Patent Application: US-40321398-A

Abstract:
the invention relates to gene delivery vehicles which comprise nucleic acid molecules encoding apoptosis - inducing proteins vp2 and / or apoptin like activity . vp2 and vp3 are viral proteins of the chicken anaemia virus . also , the invention relates to anti - tumor therapies . infection of various human tumor cells with the gene delivery vehicles of the invention will result in the induction of apoptosis in tumor cells and much reduced apoptosis , if at all , in normal diploid , non - transformed / non - malignant cells . also the invention relates to the diagnosis of cancer , and related forms of hyperplasia , metaplasia and dysplasia .

Description:
ad5 e1 - transformed human embryonic kidney ( hek ; 293 ) and human embryonic retina ( her ; 911 and per . c6 ) cell lines were grown in dulbecco &# 39 ; s modified eagle medium ( dmem ) supplemented with 10 % fetal calf serum ( fcs ) in a 5 % co2 atmosphere at 37 ° c . cell line 293 was obtained from the american type culture collection ( atcc crl 1573 ). cell lines 911 and per . c6 were obtained from fallaux et al . ( 1996 ). cell culture media , reagents , and sera were purchased from gibco laboratories ( grand island , n . y .) culture plastics were purchased from greiner ( nürtingen , germany ). human epidermal keratinocytes were isolated from foreskin and grown in the presence of a layer of mouse 3t3 fibroblasts lethally irradiated with 137 - cs . primary cultures of keratinocytes ( fsk - 1 ) were initiated in complete medium as described ( rheinwald and green , 1975 ) with minor modifications . tumorigenic keratinocytes , scc - 15 cells ( rheinwald and beckett , 1981 ), derived from squamous - cell carcinoma , were cultured in dmem / f12 ( 3 : 1 ) medium containing 5 % fetal calf serum , 0 . 4 ug hydrocortisone and 1 um isoproterenol . the human hepatoma - derived hepg2 cells ( aden et al ., 1979 ) and the human osteosarcoma - derived u2os and saos - 2 cells ( diller et al ., 1990 ) were grown in dmem ( gibco / brl ) supplemented with 10 % fetal calf serum . the spontaneously transformed keratinocyte strain hacat ( boukamp et al ., 1988 ) was a gift from prof . dr . r . fusenig , dkfz , heidelberg , germany . hacat cells were grown in dmem supplemented with 10 % fetal calf serum . murine cell lines were cultivated in dulbecco &# 39 ; s modified eagle medium with high glucose ( 4 . 5 gram per liter ) and 10 % fetal calf serum in a 5 % co2 atmosphere at 37 ° c . the ecotropic packaging cell line psi - 2 ( mann et al ., 1983 ) and the amphotropic packaging cell line pa317 have been described before ( miller , 1990a , b ). plaque assays were performed as described previously ( fallaux et al ., 1996 ). briefly , adenovirus stocks were serially diluted in 2 ml dmem containing 2 % horse serum and added to near - confluent 911 cells in 6 - well plates . after 2 h incubation at 37 ° c ., the medium was replaced by f - 15 minimum essential medium ( mem ) containing 0 . 85 % agarose ( sigma , usa ), 20 mm hepes ( ph 7 . 4 ), 12 . 3 mm mgcl2 ; 0 . 0025 % l - glutamine , and 2 % horse serum ( heat - inactivated at 560 c for 30 minutes ). small - scale production of adenovirus lots was performed as described previously ( fallaux et al ., 1996 ). briefly , near - confluent 911 ot per . c6 monolayers were infected with approximately 5 plaque - forming units ( p . f . u . _s ) per cell , in phosphate - buffered saline ( pbs ) containing 1 % horse serum . after 1 hour at room temperature , the inoculum was replaced by fresh medium ( dmem / 2 % horse serum ). after 48 hours , the nearly completely detached cells were harvested , and collected in 1 ml pbs / 1 % horse serum . virus was isolated from the producer cells by 3 cycles of flash - freeze / thawing . the lysates were cleared by centrifugation at 3000 rpm fr 10 minutes , and stored at − 20 ° c . the 911 and per . c6 produced radv stocks were screened for the presence of recombinant - competent adenovirus by5 performing pcr analysis with primers derived from the ad5 itr region ( 5 ′- gggtggagtttgtgacgtg - 3 ′) seq id no : 1 and the e1a encoding region ( 5 ′- tcgtgaagggtaggtggttc - 3 ′) seq id no : 2 as described by noteborn and de boer ( 1995 ) using a perkin elmer pcrapparatus . the presence of a 600 - bp amplified fragment indicates that replication - competent ( e1 - region containing ) adenovirus exists in the analyzed virus stock ( hoeben , unpublished results ) or by infecting hepg2 cells with radv batch . during a period of at least 10 days , the cells were monitored for potential cytopathogenic effects and by indirect immunofluorescence using a specific monoclonal antiserum directed against e1a protein . the adaptor plasmid pmad5 was constructed from pmlp10 ( levrerno et al . 1991 ) as described below , plasmid , pmlp - 10 - lin was derived from pmlp10 by insertion of a synthetic dna fragment with unique sites for the restriction endonucleases mlui , spli , snabi , spi asuii , and muni into the hindiii site of pmlp10 . the adenovirus bglii fragment spanning nt 3328 to 8914 of the ad5 genome was inserted into the muni site of pmlp - lin . from the resulting plasmid , the sali - bamhi fragment was deleted to inactivate the tetracycline resistance gene . the resulting plasmid was controlled by restriction - enxyme analysis and named pmad5 . expression of genes inserted in the multiple cloning site will be driven by the adenovirus major late promoter , which in this configuration is linked to the adenovirus immediate - early gene 1 ( e1 ) enhancer . all cav dna sequences are originally derived from the plasmid pic - 20h / cav - ecori ( noteborn and de boer , 1990 ). the expression plasmid pcmv - fs , formerly called pcmv - vp3 ( noteborn et al . 1994 ), contains cav dna sequences encoding apoptin exclusively ( nt 427 – 868 ). the plasmid pcmv - vp2mu ( noteborn , unpublished results ) contains cav dna sequences of positions 380 to 1512 . this cav dna fragment contains the coding region for vp2 flanked by 25 bp 5 ′- non - coding and 484 bp 3 ′- non - coding cav dna sequences . 106 bp downstream of the start codon for vp2 the start codon for apoptin is situated in another reading frame . to prevent the synthesis of apoptin without interfering the vp2 synthesis a mutation in the apoptin - initiation codon ( atg was changed into acg ) was introduced and in addition a point - mutation at position 549 ( t was changed into an a ), resulting in an extra stopcodon within the gene encoding apoptin . therefore , the inserted cav sequences will only express full - length vp2 protein . by indirect immunofluorescence was shown that vp2 can be produced but that apoptin was not synthesized . for the cloning of pcr - amplified dna fragments , we have used plasmid pcr - 3 . 1 , which was purchased commercially from invitrogen ( carlsbad , calif .). for the construction of a recombinant - apoptin replication - defective retrovirus , the retrovirusvector plxsn was used ( miller , 1990a , b ). all cloning steps with plasmid dnas were in principle carried out according the methods by maniatis et al . ( 1992 ). all used enzymes were commercially obtained from boehringer mannheim , germany and / or biolabs , usa . plasmid dna was purified by centrifugation in a cscl gradient and column chromatography in sephacryl s500 ( pharmacia , uppsala , sweden ). the human cell lines hacat , hepg2 , scc - 15 , 293 , 911 , and per . c6 were transfected with plasmid dna by calcium - phosphate precipitation as described by graham and van der eb ( 1973 ). normal human diploid keratinocytes ( fsk - 1 ; second passage ), u2os and saos - 2 cells were transfected with dotap ( fischer et al ., 1996 ). cells were fixed with 80 % acetone and used for immunofluorescence assays with cav - specific or adenovirus e1a - specific monoclonal antibodies and goat anti - mouse and / or goat anti - rabbit igc conjugated with fluorescein ( jackson immunoresearch laboratories inc ., west grove pa . ), as described by noteborn et al . ( 1990 ). nuclear dna was stained with 2 , 4 - diamino - 2 - phenylindole ( dapi ) or propidium iodide ( pi ). to introduce a bamhi restriction - enzyme site into the adaptor plasmid pmad5 , it was digested with the restriction enzyme clai and treated with calf intestine alkaline phosphatase . a clai - bamhi linker was treated with t4 - kinase and ligated to itself by using t4 - dna ligase and subsequently by clai digestion . the clai / bamhi / clai linker was isolated and ligated to the linearized pmad5 vector . the bacterial strain jm109 was transformed with the ligation products . by restriction - enzyme digestions , the final vector pmab was characterized . by means of the pmab vector foreign genes can be ligated into the unique bamh1 site under regulation of the adenovirus major late promoter . a schematic representation of pmad5 and pmab is shown in fig1 . to construct a adaptor vector for introducing the apoptin gene into a adenovirus , pmab was treated with bamhi and subsequently with calf intestine phosphatase . subsequently , pcmv - fs was treated with bamh1 and a 0 . 45 kb dna fragment containing the apoptin - encoding sequences was isolated . the apoptin dna fragment was ligated into the bamhi site of the linearized pmab adaptor vector . the ligation products were cloned into the baterial strain jm109 . the orientation of apoptin in pmab was determined by restriction - enzyme analysis . the pmab construct containing the apoptin gene in the 5 ′– 3 ′ orientation under the regulation of the adenovirus major late promoter will express the apoptin gene . this adaptor vector is called pmab - vp3 and will be used to generate a adenovirus vector expressing apoptin . the pmab dna plasmid containing the apoptin - encoding sequences in the 3 ′– 5 ′ orientation downstream of the adenovirus major late promoter cannot express apoptin and will be used to make a control recombinant adenovirus vector . a schematic representation of both recombinant adaptor vectors is shown in fig2 . induction of apoptosis by a cmv - plasmid versus a recombinant - apoptin adaptor vector expressing apoptin . firstly , we have examined whether the pmab - vp3 dna vector is indeed able to express apoptin in transfected cells , whereas pmab - con should not do so . to that end , human adenovirus - transformed 293 and 911 cells were transfected with pmab - vp3 , pmab - con , and as positive control with pcmv - vp3 . approximately two days after transfection , the cells were fixed and examined for expression of apoptin by means of an indirect - immunofluorescence assay . the cell cultures transfected with pcmv - vp3 and pmab - vp3 contained about 1 % of the cells reacting with an apoptin - specific monoclonal antibody , whereas cell - cultures transfected with pmab - con dna did not . these results imply that pmab - vp3 expresses apoptin and as expected pmab - con not . in an other transfection experiment , we have analysed the induction of apoptosis in 911 cells after transfection with pmab - vp3 versus pcmv - vp3 . three days after transfection , the 911 cells were harvested and examined by indirect - immunofluorescence for expression of apoptin . in addition , the cells were stained with dapi or pi , which stain intact dna strongly , but apoptotic dna weakly and / or irregularly ( telford , 1992 ). approximately 60 % of the apoptin - positive 911 cells , transfected with pmab - vp3 were apoptotic , whereas around 40 % of the apoptin - postive cells , transfected with pcmv - vp3 underwent apoptosis . these results indicate that pmab - vp3 - regulated expression of apoptin results in a similar or somewhat higher level of apoptosis induction , than apoptin expressed by pcmv - vp3 . the results are shown in fig3 . furthermore , apoptin is able to induce apoptosis in human adenovirus - transformed cells , e1b does not inhibit apoptosis induced by apoptin . in contrast , e1b is able to block apoptosis induced by a great variety of chemotherapeutic agents . these results indicate that apoptin is a very potent anti - tumor agent . recombinant - apoptin adenovirus vectors were generated by co - transfection into helper cell line 911 of adaptor plasmids pmab - vp3 carrying the coding sequences for apoptin plus some adenovirus sequences , and plasmid dna jm17 containing the entire adenovirus dna minus the e1 and e3 region ( mcgrory et al ., 1988 ). the co - transfections were transformed with calcium - phosphate - precipitated dna as described by graham and van der eb ( 1973 ). the recombinant adenovirus dna is formed by homologous recombination between the homologous viral sequences that are present in the plasmid pmab - vp3 and the adenovirus dna of jm17 dna . in a similar way , co - transfections of 911 cells were carried out with pmab - con and pjm17 dna to generate the control recombinant adenovirus that cannot express apoptin , and which will be used as adenovirus control for the apoptin - induced apoptotic effects . several hours after transfection , the 911 cell monolayers were covered with an agarose overlayer and incubated at 37 ° c . until recombinant adenovirus - induced plaques became clearly visible . the virus was harvested from plaques as pbs - horse serum stocks , as described by fallaux et al . ( 1996 ). subsequently , a part of the recombinant - virus stocks was added to 24 - wells containing fresh 911 cells . several days later , these infected 911 cells were lysed and the recombinant viruses were harvested . next , the expression of apoptin by the potential recombinant - apoptin adenoviruses ( rad - vp3 ) or its absence of expression by control recombinant adenoviruses &# 39 ; ( rad - con ) was examined . a part of the recombinant virus stocks derived from the infected 24 - wells plates were used to infect fresh 911 cells , which were grown as monolayers on glass cover slips . one day later , the infected 911 cells were fixed with aceton and analysed by immunofluorescence using the apoptin - specific monoclonal antibody 85 . 1 . five out of 5 analysed 911 cell cultures infected with putative rad - vp3 , contained cells expressing apoptin . none of the 911 cells infected with ad - con and non - infected 911 cells were positive for apoptin . these results imply that upon co - transfection of adenovirus packaging cell lines , such as 911 cells , with the required adaptor and adenovirus dna , viable rad - vp3 expressing apoptin can be generated . two stocks derived from rad - vp3 or rad - con plaques were used for purifying the rads by carrying out three subsequent plaque purifications with 911 cells or in parallel a limited - dilution assay on per . c6 cells as described by fallaux ( 1996 ). based on the above described methods resulting in the production of rad - vp3 expressing apoptin under the regulation of the adenovirus major late promoter , we also succeeded in an adenovirus vector expressing apoptin under the control of a cytomegalovirus ( cmv ) promoter . these results show that various types of recombinant adenoviruses can be produced regulating by one of its own or heterologous promoter elements . small - scale production of rad - vp3 and rad - con lots using per . c6 cells were performed as described ( fallaux , 1996 ). briefly , the procedure is described in the experimental section . by plaque - assay , the titres were determined to be approximately 10 11 - 12 per ml cleared lysate for both rad - vp3 and rad - con . the obtained titres are not lower than observed in our laboratory for other rad &# 39 ; s . by means of the pcr - analysis and infection of hepg2 with rad - vp3 and rad - con was examined whether the produced virus batches contained replication competent adenovirus ( see also experimental section ). both the rad - vp3 and rad - con batches were free of rca , as proven by both methods . we conclude that the expression of apoptin does not negatively interfere with all required steps of the adenovirus life cycle under cell - culture conditions . therefore , a gene - therapy based on an adenovirus vector expressing apoptin is feasible . due to the expression of the anti - apoptotic ad5 e1 proteins ( white , 1996 ), apoptin optimally induces apoptosis after the recombinant - apoptin adenovirus has been produced in high amounts . the fact , that an adenovirus vector expressing apoptin can be produced in human cells transformed with adenovirus type 5 ( ad 5 ) e1 proteins , such as 293 , 911 and perc6 cells , indicates that the e1 protein enables this dna virus to replicate to high titres in the presence of the apoptosis - inducing protein apoptin . these results indicate that it is also possible to generate other recombinant dna - virus vectors expressing apoptin in cell lines transformed by the adenovirus 5 e1 protein . for instance , the recombinant parvovirus vectors based on the h - 1 or mvm parvoviruses can be propagated in 293t cells , which are transformed by ad5 e1 protein ( dinsart et al ., 1996 ). the h - 1 and mvm parvoviruses specifically induce cell death in transformed cells , but not in all ( lopez - guerrero , 1997 ). parvovirus vectors expressing apoptin will be more potent in inducing tumor - specific apoptosis than parvovirus as such , due to the additional tumor - specific induction of apoptosis by apoptin ( dinsart et al ., 1996 , danen - van oorschot , 1997 ). a protocol of the production of recombinant - apoptin virus vectors based on ad5 e1 protein transformed cells , also holds true for rna - virus species , such as retroviruses . we have examined whether infection of human tumor cells with rad - vp3 will result in apoptin - induced apoptosis . to that end , human hepatoma hepg2 , osteosarcoma u2os cells , scc - 15 cells , derived from a squamous cell carcinoma , and cells from the spontaneously transformed keratinocyte cell line hacat were infected with rad - vp3 . one day after transfection , the cells were fixed and by means of immunofluorescence and dapi staining the cells were examined for apoptin synthesis , and whether they have underwent apoptosis . already , 1 day after infection almost all analysed apoptin - positive human tumor cells were apoptotic . in non - infected cultures , only a few percent of the human tumor cells were apoptotic . the results for hepg2 and u2os cells are shown in fig4 . these results indicate that rad - vp3 - expressed apoptin can induce apoptosis in different mammalian tumorigenic and / or transformed cell lines . to analyse the effect of apoptin expressed by rad - vp3 in infected normal non - transformed cells , fsk - 1 cells were infected with rad - vp3 . four days after transfection , the cells were analysed by indirect immunofluorescence using the monoclonal antibody 85 . 1 and dapi - staining . at most 8 % of the apoptin - positive cells showed a dapi - abnormal staining , indicating that they might have underwent ( apoptin )- induced apoptosis . however , 7 % of the cells that were not infected also had a aberrant dapi - stained dna pattern . the results are shown in fig4 . given the hepatotropic nature of human ad5 after systemic delivery , it is also of importance to investigate the effect of apoptin in normal diploid hepatocytes . to that end , isolated rat hepatocytes were cultured in williams e medium ( gibco / life technologies , grand island , n . y ., usa ) supplemented with insulin ( 2 mu / ml ) and dexamethasone ( 1 nx ). the cells were grown on collagen - coated culture slides ( micronic ). the primary rat hepatocytes were infected by the adenoviral vector ad - vp3 expressing apoptin , a control adenovrius expressing lacz , or mock - infected . after two days , the cells were fixed and by means of immunofluorescence and dapi - staining the percentage of apoptotic cells was examined . no difference was observed in the percentage of dead cells either expressing apoptin , lacz or mock - infected cells . these observations indicate that ( rat ) hepatocytes do not undergo apoptin - induced apoptosis , also when apoptin is synthesized by means of an adenovirus vector . these results indicate that rad - vp3 - directed expression of apoptin does not result in apoptin - induced apoptosis in normal non - transformed human cells , in contrast to transformed / tumorigenic human cells . to examine the effect of the direct sequences in front of the apoptin atg - initiation codon , we have made two pcr - 3 . 1 - apoptin constructs . pcr - vp3ori contains the original direct upstream sequences ( 5 ′- tttcaa - 3 ′) seq id no : 3 of the atg - codon , whereas the other one , pcr - vp3mu contains the direct upstream sequence ( 5 ′- gccaac - 3 ′) seq id no : 4 . by means of an in - vitro transcription / translation wheat - germ assay , it was determined that the apoptin expression of the pcr - vp3mu was at least 5 times more than observed for pcr - vp3ori . these data indicate that the nature of the direct upstream sequences of the apoptin - atg influences the synthesis of apoptin . construction of ( viral ) vectors with the direct upstream sequence ( 5 ′- gccaac - 3 ′) seq id no : 4 in front of the atg - codon of apoptin , will result in a higher apoptin production and indirectly in an increased apoptin - induced apoptosis . important to mention is also that the amino - acid sequence of apoptin is not altered as predicted to be necessary for increased translation efficiencies according the “ kozak rule ” ( caventer and stuart , 1991 ). according to this rule , we should have changed the nucleotide at position + 4 from an a into a g , resulting into a different second amino acid of apoptin which would have changed its activity . to examine whether a part of the apoptin protein is essential for its apoptotic activity , a plasmid was constructed encoding chimeric proteins , consisting of the green - fluorescence protein ( gfp ; rizzuto , 1995 ) and the n - terminal 71 amino acids of apoptin ( n - apoptin ) or its c - terminal 50 amino acids ( c - apoptin ). human transformed cells , such as saos - 2 cells ( zhuang , 1995 ) were transiently transfected with the plasmids expressing chimeric gfp / n - apoptin or gfp / c - apoptin . only , the saos - 2 cells expressing the gfp / c - apoptin underwent apoptin - specific apoptosis . this coincides with the fact that c - apoptin ( linked to gfp ) can enter the nucleus . these results indicate that a part of apoptin can also be sufficient to induce apoptosis in ( human ) tumorigenic / transformed cells . therefore , one can also develop an effective ( gene ) therapy based on ( virus ) vectors expressing only that part of apoptin . furthermore , these data indicate that a part of apoptin contains its apoptotic acitivity when covalently linked to a foreign protein . co - expression of vp2 and apoptin in human tumor cells synergistically increases the induction of apoptosis to examine the effect of co - expression of vp2 and apoptin on the induction of apoptosis , saos - 2 cells were ( co )- transfected with pcmv - fs , expressing apoptin and / or pcmv - vp2mu , expressing vp2 . the cells were fixed with aceton at various time intervals after transfection . by indirect immunofluorescence , the vp2 - positive cells were determined with monoclonal antibody cvi - cav - 111 . 1 ( noteborn and koch , 1996 ) and the apoptin - positive cells with monoclonal antibody cvi - cav - 85 . 1 . at day 3 after transfection , only 3 % of the vp2 - expressing cells underwent apoptosis , and only about 10 % of the apoptin - expressing cells . in contrast , about 40 % of the saos - 2 cells expressing both vp2 and apoptin were already apoptotic also 4 days after transfection , the percentage of vp2 / apoptin - positive cells that underwent apoptosis was significantly higher than in cells expressing apoptin or vp2 alone . these results show that vp2 enhances the apoptin - induced apoptosis and are shown in fig5 . to construct a recombinant adenovirus expressing the viral protein 2 ( vp2 ) of chicken anemia virus , the adaptor plasmid pmab - vp2 was made . a 1 . 1 - kb bamhi fragment was isolated from the plasmid pcmv - vp2mu containing all vp2 coding sequences , but with 2 point mutations within the apoptin - coding region ( see experimental section ) and ligated into the bamh1 - linearized and calf intestine alkaline phosphatase - treated adaptor vector pmab . the final construct pmab - vp2 was characterized by restriction - enzyme and sequence analysis and shown in fig6 . by co - transfection of 911 cells with pmab - vp2 dna and pjm17 dna , rad - vp2 was made . the co - transfection and all other required steps needed for characterization , purification and production of rad - vp2 , were carried out as described for rad - vp3 and rad - con . by indirect immunofluorescence using monoclonal antibody cvi - cav - 111 . 1 , it was shown that 911 and per . c6 cells infected with rad - vp2 , indeed could express vp2 protein . to generate plasmid pl - vp3 - sn ( see fig7 ), a bamhi fragment carrying the apoptin - coding sequences were inserted in the unique bamhi site of plxsn . with restriction - enzyme analysis the proper orientation of the insert was confirmed . to test the integrity of the insert the plasmid pl - vp3 - sn was transfected with the calcium phosphate co - precipitation technique in cos - 7 and hepg2 cells . four days after transfection , the cells were fixed and analyzed with monoclonal antibody 85 . 1 for the expression of the apoptin protein . in appoximately 1 – 2 % of the cells , apoptin expression could be detected . the majority of the cells underwent apoptosis , as determined by dapi staining . these data show that the proviral ltr promoter is capable of driving the expression of the apoptin protein , that its gene is intact in the dna construct and that in transfected hepg2 and cos - 7 cells the expression of apoptin induces apoptosis . to generate viruses the plasmid pl - vp3 - sn was transfected into psi - 2 cells and into pa 317 cells with the calcium phosphate co - precipitation technique . fourty - eight hours after transfection , the supernatant of the cells wass harvested and dilutions were used to infect hepg2 cells ( the pa317 supernatant ) and nih3t3 cells ( the psi - 2 supernatant ) in the presence of 4 ug / ml polybrene . four days after infection , the cells were fixed and analysed for apoptin expression by staining with the monoclonal antibody 85 . 1 . approximately 1 % of the cells were found to express apoptin . the majority of the apoptin - positive hepg2 cells were apoptotic . these data demonstrate that the cells had been transduced with the l - apoptin - sn retroviruses . in addition , it demonstrates that a single copy of the provirus is sufficient to express sufficient amounts of the apoptin protein to be detected by immunofluorescence , and this amount is sufficient to induce apoptosis in a human tumor cell line , namely the hepatoma cell line hepg2 . taken together , these data demonstrate that retrovirus vectors carrying the apoptin gene can be generated and can be used to induce apoptosis in human tumor cells . it formally proves that neither the apoptin gene nor its expression interfere with essential steps in the retrovirus life cycle . it also demonstrates that apoptin - containing retroviruses can be produced batch - wise in quantities sufficient to be used to transduce human tumor cells in tissue culture . these results further imply that apoptin expression , and consequently apoptin - induced apoptosis in ( human ) tumor cells , will also be possible by means of ( retro )- virus - derived vector systems , such as plasmoviruses ( noguiez - hellin , 1996 ). the critical step for such a recombinant - apoptin plasmovirus system is whether the retrovirus replication is not blocked by the expression of apoptin . we have provided evidence that this indeed is not the case , for the above - described recombinant - apoptin retrovirus implying successful production of recombinant - apoptin plasmovirus . the cellular location of apoptin is different in tumorigenic / transformed human cells in comparison to normal non - transformed cells . furthermore , another marker is the specific ability of apoptin to induce apoptosis in tumorigenic / transformed cells and not in normal cells . by infecting cells with rad - vp3 and analyzing the apoptin location and / or induction of apoptosis within these cells , one is able to prove whether a cell is malignant or not . primary cells are isolated from ( suspicious ) tissue and cultured in the required medium . the cells are infected with rad - vp3 and in parallel with rad - con , and subsequently analyzed . for instance , by using an immunofluoreacence assay based on monoclonal antibodies specific for apoptin , 85 . 1 . the cells will be checked for having apoptin in the cytoplasm ( normal cells ) or in the nucleus ( transformed cells ). in addition or instead of , the percentage of apoptotic cells will be estimated . if the percentage of apoptotic cells is significantly higher for rad - vp3 - than for rad - con - infected cells , these cells have become malignant . under tissue culture conditions , apoptin expressed by the recombinant adenovirus rad - vp3 in normal cells , e . g . derived from human or rodent origin , does not induce apoptosis . in the experiment described below , we have examined whether expression of apoptin by means of the recombinant adenovirus vector rad - vp3 in healthy rats does not result in acute toxicity . the used rad - vp3 vector and a control rad vector were both grown on per . c6 cells and by pcr analysis proven to be negative for replication - competent adenovirus ( rca - free ). the rad &# 39 ; s were purified by means of cscl - gradient centrifugation . male wag / rij rats ( harlan , the netherlands ) with a body weight of about 200 gram were injected with recombinant adenovirus expressing apoptin ( rad - vp3 ; 2 . 5 × 10 9 plaque forming units , pfu ), with control recombinant adenovirus expressing the geneproduct luciferase ( rad - luc ; 2 . 5 × 10 9 ). both adenovirus vectors were resuspended in phosphate - buffered saline , containing 0 . 1 % bovine serum albumin , and 10 % glycerol ( pbs +). this solution without adenovirus vector was also injected in rats and serves as additional negative control . two rats were injected intravenously , intra - peritoneally or subcutaneously , either with rad - vp3 , rad - luc or pbs + suspension . the first method to examine a possible toxic effect of ad - vp3 - expressed apbptin was to determine the general health condition and in particular the body weight of the treated rats , which was done every day following the injections . all rats were in good health condition during the experiment . the body weight was not significantly different in the various groups . after 1 week , all examined rats , including those injected with rad - vp3 , had gained body weight indicating that none of the animals was suffering an acute toxicity due to one of the treatments with rad - vp3 . to further establish the absence of acute toxicity the following determinations were carried out . two hours before sacrifice , all rats were injected with brdu . after sacrifice , several tissues ( liver , kidney , intestines , heart , lung , spleen , gonads and penis ) were pathologically examined directly and / or collected for further histopathologic analysis ( see below ). macroscopic analysis showed that none of the ad - vp3 - treated rats had organs with significant pathological effects . the main target of intravenously injected ( recombinant ) adenovirus ( vector ) is the liver and to some extent the spleen . therefore , any toxic effects of apoptin will be observed in the liver . a panel of experiments were carried out to examine the presence of ad - vp3 dna , apoptin expression by ad - vp3 and a possible cyto - pathological effect in the liver . first , we have examined by southern blot analysis whether isolated dna from livers of ad - vp3 - treated rats contained the apoptin dna at the day of sacrifice , which means 8 days after injection . as negative controls , the dna from the livers of ad - luc - treated rats were examined in parallel . before , loading the isolated dna on a agarose gel the dna was digested with bamhi , which results in a apoptin dna fragment of about 0 . 5 kbp . the southern blot was hybridized with a 32 p - labeled apoptin - dna probe . the apoptin bamhi - dna fragment was clearly visible on the southern blot in case of dna derived from the ad - vp3 - treated animals , and as expected absent in the lanes containing the dna isolated from livers of rats treated with the control rad - luc . to examine the amount of ad - vp3 copies in the liver , various amounts of apoptin dna were loaded on parallel on the southern - blot and hybridized with the labeled apoptin - dna probe . even eight days after intravenously infection , 0 . 25 ad - vp3 copies per cell could be determined , which indicates a very significant transduction of ad - vp3 in the liver . by means of immunostaining of paraffin sections of livers treated with ad - vp3 or control livers using the apoptin - specific monoclonal antibodies cvi - cav - 85 . 1 or cvi - cav - 111 . 3 ., we have shown that about 20 – 30 % of the liver cells of the ad - vp3 - treated animals had expressed apoptin . the liver sections of the control rats were negative for apoptin . to examine the possible cyto - toxic effects on ad - vp3 - expressed apoptin on liver cells , two different methods were carried out . first , the liver sections of all ad - vp3 - treated rats and those of both types of control animals were stained with haematoxyline - eosine ( he ). for all examined liver sections no morphological pathological changes could be observed , indicating that apoptin expression is not toxic for rat liver cells . damaging effects can be seen by means of brdu - labeling that detect newly divided liver cells . in case of ad - vp3 - containing liver the amount of brdu - labeled liver cells was to a similar extent ( about 2 %) as control ad - luc - treated rat livers . therefore , apoptin expression , as such , has no significant toxic effect in vivo . both macroscopic , as well as , histological analysis in combination with biochemical and immunological data revealed that expression of apoptin has no ( acute ) toxic effect in an in - vivo model . these results indicate that a therapy based on expression of apoptin by use of a gene - delivery vehicle or by other methods will have limited negative side effects . ad - vp3 - regulated expression of apoptin results in the induction of apoptosis in human transformed cells under tissue - culture conditions . for instance , ad - vp3 - driven apoptin expression results in induction of apoptosis in the human hepatoma - derived cells hepg2 . thusfar , no in - vivo anti - tumor activity of apoptin ( e . g . expressed by ad - vp3 ) was examined . therefore , we have determined whether ad - vp3 - regulated apoptin expression will result in an anti - tumor activity in an in - vivo model . to that end , male nude balb / c / nu / nu mice were injected subcutaneously with 1 × 10 6 human hepg2 cells per side . at least at two or three locations per mouse human hepatoma cells were injected . three weeks after injection clear hepatoma tumors had developed , were subcutaneously visible and had a mean size of at least 5 × 5 mm . the rad - vp3 and control rad - con1 vectors , suspended in phosphate - buffered saline , 5 % sucrose and 0 . 1 % bovine serum albumin , were intra - tumorally injected . the used rad - vp3 vector expressing apoptin and the control vector rad - con1 containing the apoptin gene in the 3 ′– 5 ′ ( reverse or anti - sense ) orientation opposite to the ad mlp promoter were both grown on per . c6 cells . both batches of recombinant adenoviruses were proven to be rca - free by means of pcr analysis . the rad &# 39 ; s were purified by use of cscl - gradient centrifugation . per tumor 7 × 10 9 pfu rad particles in 40 micro - liter suspension were injected . per type rad vector 6 mice with 2 to 3 hepg2 tumors were treated . as additional control , a group of 4 nude mice containing hepg2 tumors were intra - tumorally injected with phosphate - buffered saline containing 5 % sucrose and 0 . 1 % bovine serum albumin ( pbs +- group ). to examine the possible anti - tumor effect of ad - vp3 expressed apoptin in the human hepg2 tumors , the size of the subcutaneous tumors were measured during the experiment which continued till 7 days after injection of the rad - vp3 and control suspensions . both the pbs + group and the group treated with control radcon1 showed a mean progressive increase of hepg2 tumor size . in contrast , to the group that was intra - tumorally treated with radvp3 , which showed a reduced tumor size . at seven days after injection , the nude mice were sacrificed . the tumors were isolated and macroscopically examined . it is clear that the hepatoma tumors treated with the control rad - con1 vector or pbs + showed to be heavily vascularized hepg2 - tumor tissue and had been become bigger after treatment . a complete different pattern was observed with the rad - vp3 - treated hepg2 tumors . the residual tumor mass had a pale appearance , due to a reduced tumor vascularization . the tumors had become reduced in size after treatment with rad - vp3 . the tumors contained also white bubble - like structures , which is indicative for dead cells . besides the apoptin - induced tumor regression , no negative effect of apoptin - expression on the organs ( in particular liver and spleen were examined ) could be observed . in conclusion , tumors treated with rad - vp3 showed a reduced tumor size , whereas the controls did not . this implies that expression of apoptin has an anti - tumor activity in an in - vivo model . the fact that ad - vp3 - expressed apoptin can induce tumor regression in a fast - growing tumor as hepg2 proves the strong anti - tumor potential of apoptin . the fact that apoptin reduces tumor growth in nude mice shows that expression of apoptin itself ‘ kills ’ the tumor cells without an additional immune response . the described toxicity and anti - 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