Antitumor effects of a recombinant fowlpox virus expressing Apoptin

in vivo and in vitro

Xiao Li1, Ningyi Jin1*, Zhiqiang Mi1, Hai Lian1, Lili Sun2, Xuemei Li1 and Hongling Zheng1

1Genetic Engineering Laboratory of PLA, The Eleventh Institute of Academy of Military Medical Sciences of PLA, Changchun,People’s Republic of China2Otorhinolaryngology Division of the Second Hospital of JiLin University, Changchun, People’s Republic of China

Apoptin is a chicken anemia virus-derived, p53-independent, bcl-2-insensitive apoptotic protein with the ability to specificallyinduce apoptosis in tumor cells. To explore the use of the Apoptingene in cancer gene therapy, we constructed a recombinant fowl-pox virus expressing the Apoptin protein (vFV-Apoptin) and com-pared the tumor-killing activity of the recombinant virus with thatof wild-type fowlpox virus in the human hepatoma cell lineHepG2. We found that although cells were somewhat resistant tothe basal cytotoxic effect of wild-type fowlpox virus, infection withvFV-Apoptin caused a pronounced, additional cytotoxic effect.Furthermore, cell death and disruption of tumor integrity wereapparent in the vFV-Apoptin-infected cells. We also testedwhether fowlpox virus-mediated expression of Apoptin in tumorcells could stimulate an antitumor effect by injecting aggressivesubcutaneous tumors derived from H22 mouse hepatoma cells inC57BL/6 mice with vFV-Apoptin. We found that fowlpox virus-mediated intratumoral expression of the Apoptin gene can induceprotective and therapeutic antitumor effects and significantlyincrease survival. Taken together, these data indicate that infec-tion of tumors with fowlpox virus expressing Apoptin inhibits tu-mor growth, induces apoptosis and may be an effective cancertreatment.' 2006 Wiley-Liss, Inc.

Key words: fowlpox virus vector; Apoptin; cancer; gene therapy

The applicability of cancer therapies is not only determined bytheir efficiency in eliminating tumor cells; specificity is an equallyimportant prerequisite. Apoptin, the product of the chicken anemiavirus VP3 gene, shows specificity and efficiency toward a widerange of transformed and malignant cells of avian, murine andhuman origin including carcinomas, sarcomas, melanomas, lym-phomas and leukemias, while sparing nontransformed primarycells.1–3 However, the mechanism by which Apoptin distinguishesmalignant cells from their normal counterparts has not yet beenfully elucidated. Apoptin’s apoptosis-inducing activity may corre-late with the subcellular localization of the protein. In normalcells, Apoptin resides predominantly in the cytoplasm but local-izes to the nucleus in transformed cells.4,5 Intriguingly, severalinhibitors of upstream caspases or the p53 pathway failed to affectApoptin-induced cell death.6 Moreover, overexpression of antia-poptotic genes such as bcl-2, BAG-1 or bcr-abl did not protectcells from Apoptin-induced apoptosis.7–10 Indeed, apoptosis iseven stimulated by bcl-2 overexpression.11 Although Apoptin-induced apoptosis involves caspase-3, the protein bypasses mostof the upstream components of the apoptotic pathway, making itresistant to mutations in this pathway.12 Thus, tumors containinglesions in any of these antiapoptotic genes or pathways should stillbe susceptible to Apoptin, in contrast to conventional therapiesthat rely on the intact cellular apoptotic machinery such as chemo-therapy or radiation. Furthermore, because of its small size, theApoptin gene can be inserted into various vectors such as parvovi-ruses, papovaviruses and adenoviruses,13–15 making it attractivefor cancer gene therapy.

The 2 major viral families used for immunization againsthuman cancer antigens are adenoviruses and poxviruses. In priorstudies, a transgenic adenovirus expressing Apoptin had a sig-nificant antitumor effect in vivo15 with no adverse toxic effects.

However, adenovirus transgene delivery to tumors has severalserious well-known drawbacks, such as low efficiency of transduc-tion in vivo, low intratumoral spreading, immunogenicity or pre-existing immunity, clearance from the bloodstream and a lack oftumor specificity.16–18 Fowlpox virus, a member of the avipoxfamily that includes canarypox virus, is an attractive vector forcancer gene therapy because it has a wide host and cell type range,can incorporate large amounts of foreign DNA without loss of ac-tivity, is highly stable, can be readily grown to high titers underGood Manufacturing Practice conditions and, after infecting mam-malian cells, can undergo efficient posttranslational processing ofthe inserted transgene.19–22 Moreover, this avipox virus isextremely safe because of its ability to infect but not multiply inhuman tissues.20 Given these advantages and the absence of anti-bodies against fowlpox viruses in most humans, we have pro-moted the use of fowlpox viruses for cancer gene therapy.

In our study, we constructed a recombinant, replication-defi-cient fowlpox virus containing the Apoptin gene (further referredto as vFV-Apoptin) and tested its antitumor effect both in vitro andin vivo. Our in vitro data show that infection of human hepatomacells with vFV-Apoptin results in a significant induction of apopto-sis. To determine whether Apoptin can cause tumor regression, weperformed multiple injections of vFV-Apoptin into subcutaneoushepatomas in mice, which effectively controlled tumor growth.

Material and methods

Cell lines and cell culture

Specific pathogen-free primary chick embryo fibroblasts (CEF)cells were obtained from the Animal Center of the Harbin Veteri-narian Institute of the Chinese Agriculture Academy of Sciencesand maintained in Dulbecco’s minimum essential medium supple-mented with 2% fetal bovine serum (Gibco) plus 100 U/ml peni-cillin and 100 mg/ml streptomycin. Mycoplasma-free HepG2human hepatoma cells (American Type Culture Collection, HB-8065) negative for mouse antibodies were grown in Dulbecco’smodified Eagle’s medium (Gibco) supplemented with 10% fetalcalf serum at 37�C/5% CO2. H22 murine hepatoma cells (synge-neic to C57Bl/6 mice) were obtained from the China Center forType Culture Collection and grown in RPMI 1640 complete me-dium (Gibco) containing 10% fetal bovine serum plus 100 U/mlpenicillin and 100 mg/ml streptomycin.

Construction of recombinant fowlpox virus expressing Apoptin

The recombinant fowlpox virus expressing Apoptin (fur-ther referred to as vFV-Apoptin) was generated as described

Grant sponsor: National 973 Project of China; Grant number:G199011902.*Correspondence to: Genetic Engineering Laboratory of PLA, The

Eleventh Institute of Academy of Military Medical Sciences of PLA, QingLong Road 1068, 130062 Changchun City, PRC.Fax:1186-0431-7981365. E-mail: skylee528112@yahoo.comReceived 17 May 2006; Accepted after revision 28 June 2006DOI 10.1002/ijc.22215Published online 11 October 2006 in Wiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 119, 2948–2957 (2006)' 2006 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

previously.23 In brief, the plasmid pVAX1-Apoptin24 was digestedwith EcoRI to obtain the 366 bp fragment containing the Apoptingene, which was ligated into our shuttle plasmid pUTA223 to cre-ate pUTA2-Apoptin. To generate the recombinant fowlpox virusby homologous recombination, 80 % confluent CEF cells werethen infected with 1 multiplicity of infection (MOI) of 282E4fowlpox virus strain (Animal Pharmaceutical Factory, Nanjing,China) and, after 2 hr incubation at 37�C, transfected with 10 mgpUTA2-Apoptin by using 10 ml Lipofectin reagent (GIBCO). Twodays after cotransfection, the virus was released from cells by 3freeze-thawing cycles and used for further infection and selectionof the recombinants. Recombinant plaques were screened in thepresence of 40 mg/ml of 50-bromo-20-deoxyuridine (Br-dU)(Sigma) and then subjected to 4 cycles of plaque purification onsecondary CEF cells. One clone was selected for high expressionof the Apoptin gene as determined by both Western blot andreverse transcription-polymerase chain reaction (RT-PCR).

Identification of recombinants by RT-PCR

Total cellular RNA was extracted from the vFV-Apoptin-infected cells by the Trizol method (Life Technologies). As posi-tive and negative controls, expression of Apoptin mRNA extractedfrom pV-Apoptin-transfected or wild-type fowlpox virus-infectedcells, respectively, was also determined. For the RT-PCR analysis,3 mg of total RNA were reverse transcribed by using Moloneymurine leukemia virus reverse transcriptase (Promega). RNA-equivalent cDNA (500 ng) was amplified by PCR using Taq DNApolymerase (Life Technologies) for 30 cycles under the followingconditions: 95�C for 5 min, 55�C for 30 sec and 72�C for 1 min.The primers used for the cDNA amplification were 50 primer, 50-CGG AAT TCA TGA ACG CTC TCC AAG AAG-30 and 30primer, 50-GCC ATA CTT AGT TAC TCT AAT CGG-30.

Western blot analysis

After 24–36 hr of infection, vFV-Apoptin-infected cells werewashed with cold phosphate-buffered saline), scraped into 400 mllysis buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA,0.1% Triton X-100, 20 mM b-glycerophosphate, 5 mM NaF,1 mM phenylmethylsulphonylfluoride, 1 mg/ml leupeptin, 200 mg/ml trypsin inhibitor, 1 mg/ml pepstatin), transferred to a microfugetube and kept on ice for 30 min. Samples were boiled in samplebuffer (50 mM Tris, pH 7.4, 100 mM dithiothreitol, 2% sodiumdodecyl sulfate (SDS), 0.1% bromophenol blue, 10% glycerol) for3 min and cell debris was removed by centrifugation at 12,000g at4�C for 10 min. Samples were loaded onto 12% SDS polyacryl-amide gels, separated by electrophoresis and electroblotted ontoHybond-C membranes (Amersham). The membrane was thenblocked with 5% milk in Tris-buffered saline. Blots were incu-bated with mouse polyclonal anti-Apoptin antibody (a kind giftfrom Yang, X. F., Yangzhou University, China) for 2 hr followedby incubation with goat anti-mouse IgG antibody (Santa Cruz Bio-technology) labeled with alkaline phosphatase for 2 hr. Signalswere visualized by chemiluminescence using 4-nitroblue tetrazo-lium chloride (NBT) and 5-bromo-4-chloro-3-indolylphosphate(BCIP) solutions (0.3% NBT, 0.3% BCIP, 100 mM Tris, 100 mMNaCl, 50 mM MgCl2; pH 9.5). Extracts of pV-Apoptin-transfectedcells was used as a positive control and fowlpox virus-infectedand noninfected cells were used as negative controls.

Genetic stability of the recombinant virus

To evaluate the stability of the recombinant virus, vFV-Apoptinwas inoculated into CEF cells at a MOI of 1 and passaged sequen-tially.25,26 The inserted genes were detected by PCR and theproteins of interest were analyzed by Western blot as describedearlier.

Production and titration of virus stocks

CEF cells growing in serum-free medium were infected with 1MOI of vFV-Apoptin. After 48–72 hr of infection, the medium

was removed and the cells were washed with Hank’s balanced saltsolution (HBSS). After 3 freeze/thaw cycles, cell debris werescraped from the plates, centrifuged and the supernatant wasrecovered. The clarified virus suspensions were pooled and the vi-rus was purified by ultracentrifugation at 100,000g for 2 hr at 4�C.Titers of recombinant and wild-type virus were determined by pla-que assays and expressed as plaque-forming units (pfu) per millili-ter virus suspension.27 The titers of wild-type FPV virus stocksdetermined by plaque assays were identical. On average, titers ofvFV-Apoptin and FPV were 7–83 109.

MTT colorimetric assay

The MTT (a tetrazolium dye, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue, Sigma) colorimetricassay was performed to detect tumor cell viability after infectionaccording to the method of Yang et al.28 HepG2 cells were seededin 96-well plates (1 3 104 cells/well) 1 day before cells wereinfected with various concentrations (1 MOI, 10 MOI and 100MOI) of vFV-Apoptin or fowlpox virus. Tumor viability wasmeasured every 12 hr over a 72 hr period by treating cells with20 ml MTT (5 mg/ml) and incubating for 4 hr at 37�C to allowMTT metabolization. The culture media were removed and thecrystals formed were dissolved by adding 100 ml/well dimethyl-sulfoxide. The absorbance at 490 nm was measured witha enzyme-linked immunosorbent assay (ELISA) plate reader(Molecular Devices Spectramax 190, Sunnyville, CA). Untreatedcells were used as controls and all measurements were performedin triplicate. The percent cell survival was expressed with respectto control values (untreated cells) using the following formula:[100(Experiment)/(control)].29,30

Ethidium bromide/Acridine orange staining and microscopy

To determine the percentage of live, necrotic and apoptoticcells, the ethidium bromide (EB) and Acridine orange (AO) stain-ing assay was performed as described previously.31,32 Briefly,vFV-Apoptin- and fowlpox virus-infected HepG2 cells were tryp-sinized and washed 3 times in HBSS. A 250 ml aliquot was addedto a microcentrifuge tube and 2 ml of EB and 2 ml of AO wereadded and vortexed. Twenty microliters of the sample was thenplaced on a microscope slide with cover slip and images of repre-sentative cells were made with a digital video camera connectedto a fluorescence microscope VANOX (Olympus). The imagesfrom the microscope were captured with the Image-Pro Plus (ver-sion 5.0.2) software program and analyzed the following day.Tests were done in triplicate and at least 500 cells were scoredeach sample to determine the presence of normal, apoptotic ornecrotic chromatin.

Murine tumor model and intratumoral treatment protocol

Six- to eight-week-old female C57BL/6 mice were obtainedfrom the Experimental Animal Center of the Academy of MilitaryMedical Sciences of China and housed in a pathogen-free facilityfor all experiments following Chinese government guidelines. Allexperiments were approved in advance by the Animal WelfareCommittee of China.

H22 cells were harvested by trypsinization and resuspended inHBSS. The viable cell number was determined by trypan blueexclusion and the cell concentration was adjusted to 5 3 107 cells/ml in serum-free HBSS. Within 1 hr of harvesting, 100 ml of cellsuspension per flank were injected subcutaneously into the rightflanks of the mice with a 25-gauge needle.13 To lessen stress onthe mice, the cells were kept at 37�C between harvest and injec-tion. Mice were injected intratumorally on days 8, 15 and 22 aftertumor implantation. The 3 treatments were (i) vFV-Apoptin alone(108 pfu/mouse in saline), (ii) FPV alone (108 pfu/mouse in saline)and (iii) saline. Each group had 10 mice, and all injections wereperformed in 50 ml. Tumor size was measured using calipers every3 days. Tumor volumes were calculated as follows: [0.52(smallestdiameter)2(largest diameter)].33

2949ANTITUMOR EFFECTS OF vFV-APOPTIN

Measurement of IgG antibody against recombinant fowlpoxvirus vector

Serum antibody responses to the fowlpox virus vector weredetermined by ELISA.34,35 Briefly, fowlpox virus antigen was pre-pared as a sucrose-cushioned cell lysate of the 282E4 strain offowlpox virus grown on chick embryo dermal cells. Serial 3-folddilutions of mice sera were reacted with fowlpox virus antigencoated onto Nunc maxisorp plates. Plates were then treated withalkaline-phosphatase-conjugated goat anti-mouse IgG (Sigma)and then developed with ortho-phenylenediamine peroxidase sub-strate (Sigma). The A450 nm of each sample was determinedusing an ELISA plate reader, subtracting the A450 nm of controlsamples that lacked sera. Titers were defined as in the highest dilu-tion with A450 nm above background.

Statistical analysis

The statistical significance of differences was evaluated usingthe analysis of t test with repeated measures using SPSS software(version 10.1). All p-values reported are 2-sided and statistical sig-nificance was accepted as p < 0.05. Log rank tests were used forsurvival analysis. All animals are shown in the Kaplan-Meier plot.In all cases, the investigator responsible for treatment and mea-surement was ‘‘blinded’’ to the experimental status of the mouse.

Results

Production and characterization of the recombinantfowlpox virus

The shuttle plasmid pUTA2, which consists of the combinedpromoter ATI-P7.5 (ATI promoter of cowpox virus and 20 tan-demly repeated mutant P7.5 early promoters of vaccinia virus),has been reported previously.23 To generate the recombinant fowl-pox virus expressing Apoptin, the recombinant plasmid pUTA2-Apoptin containing the Apoptin gene was constructed (Fig. 1a).The restriction endonuclease digestion pattern showed that theApoptin gene was inserted correctly into the vector (data notshown). The recombinant plasmid pUTA2-Apoptin and fowlpoxvirus were then cotransfected into CEF cells. Recombinants werescreened with Br-dU and subjected to plaque purification asdescribed in Material and methods. The correct transcription andtranslation of the Apoptin gene was determined by RT-PCR (Fig.1b) and Western blot analysis (Fig. 1c) of the vFV-Apoptin-infected CEF cells. pVAX1-Apoptin-transfected CEF cells wereused as a positive control and fowlpox virus-infected CEF cellswere used as a negative control.

Genetic stability of the recombinant virus

The recombinant virus was inoculated into CEF cells and pas-saged for 8 generations to determine its stability. The genomicDNA and cellular lysate were then analyzed for the presence ofthe inserted genes and for protein expression. The recombinantvirus could transcribe (Fig. 1d) and express the proteins (Fig. 1e)after 2, 4, 6 and 8 passages in CEF cells, indicating that the re-combinant virus was genetically stable and could express foreignproteins after many passages.

Inhibition of cell growth by Apoptin

With the aim of developing a strategy that could effectively killtumor cells, 3 different MOIs and various lengths of infectionwere compared for their effect on cell viability (Figs. 2a and 2b).The MTT colorimetric assay was performed to detect tumor cellviability after infection as described in Material and methods.When treated with 3 different MOIs of fowlpox virus or vFV-Apoptin, the growth of the hepatoma cell line HepG2 was inhib-ited by 4–7% after 24 hr. As expected, with longer infection times,growth of the fowlpox virus- or vFV-Apoptin-infected cells wasinhibited. However, fowlpox virus-infected cells graduallyresumed growth after 48 hr (Fig. 2a and the table below). In con-trast, when HepG2 cells were treated with 10 MOI or 100 MOI of

vFV-Apoptin, cell growth was inhibited by 25–30% and 70–80%after 36 and 48 hr, respectively, and the growth of cells treatedwith 100 MOI was eventually completely blocked (Fig. 2b and thetable below). As shown in the table of Figure 2a, the MOI of fowl-pox virus had little effect on cell viability. There was no signifi-cant difference in the growth of fowlpox virus cells infected with100 MOI or 10 MOI, and when 100 MOI fowlpox virus-infectedcells were compared with 1 MOI fowlpox virus-infected cells, asignificant decrease (p < 0.05) in cell growth was only observedat 36, 48 and 60 hr. In contrast, when HepG2 tumor cells wereinfected with vFV-Apoptin at a MOI of 100, their growth was sig-nificantly inhibited when compared with infection with a MOI of10 or 1 (shown in the table of Fig. 2b). In conclusion, vFV-Apoptincould effectively restrain the growth of cultured HepG2 cells. Theinteraction of infection time and MOI was complex and synergistic,and cell viability showed a nonrigorous dependent relationship withboth factors. Therefore, we performed the following in vitro experi-ments 48 hr after infection at a MOI of 100.

Morphological change of treated cells

Chromatin condensation and nuclear fragmentation remain thehallmarks of apoptotic cells36–38 and as a rule, classification of celldeath should always include morphological examination coupledwith at least 1 other assay.38 Fluorescence light microscopy withdifferential uptake of fluorescent DNA binding dyes (such as EB/AO staining) is a method of choice for its simplicity, rapidity andaccuracy. In such an assay, the apoptotic index and cell membraneintegrity can be assessed simultaneously, and the lack of a cell fix-ation step avoids a number of potential artifacts.38 AO permeatesall cells and makes the nuclei appear green, while EB is only takenup by cells when cytoplasmic membrane integrity is lost and stainsthe nucleus red, with EB dominating over AO. Thus, live cellshave a normal green nucleus; early apoptotic cells have a brightgreen nucleus with condensed or fragmented chromatin; late apo-ptotic cells display condensed and fragmented orange chromatinand cells that have died from direct necrosis have a structurallynormal orange nucleus.38 Using the EB/AO method, we also quan-tified the percentage of live, necrotic and apoptotic cells aftervFV-Apoptin treatment (Fig. 3). Figure 3a compares the resultsfor both control and treated HepG2 cells. The proportion of live,necrotic and apoptotic cell populations in both control and treatedHepG2 cells was quite different. As shown in the middle figure ofFigure 3b, infection with fowlpox virus was slightly cytotoxic,and the main change in morphology indicated necrosis (p < 0.01)but not apoptosis (p > 0.05) (Fig. 3c), while infection with vFV-Apoptin increased the proportion of apoptotic cells (p < 0.01)(Fig. 3c) and significantly inhibited their growth (p < 0.01, vs. livecells) (right figure of Fig. 3b). In conclusion, although the fowlpoxvirus vector had a significant in vitro antitumor effect via induc-tion of necrosis, vFV-Apoptin could significantly restrain thegrowth of HepG2 cells by inducing apoptosis.

Antitumor effect of vFV-Apoptin in vivo

To evaluate the potential of Apoptin gene therapy, we treatedtumor-bearing mice with 3 intratumoral injections/tumor every 10 days,each injection comprising 1 3 108 pfu vFV-Apoptin, 1 3 108 pfuFPV or saline. At the time of treatment, the tumors had a volume of200–250 mm3. Each tumor was measured twice a week. To opti-mize the treatment, the inoculation was administered to the co-ver-tical directions of the tumor and in multiple locations. The growthkinetics of the tumors following treatment is shown in Figures 4a–4c. During treatment, the tumors in both virus-infected groupsdecreased in size, but soon after the end of treatment, the fowlpoxvirus-infected tumors gradually resumed their growth (Fig. 5b),whereas most of the vFV-Apoptin-infected tumors did not (Fig. 4c).The majority of the mice infected with vFV-Apoptin showed a par-tial or complete response to treatment (Figs. 4c and 4d). However,there were also several nonresponders to the vFV-Apoptin treat-

2950 LI ET AL.

ment, who died early in the experiment along with animals fromthe saline- and fowlpox virus-treated groups (Figs. 4a and 4d). Ani-mals expired when their tumor reached a size of 2,000–2,500 mm3

(Figs. 5a and 5b), and all saline-treated animals died between 21and 39 days after intratumoral injection. After 42 days, all fowlpoxvirus-infected mice died, whereas 8/10 of vFV-Apoptin-infectedanimals were still alive at that point (Fig. 4d). Individual tumors ofboth virus-infected groups showed a ulcerate appearance during thetreatment. The mean survival was 34 days for fowlpox virus-infected mice when compared with 27 days for saline-treated mice(inserted table in Fig. 4d). However, tumor-bearing mice treatedwith vFV-Apoptin survived much longer than the mice in bothother groups and the mean survival of vFV-Apoptin-infected micewas > 60 days (inserted table in Fig. 4d). At the end of this experi-

ment, 1 in 3 vFV-Apoptin-infected mice who survived was com-pletely tumor-free. In conclusion, inoculation with vFV-Apoptininduces an antitumor response stronger than inoculation withfowlpox virus or saline and confers significant survival benefitsand reduction in tumor size in vivo.

Antibody titers against fowlpox virus in mice

To evaluate the antivector immunity, we measured antibodytiters to fowlpox virus in the sera of mice after tumor transplanta-tion but before these mice had received any inoculations withvirus. No animals had detectable (<100 titer) levels of antibodyagainst fowlpox virus, but all of the mice developed detectablelevels of antifowlpox virus antibody (median titer 450) 7 days

FIGURE 1 – Generation andcharacterization of vFV-Apoptin.(a) Schematic diagram depictingthe organization of the shuttle plas-mid pUTA2 and the recombinantplasmid pUTA2-Apoptin. The ex-pression cassette with the Apoptingene under the control of the com-bined promoter ATI-P7.5 is flankedby homology regions (FPV-TK gene)to allow homologous recombination.P7.5: 20 tandemly repeated mutantP7.5 vaccinia virus early promoters,ATI: cowpox virus ATI promoter.The expression vector was subse-quently inserted into the fowlpoxvirus genome, generating the re-combinant fowlpox virus vFV-Apoptin.(b) RT-PCR of the Apoptin gene.CEF cells were infected with theindicated recombinant fowlpox vi-rus at an MOI of 10 pfu/cell andApoptin transcription was analyzed48 hr later as described in Materialand methods. (c) Western blot todetect Apoptin protein from CEFcell supernatants and lysates. To an-alyze Apoptin expression, CEF cellswere infected with the indicatedrecombinant fowlpox virus at anMOI of 10 pfu/cell and cell lysateswere analyzed 48 hr later as de-scribed in Material and methods.(d) RT-PCR analysis of recombi-nant virus after passage. CEF cellswere infected with indicated recombi-nant fowlpox virus and Apoptin cDNAwas detected by RT-PCR. (e) West-ern blot analysis of recombinantvirus after passage. CEF cells wereinfected with indicated recombinantfowlpox virus and the proteins weredetected by Western blot.

2951ANTITUMOR EFFECTS OF vFV-APOPTIN

after the first vaccination with fowlpox virus. Seven days after thesecond dose, the median titer rose to 2,200 and descended to 1,400seven days after the 3rd vaccination. As shown in Figure 6, theinduced responses were moderate overall, particularly after thefirst and last vaccinations.

Discussion

In our study, we describe the generation of a recombinant fowl-pox virus vector expressing Apoptin and its effects on transformedcells in vitro and in vivo based on its tumor-specific apoptosis-

FIGURE 2 – Assessment of inhi-bition of tumor cell growth byvFV-Apoptin in vitro. (a) Effectsof different fowlpox virus MOIsand infection times on HepG2 cellviability. Data were mean 6 SD.*p < 0.05, compared with 24 hrafter 100 MOI FPV infection; **p< 0.05, compared with 36 hr after100 MOI FPV infection. (b) Effectsof different vFV-Apoptin MOIs andinfection times on HepG2 cell via-bility. Data were mean 6 SD. *p <0.05, compared with 24 hr after100 MOI vFV-Apoptin infection;**p < 0.01, compared with 36 hrafter 100 MOI vFV-Apoptin infec-tion; ***p < 0.05, compared with48 hr after 100 MOI vFV-Apoptininfection. Inhibition of growthwas measured every 12 hr by theMTT assay.

2952 LI ET AL.

inducing activity. With this approach, we achieved in some caseseven complete regression of tumors treated with vFV-Apoptin,thus providing proof of principle that this recombinant virus canbe used as an antitumor agent.

Efficacy and specificity is an important prerequisite for success-ful cancer therapy.39 Apoptin can induce apoptosis in cell lines

derived from a great variety of human tumors, including hepato-mas, lymphomas, cholangiocarcinomas, melanomas and breast,lung and colon carcinomas. In contrast, Apoptin does not induceapoptosis in normal, nontransformed cells such as fibroblasts, ke-ratinocytes or smooth muscle cells.40 The manner by which Apop-tin is able to distinguish between tumor and normal cells remains

FIGURE 3 – Morphological changes of HepG2 cells infected with vFV-Apoptin by EB/AO staining. (a) Cell images. ‘‘L’’ indicates live cells; ‘‘A’’indicates apoptotic cells and ‘‘N’’ indicates necrotic cells. Images at3 400 magnification were used to show morphological changes and3 100 mag-nification was used to quantify cell numbers. (b) Quantification of live, necrotic and apoptotic cells. The mean cell numbers, standard deviation andp values are indicated in the table below the corresponding graph. The mean numbers of control cells, fowlpox virus-infected cells and vFV-Apoptin-infected cells were 522, 600 and 573, respectively. Data were mean6 SD. (c) Statistical analysis of morphological differences among groups.

2953ANTITUMOR EFFECTS OF vFV-APOPTIN

to be elucidated. Surprisingly, it has been reported that bcl-2 facil-itates and accelerates apoptosis rather than inhibiting it,7,41,42 sug-gesting that Apoptin-induced apoptosis does not feed into the clas-sical apoptotic pathway. For instance, studies with an adenovirus

expressing p53 showed that expression of p53 in establishedtumors can induce apoptosis and tumor regression in vivo.43 Bothpreclinical and clinical studies suggest a low toxicity for nontrans-formed cells when they are forced to express exogenous p53 atlevels sufficient to kill tumor cells.44–46 However, the expressionof wild-type p53 is most effective in cells lacking functional p53.Tumor cells expressing wild-type p53 are only marginallyaffected. In vitro, Apoptin does not seem to discriminate betweenp532 and p531 cells.6 These data make it therefore reasonable toanticipate that Apoptin can be explored for cancer gene therapy.

Investigation of the antitumor effect of fowlpox virus-mediatedexpression of Apoptin at the cellular level shows that a singlevFV-Apoptin treatment at 100 MOI completely inhibits the growthof HepG2 cells, whereas treatment at 10 MOI or 1 MOI are lesseffective. In contrast, fowlpox virus-treated tumor cells resumedproliferation after treatment with any MOI dose as determined bythe MTT assay. As shown in Figure 2b, because no replicatingvirus is produced, any tumor cell that escapes viral infection willcontinue to proliferate provided that there is not too much sur-rounding damage. The vFV-Apoptin-treated tumors can be dividedinto 3 distinct groups: those with complete tumor regression, thosewith a significant delay in tumor growth and those with tumorgrowth kinetics similar to tumors treated with fowlpox virus.Thus, differences in response to Apoptin treatment are probablythe result of the MOI dose and/or fowlpox virus dispersionthroughout the tumor cells.

As shown in Figure 3, infection of HepG2 cells with a fowlpoxvirus vector may inhibit cell growth by causing necrosis. Weexpected, therefore, that the effects of the recombinant fowlpoxvirus expressing the Apoptin gene might be masked by necrosiscaused by the vector itself. However, the effects of fowlpox virusdid not impede the Apoptin-induced apoptosis of vFV-Apoptin.After vFV-Apoptin infection, 45% of the Apoptin-expressingtransformed cells become apoptotic after merely 36–48 hr, with25% of infected cells exhibiting necrosis. In contrast, although25% of cells were necrotic 48 hr after fowlpox virus infection, thetumor cells eventually resumed growth. Although the fowlpox vi-rus infection somehow stimulates necrosis, this does not appear torender tumor cells sensitive to Apoptin.

Vaccinia virus is an alternative vector that has shown encourag-ing results in animal and human studies. There is further evidencethat attenuated poxviruses have potential use as safer alternativesto current replication-competent vaccinia virus. The NYVAC vec-tor was derived from vaccinia virus through 18 deletions of genesencoding for virulence factors and human host range replication.47

Recombinant NYVAC has been shown to be a safe vaccine candi-date in humans for Japanese encephalitis B47 and malaria(NYVAC-Pf7 has 7 malaria genes inserted into the NYVAC ge-nome encoding proteins from all stages of the parasite’s life cycle).The vaccine was safe and well tolerated but variably immuno-genic.48 Avipox viruses represent an alternative to orthopox virusesin the design of vaccines and share many of their desirable featuresas vaccine candidates. Fowlpox vaccines have been shown to be safe

FIGURE 4 – Antitumor effect of vFV-Apoptin in vivo. This experimentwas performed twice and essentially identical results were obtained. (a,b, c) Tumor growth kinetics after treatment with vFV-Apoptin. C57BL/6mice were implanted subcutaneously with H22 cells and tumor growthwas determined every 3 days. When the tumor reached a volume of200 mm3, mice received multiple injections with either saline (a), FPV(b) or vFV-Apoptin (c). The size of each tumor was measured until themouse died. (d) Analysis of survival. Mice treated with vFV-Apoptinsurvived longer than the mice in the other 2 groups and the mean sur-vival of vFV-Apoptin-infected mice was > 60 days (p < 0.005). Thirty-nine days after the beginning of the treatment, 90% of the animalsinfected by vFV-Apoptin (solid line) were alive, while 70% of fowlpoxvirus- (long dashed line) and 100% of saline-treated (dotted line) micehad died. Tumor-bearing mice treated with saline had a mean survival of27 days, while the mean survival for fowlpox virus-treated mice was34 days. The inserted table was statistical survival analysis.

2954 LI ET AL.

in macaques49 and in human clinical trials of cancer vaccines.19,35,50

ALVAC (canarypox) vectors have been extensively studied for sev-eral infectious diseases including human immunodeficiency virus(HIV),51 rabies,52 cytomegalovirus53 and Japanese encephalitis B47

and have been shown to be safe and immunogenic. As with otherpoxviruses, ALVAC-based vaccines produce frequent mild local andsystemic reactions and are generally well-tolerated. In a large studyof an ALVAC-HIV construct, extensive laboratory testing indicatedno alteration in hematological, renal, hepatic or immunologicalfunction.51 A direct comparison of NYVAC and ALVAC vectorsexpressing Japanese encephalitis B antigens showed similar reac-tivity between the 2 vaccines47 and this is believed to be compa-rable to that of existing vaccines licensed for use in adults suchas the pneumococcal polysaccharide vaccine.51,54 In the in vivoexperiments described here, we did not observe any toxic effectsafter intratumoral injection of vFV-Apoptin. Thus, there is a greatpotential for improving the safety and efficacy of these poxvirusvaccine delivery systems with wide application for a variety ofhuman and veterinary pathogens as well as for therapeutic vac-cines for chronic viral infections and malignant diseases.

The animal studies presented in this article demonstrate thelow toxicity and effectiveness of Apoptin in vivo, confirming andextending the results of the in vitro studies. Soruri et al.55

have shown that apoptotic tumor cells can trigger dendritic cellsto process and present responding T lymphocytes, resulting in acytotoxic response and inducing apoptosis in the unaffected tumorcells. The antitumor effects of Apoptin in immune-competent ani-mals have also been evaluated.13 In this test for toxicity and anti-tumor activity, tumors arising from mice hepatoma cells trans-planted into C57BL/6 mice were infected with vFV-Apoptin. Wefound that Apoptin had a significant antitumor effect after 3 intra-tumoral injections without visible toxicity, despite the fact that thisapproach likely leads to infection of only a part of the tumor.Indeed, the characteristic architecture of hepatomas, with their par-titioned lobular structure, does not allow an even distributionthroughout the tumor after injection.13 Thus, it is plausible thatcomplete regression occurred only in tumors in which all lobesreceived sufficient vFV-Apoptin. The partially responding tumorswere most likely only transduced in certain areas; although sub-stantial tumor cell death and disruption of tissue integrity contrib-uted to a delayed outgrowth of these H22 tumors, there was notenough viral dispersion to completely eliminate the tumor. In thecase of the nonresponders, only a minor percentage of tumor cellsmay have been infected, resulting in a rapid outgrowth of the non-transduced cells. The infiltration injection was then performed in 2perpendicular directions of the tumor in our study. This in vivo

FIGURE 5 – Mouse hepatomas inmice before and after Apoptin genetherapy. Subcutaneously grownH22 tumors were injected 3 timeson alternating days with 1 3 109

pfu fowlpox virus, 1 3 109 pfuvFV-Apoptin or saline. (a, b)Examples of H22 tumors treatedwith fowlpox virus or saline. (c, d)Tumor regression after vFV-Apop-tin treatment. Examples of com-plete and partial remission of H22tumors achieved after Apoptingene therapy.

FIGURE 6 – Anti-fowlpox anti-body titers in mice after immuniza-tion with wild-type or recombinantfowlpox virus. Antibody titerswere determined 7 days after eachinoculation. The endpoint antibodytiter was the highest dilution result-ing in an OD value greater than thepositive cut-off.

2955ANTITUMOR EFFECTS OF vFV-APOPTIN

experiment showed a significant increase in survival in tumor-bear-ing mice treated with vFV-Apoptin, provided that a substantial per-centage of tumor cells were transduced with Apoptin.

Taken together, gene therapy with Apoptin offers unique advan-tages over current approaches for cancer therapy. The fact thatApoptin does not need a functional p53 pathway, is not hinderedby the commonly occurring blockage of apoptosis by bcl-2 or bcr-

abl, apparently acts downstream of most other factors, and hasunparalleled sensitivity suggests that it will be applicable to a widerange of tumors. In addition, new delivery methods are beingdeveloped to increase the safety and efficacy of spread throughoutthe body. These features and the fact that vFV-Apoptin shows amarked antitumor effect and improves survival warrant furtherevaluation for its implementation in clinical trials.

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