted toa volume of 100 �l and analyzed in a FACS Calibur flow cytometer,data were analyzed with CELLQuest Pro Software (Becton Dickison,Heidelberg, Germany).

2.9. Biological therapeutic activity of the IL-2 expressed byNDV/IL-2

MCF-7 cells were infected with the recombinant NDV/IL2 or NDVand cultured at 1 × 105 cells/ml, the supernatants were taken after24, 48, 72 and 96 h of infection. They were than added in serial dilu-tions to 1 × 104 IL-2-dependent CTLL cells for 48 h and then 4 h inthe medium containing 5 �Ci/ml 3H-labeled thymidine. Radioac-tivity was recorded in a beta-counter measured and expressed incounts per minute (cpm) as a sign of the proliferation of these cellsduring 4 h of pulse.

2.10. Temperature stability of the expressed gene product IL-2

MCF-7 cells were infected with 100 HU recombinant virusNDV/IL-2 for an hour at 37 ◦C, and then cells were washed and dis-tributed equally on four cell culture bottles. After stored for 24,48, 72 and 96 h, the supernatants were harvested and centrifuged.Each aliquot of the supernatant was stored on the different temper-

◦ ◦

atures including at −20 C (frozen samples), 4 C (chilled samples)and 37 ◦C for 16 days. The analysis of the remaining amounts ofprotein was measured by the IL-2 specific ELISA.

3. Results

3.1. Recovery of the recombinant virus NDV/IL-2

The IL-2 gene ORF flanked by NDV gene start and gene endsequence was inserted into the position between the HN and Lgenes. This transcriptional cassette was inserted into the newlycreated SpeI and BsiWI sites between the HN and L genes (Fig. 1).In the recovered virus, the inserted IL-2 gene would be expressedas a monocistronic mRNA under the control of NDV transcrip-tion system. For virus recovery, the supernatant of the transfectedmonolayers was transferred into 10-day-old SPF embryonatedchicken eggs for efficient virus propagation. After incubation for5 days, the allantoic fluid was harvested and analyzed in a rapid-plate HA test using chicken erythrocytes. The presence of theIL-2 gene in the genome of recovered virus was verified byRT-PCR.

Fig. 2. Growth curve for recombinant NDV and NDV/IL-2. (A) The virus titers in thesupernatant were determined by plating tenfold dilutions on CEF cells at 0, 24, 36, 48,72 h post-infection and were shown as log10 TCID50/ml. The results represent meanvalues from three independent experiments. (B) Replication fitness of NDV/IL-2.The allantoic cavities of 10-day-old embryonated SPF chicken eggs were inoculatedwith 100 EID50 of recombinant virus. The allantoic fluid was harvested after 4 daysincubation. The amount of virus was determined in duplicate by end-point titrationas described in Section 2.

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78 H. Zhao et al. / Virus R

different time points. Furthermore, the allantoic cavities of 10-day-old embryonated SPF chicken eggs were inoculated with 100 EID50of recombinant virus. The allantoic fluid was harvested after 4 days

incubation. The amount of virus was determined in duplicate byend-point titration. Thus, replication fitness of NDV/IL-2 indicatedthat the amount of the recombinant virus remains similar after 10passages (Fig. 2B).

3.3. Expression of IL-2 by tumor cells infected with NDV/IL-2 andSPF chicken eggs

Tumor cell lines MCF-7, HT29 and Jurkat, and the immortalizedepithelial cell line MCF-10 were used to define whether the IL-2gene integrated in the NDV genome leads to the production of thetherapeutic IL-2 after infection of tumor cells in a soluble form.As shown in Fig. 3, each cell lines infected with 100 HU NDV/IL-2 expressed the desired gene product IL-2, while no gene productof IL-2 could be detected in the control that was infected with theNDV. The expression level of IL-2 differed in different tumor celllines. The MCF-7 cells expressed the highest amount of IL-2 afterinfection, followed by MCF-10 and Jurkat cells. With infected HT29cells was found the lowest amount of IL-2. The MCF-7 breast cancercell line was selected as model for the further assays (Fig. 3A).

Fig. 3. Expression of the IL-2 in tumor cell lines after recombinant NDV/IL-2 infec-tion. (A) Different cell lines MCF-7, MCF-10, HT29, and Jurkat were incubated with100 HU NDV/IL-2 or NDV as controls for 1 h at 37 ◦C, the supernatants were analysisin ELISA assay specific for IL-2. (B) Kinetics of expression of IL-2. MCF-7 tumor cellswere infected with recombinant NDV for 1 h and washed after 24, 48, 72, and 96 hafter the infection, the expression of IL-2 was analyzed at those different time pointsby ELISA assay.

Fig. 4. Biological activity of the IL-2 expressed by the recombinant NDV/IL-2. MCF-7cells were infected with NDV and NDV/IL2, the supernatants were taken after 24,48, 72 and 96 h of infection. After serial dilutions, the supernatants were incubationwith 1 × 104 IL-2-dependent CTLL cells for 72 h. The proliferation of the CTLL cellswas determined by measuring uptake of radioactive 3H-labeled thymidine during4 h of pulse.

Time course of IL-2 expression in MCF-7 cells in response to theNDV/IL-2 infection was further investigated. The IL-2 expressionhas already shown at 24 h after infection, and it appeared to reachthe maximum at 48 h after infection. Afterwards, the concentra-tion of IL-2 appeared almost constant (Fig. 3B). This observationcan be important for future applications, as the foreign gene prod-uct induced by the infection with such recombinant NDV will bepresent at an early stage in a large quantity.

SPF chicken eggs were infected with each virus at an EID50 of100 to quantify the IL-2 expression. After 4 days of incubation,allantoic fluids were harvested and ELISA assays were carried out.Allantoic fluids from eggs infected with NDV were analyzed in par-allel as negative controls. The IL-2 expression reached 2.92 �g/ml(data not shown) and no IL-2 expression was found in uninfectedeggs or in eggs infected with NDV. Sequencing analysis for the IL-2gene expression unit in rNDV/IL-2 after ten passages in eggs hasconfirmed the stability of IL-2 gene expression in rNDV/IL-2.

3.4. Biological activity of the expressed IL-2

In order to prove that the IL-2 expressed by NDV/IL-2 viruspossesses the biological activity for further experiments, the CTLL

cells (IL-2 dependent) were applied. Proliferation was observed ina dilution-dependent fashion when they were cultured in mediumthat contained the vital IL-2, while no proliferation was observedin the negative control (Fig. 4). The supernatants containing the IL-2 protein produced by NDV/IL-2 during the infection have beenshown to stimulate the growth of IL-2-dependent CTLL cells,which clearly demonstrated the biological activity of IL-2 proteinexpressed by recombinant NDV/IL-2 virus.

3.5. Apoptosis analysis

As the recombinant virus NDV/IL-2 was designed for the ther-apeutic cancer treatment, we measured apoptosis in target cancercell lines MCF-7 (breast carcinoma cells). Both recombinant NDV/IL-2 and NDV showed the capacity of inducing apoptosis events after96 h of infection (Fig. 5). Only a slight difference has been observedbetween these two recombinant viruses, both are much higher thanthe negative control MCF-7 cells in the absence of virus, which indi-cating the apoptosis observed is a result of viral killing, more thanspontaneous cell death.

H. Zhao et al. / Virus Resear

Fig. 5. Proapoptotic activity of rNDV/IL-2, NDV. MCF-7 cells was applied as the nega-tive control, apoptosis was measured by Niolettie staining in 5 × 105 MCF-7 cells persample 48, 72 and 96 h after infection. The percentage of propidium iodide-positivecells was calculated at each time.

3.6. Temperature stability of the expressed IL-2

After expression and biological activity of the IL-2 could beshown, it is important for future clinical applications to checkwhether the IL-2 gene product is stable at body temperature. Asshown in Table 1, the IL2 expression is stable for about 16 days at

the body temperature. The expressed levels of IL-2 protein werefound between 23% and 28% of the volumes of the standard condi-tions. Therefore, the gene product IL-2 remains stable long enoughin the living organism once it is expressed.

4. Discussion

NDV as a non-segmented negative-strand RNA virus (NNSV)is an ideal candidate for cancer therapy. It has a number ofadvantages of attractive candidates as viral vectors. NDV is a birdvirus, and antigenically different from common human pathogens.It does not integrate into the human genome that contributesto its potential safety; NNSV recombination is extremely rare;NNSV genome can stably express foreign genes; the expressionlevel of a foreign gene can be modified by altering its positionin the gene order. In addition, NDV has a broad range of dif-ferent anti-tumor activities and long period of laboratory andclinical research; its replication properties show tumor selectiv-ity. At the start of this work, there was no report on geneticallyengineered NDV expressing cytokine genes for cancer therapy.

Table 1Stability of the expressed IL-2 protein after stored at −20, 4 and 37 ◦C for 16 days

Supernatants (hoursafter infection)

Incubationtemperature (◦C)

Recovered IL-2concentration (ng/ml)

24 −20 37.34 39.9

37 27.8

48 −20 48.74 46.3

37 34.9

72 −20 44.64 42.5

37 34.3

96 −20 454 43.9

37 34.3

Results represent mean values from three independent experiments.

ch 136 (2008) 75–80 79

However, other viral agents have been reported to enhance theirtherapeutic efficacy by expressing cytokines which has been cho-sen as it can selectively stimulate human T cells as a powerfuldrug for treating tumor (Singh and Billeter, 1999; Krauzewicz andGriffin, 2000; Shah et al., 2003; Parato et al., 2005; Hu et al.,2006).

Previous investigation showed NDV as an ideal vector forthe expression and delivery of foreign genes for vaccination andgene therapy (Zhao and Peeters, 2003). In present study, the IL-2 gene flanked by NDV transcription gene start and gene endsequences were inserted between HN and L, as we wish that theIL-2 expression is stable and relatively high. In addition, we haveshown clearly the stability of IL-2 gene expression in rNDV/IL2after 10 passages in embryonated SPF eggs. Replication kineticsof the recombinant NDV was not distinguishable from that ofthe parent strain, which showed that the insertion of the foreigngene in the genome resulted in no significant delay in replica-tion.

As rNDV/IL2 is considered as a potential candidate for a viralvector in cancer therapy in human, we have further analyzed theability of rNDV/IL2 to express IL-2 in several human tumor celllines, including the human breast carcinoma cell line MCF-7, thehuman colon-adenocarcinoma cell line HT29, and human Jurkatcell line. IL-2 expressed by tumor cells infected with rNDV/IL-2was stable up till 16 days, at body temperature, and with biologi-cal activity. Apoptosis analysis revealed that rNDV/IL-2 had similarcytotoxic effect on tumor cells as its parent stain, which demon-strating that inserting this foreign cytokine gene in NDV to trackthe virus does not compromise its oncolytic effect. Our observationis similar with the recombinant NDV inserted with the report geneEGFP (Elankumaran et al., 2006). Expression kinetics indicated thatthe expression level of IL-2 was already high at 24 h after infec-tion and reached the highest level at 48 h after infection. This earlypresent of the foreign gene product could be very useful for futuretherapeutic cancer treatment.

Based on the encouraging clinical reports that clearly provedthe effectiveness of NDV against tumor and its safety in normaltissues, scientists began to investigate the mechanism behind itsoncolytic effect. It has been recently found that its apoptosis-inducing effect does not depend on the presence of functionalp53 protein in the infected tumor cells but is presumably exertedthrough ER-mediated cellular stress mechanism (Fabian et al., 2001,2006, 2007). Its oncolytic effect was not affected by tissue ori-gin of the infected cell lines including pancreas, glioblastoma,

melanoma, etc. It has been shown that the spread of NDV infec-tion drastically differed in normal cell lines than in tumor celllines. The outcome of NDV infection appeared to depend on theresponse of uninfected cells to beta interferon (IFN), tumor cellshas a less-than-optimum anti-viral response because of a lesionin their IFN signal transduction pathway (Krishnamurthy et al.,2006). In our lab, we have also generated the recombinant NDVwith inserted GM-CSF gene to increase the anti-tumor activity. Wehave found the activation of innate immune cells such as mono-cytes and dendritic cells by rNDV/GM-CSF, and enhancement ofIFN-� production by peripheral blood mononuclear cells (Janke etal., 2007).

In conclusion, our study suggests that a non-virulent NDV LaSota used to express the IL-2 gene as a promising oncolytic agent.We have evaluated the potential biomedical applications, the IL-2 protein expressed by this recombinant NDV/IL-2 has biologicalfunction and remains stable at body temperature. Apoptosis analy-sis also confirmed that it is suitable to express and deliver IL-2 genefor cancer therapy. Though further fundamental research studiesneed to be done to discover the mechanisms, our work stronglysupport its application as a viral agent for tumor therapy.

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simple method for measuring thymocyte apoptosis by propidium iodide staining

80 H. Zhao et al. / Virus R

Acknowledgment

We thank Margit Schraders for excellent technical assistance.

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