Patent Application: US-12647402-A

Abstract:
the invention relates to the field of apoptosis . the invention provides novel therapeutic possibilities , for example novel combinatorial therapies or novel therapeutic compounds that can work alone , sequentially to , or jointly with apoptin , especially in those cases wherein p53 is non - functional and / or bcl - 2 is over - expressed and / or functional caspase - 3 is lacking . the invention further describes that a proteinaceous substance according to the invention induces apoptosis tumor specific and does not or at least not significantly induce apoptosis in normal cells . the invention comprises an anti - tumor agent , which specifically kill tumor cells and not normal cells .

Description:
the invention provides a method for inducing apoptosis through interference with the function of this newly discovered apoptosis - related tap protein or functional equivalents or fragments thereof and / or the induction of apoptosis by means of ( over ) expression of tap or related gene or functional equivalents thereof . the invention also provides an anti - tumor therapy based on the interference with the function of tap - like proteins and / or its ( over ) expression . the invention provides a therapy for cancer , auto - immune diseases or related diseases which is based on tap - like proteins alone or in combination with apoptin and / or apoptin - like compounds . dna cloning the various cloning steps have been carried out according to the methods described by maniatis et al ., 1982 . for drug selections luria broth ( lb ) plates for e . coli were supplemented with ampicillin ( 50 microgram per ml ). sequence analysis : the clones containing the sequences encoding tap proteins were sequenced using dideoxy ntps according to the sanger - method , which was performed by baseclear , leiden , the netherlands . the used sequencing primer was a 20 - mer of the dna - sequence 5 ′- ggatccctagcaggtctgcg - 3 ′. primary rat hepatocytes primary rat hepatocytes were isolated from collagenase - perfused livers as standard and cultured in williams e medium ( gibco life technologies , grand island , n . y ., usa ) supplemented with insulin ( 2 mu / ml ) and dexamethasone ( 1 mm ). the cells were grown on collagen - coated culture slides ( micronic , lelystad , the netherlands ). transfection methods plasmid dna was purified by qiagen - column chromatography according to the instructions of the manufacturer ( westburg bv , leusden , the netherlands ). the various mammalian cell lines were grown in the appropriate culture medium supplemented with penicillin and streptomycin ( gibco / brl ). the cells were transfected by calcium - phosphate precipitation as described ( graham and van der eb , 1973 ) or transfected with fugene , according the instructions provided by the manufacturer ( roche , almere , the netherlands ). micro - injection of plasmid dna encoding tap and control genes . 1 5 cells were micro - injected in the cytoplasm with plasmid pcmv - tap ( cloning as described herein ), pcmv - lacz or pcmv - apoptin ( danen - van oorschot et al ., 1997 ) at a concentration of 50 ng per ul using an eppendorf micro - injector with the injection pressure of 0 . 5 psi . the cells were co - injected with dextran - rhodamine ( mw : 70 kda ; molecular probes , leiden , the netherlands ) to be able to later identify injected cells . the cells were incubated at 37 ′ c after injection until the cells were fixed . immunofluorescence assays cells were fixed with 80 % acetone and used for immunofluorescence tests with mye - tagged - and / or apoptin - specific monoclonal antibodies with fluorescein - isothiocyanate ( noteborn et al ., 1998 ). apoptosis assays the apoptotic cells were initially determined by means of dapi staining ( telford et al ., 1992 ). dapi stains intact cellular dna strongly and regularly , whereas it stains apoptotic dna weakly and / or irregularly . the tunel assay was carried out as described by pietersen et al ., 1999 with the use of an in - situ cell death detection kit ( roche , almere , the netherlands ). western - blot analysis the cells were harvested after transfection and lysed in 2 × sample buffer [ 100 mm trishci ( ph 6 . 8 ), 4 % sds , 20 % glycerol , 0 . 2 % bromophenol blue , 2 . 5 % beta - mercaptoethanol ]. proteins were fractionated on 15 % sds - polyacrylamide gels and electroblotted onto immobilon - p membranes ( millipore ). parts of the blots were then incubated with mab 9e10 ( 1 : 5000 ) to myc - tag to detect tap . positive signals were visualized by enhanced chemiluminescence according to the manufacture &# 39 ; s protocol ( amersham , the netherlands ). results and discussion construction of a plasmid containing the tap gene . to examine whether tap protein harbors apoptotic activity we have to make a dna construct containing the complete tap dna sequences , which are shown in fig1 . in addition , the stop codon has been also indicated . the presented tap dna sequence synthesizes a putative protein of 105 aminoids ( fig2 ). furthermore , at the n - terminal end of the tap gene , a myc - aci tag ( amino acids : eqkliseedl ) has been linked which is in frame with the tap gene product . this myc - tag enables the recognition of the tap proteins by means of the myc - tag - specific 9e10 antibody . the dna construct containing the tap dna sequences , myc - tag dna sequences and bamhi restriction - enzyme sites has been made by using a panel of dna oligomers as shown in fig3 . all steps for making the required approximately 0 . 36 kb dna fragment was carried by the midland certified reagent co ., midland , tex ., usa . in principle the synthesized dna oligomers were synthesized and purified by preparative polyacrylamide gel electrophoresis and 5 ′- phosphorylated to permit ligation . subsequently , the various oligomers were annealed and then ligated en masse in a thermostable ligation reaction . the long internal overlaps annealed stably and are efficiently ligated , whereas the cohesive termini tend to remain disassociated . as a consequence , the ligation product could be directly purified by preparative low - melting - point agarose gel electrophoresis using the freeze - squeeze method . the appropriated approximately 0 . 36 kb dna fragment was made visible in the gel with long wavelength uv light . next , the dna band was excised and purified and ligated in bamh1 - linearized puc19 plasmid dna , which was treated with calfintestine alkaline phosphatase . the final product was analyzed by bamh1digestion for containing an approximately 0 . 36 kb dna fragment and sequence analysis . the plasmid dna containing the myc - tagged tap dna sequences has been called “ pmn - tap ”. construction of an expression vector for the identification of tap protein in human and other mammalian cells . to study whether the cloned myc - tagged tap gene encode protein products , we have carried out the following experiments . the dna plasmid pcdna3 . 1 ( invitrogen , groningen , the netherlands ) contains the cytomegalovirus ( cw promoter enabling high levels of expression of foreign genes in transformed human and other mammalian cells , such as saos - 2 and cos cells . the pcdna3 . 1 construct expressing myc - tagged tap dna was constructed as follows . the pmn - tap clone was digested with the restriction enzyme bamh1 and the requested approximately 0 . 36 kb dna insert was isolated . the expression vector pcdna3 . 1 digested with bamh1 and treated with calf intestine alkaline phosphatase and ligated to the isolated myc - tagged tap dna insert . by restriction - enzyme digestions and sequence analysis , the pcdna3 . 1 construct containing the myc - tagged tap dna in the correct orientation was identified . the final construct containing the tap sequences is called pcmv - tap . the synthesis of the myc - tagged tap protein was analyzed by transfection of cos cells with plasmid pcmv - tap . as negative control , cos cells were mock - transfected . two days after transfection , the cells were lysed and western - blot analysis was carried out using the myc - tag - specific antibody9e10 . the cos cells transfected with pcmv - tap were proven to synthesize a specific myc .- tag ed tap product with the expected size of approximately 12 @ gkda . as expected , the lysates of the mock - transfected cos cells did not contain a protein product reacting with the myc - tag - specific antibodies . these results indicate that we have been able to construct a plasmid that is able to produce a protein product derived from the putative tap open reading frame . over - expression of the novel tap protein in human hepatoma hepg2cells induces the apoptotic process . next , we have examined whether tap carries apoptotic activity . first , we have analyzed the cellular localization of the novel tap protein in human hepatoma - derived hepg2 cells , which are synthesizing functional tumor suppressor protein p53 ( pietersen et al ., 1999 ). to that end , the hepg2 cells were transfected with plasmid pcw - tap encoding the myc - tagged tap protein , as described by danen - van oorschot ( 1997 ). as a negative control , hepg2 cells were transfected with a plasmid encoding myc - tagged lacz . two days after transfection , the cells were fixed . by indirect immunofluorescence using the myc - tag - specific antibodies 9e10 and dapi , which stains the nuclear dna , it was shown that the tap protein was present in the cytoplasm of the cell . finally , we examined whether ( over )- expression of our novel tap protein results in induction of apoptosis . to that end , we have analyzed the tap - positive cells for dapi - staining . four days after transfection , almost all tap - positive cells were aberrantly stained with dapi , which is indicative for induction of apoptosis ( telford , 1992 , danen - van oorschot , 1997 ). at this time point , tap was localized in the nuclear structures of the transfected hepg2cells . the nuclear dna of the cells expressing lacz was not aberrantly stained with dapi . therefore , the obtained results ( fig4 ) indicate that tap can induce apoptosis in human tumor cells . over - expression of the novel tap protein in human osteosarcomau20s cells expressing wild - type p53 versus human osteosarcoma saos - 2 and hepatoma hep3b cells lacking functional p53 . during tumor development , most of the tumors will lack functional tumor suppressor p53 . tumor cells lacking functional p53 are in general poor responders to ( chemo ) therapeutic agents . in the above described experiment , we have shown that tap can induce apoptosis in cells containing functional tumor suppressor protein p53 . therefore , it is also of importance to prove whether tap can induce apoptosis in human tumor cells in the absence of functional p53 . to that end , we have transfected human osteosarcoma - derived saos - 2cells and human hepatoma - derived hep3b cells synthesizing no tumor suppressor p53 ( zhuang et al . 1995a , 1995b ) with plasmid pcmv - tap - encoding myc - tagged tap protein , u20s cells expressing wild - type p53 were used as a control in this experiment . as control , the three different cell lines were transfected with the plasmid pcniv - lacz encoding myc - tagged lacz . four or five days after transfection , the cells were fixed and analyzed for myc - tagged tap expression by means of immunofluorescence using the myc - tag specific antibody 9e10 and apoptotic activity by dapi staining . in parallel , transfected saos - 2 , u20s and hep3b cells were fixed and examined for induction of apoptosis by means of a tunel assay ( pietersen et al ., 1999 ). an equal significant amount of saos - 2 and u20s cells were stained aberrantly with dapi whereas the cells containing myc - tagged lacz were not , which is indicative for induction of tap - specific apoptosis to a similar level in p53 - minus saos - 2 versus p53 - positive u20s cells . in addition , saos - 2 and u20scell cultures transfected with pcnw - tap showed a significant higher amount of tunel - positive cells than saos - 2 cell cultures transfected with pcmv - lacz . furthermore , the tap - positive hep3b cells underwent also apoptosis as assayed both with the dapi - staining method as well as with the tunel assay . the level of apoptosis induction was similar to the level of apoptosis found induction in hepg2 cells . the results ( fig4 ), obtained with tap synthesis in both hepg2 versus hep3b and saos - 2 versus u20s cells , prove that tap can induce apoptosis independent of p53 . therefore , tumors or tumorous cells having functional p53 or not can be treated with tap - synthesizing vectors . over - expression of the novel tap protein in human lymphoma dohh - 2 cells induces the apoptotic process . besides non - functional p53 , over - expression of anti - apoptosis proteins such as bcl - 2 hamper conventional anti - cancer therapies . therefore , we have also examined whether tap can induce apoptosis in human tumor cells over - expressing bcl - 2 such as dohh - 2 cells ( zhuang et al ., 1995 ). to that end , dohh - 2 cells were transfected with pcmv - tap or pcmv - iacz and analyzed as described for the hepg2 cells . only the dohh - 2 cells synthesizing tap protein were shown to be stained aberrantly with dapi . therefore , we conclude that tap also induces apoptosis in cells over - expressing bel the results are shown in fig4 . co - expression of tap and apoptin induces apoptosis in human tumor cells in a very efficient way . the cav - derived protein apoptin induces apoptosis upon nuclear localization as seems also to be the case for tap - induced apoptosis . however , the tap protein is present in a more diffused way in the cytoplasm in comparison to apoptin . furthermore , tap seems to enter the nucleus shortly before the cells undergo apoptosis , whereas apoptin does earlier ( see above and below ). in the following transfection experiments ( carried out as described above ), we have examined the rate of apoptosis induction in human tumor cells synthesizing both tap and apoptin in comparison with tumor cells synthesizing only tap or apoptin . all 3 examined human tumor cell lines hepg2 , saos - 2 and dohh - 2underwent a significant higher level of apoptosis after co - expression of apoptin and tap proteins in comparison with the cells expressing apoptin or tap separately . the results are shown in fig4 . tap induces apoptosis in cells lacking essential factors of the caspase - regulated apoptosis execution pathway . essential factors in the apoptotic machinery are the specific proteases called caspases ( danen - van oorschot et al ., 2000 and references herein ). apoptin was shown to be able to induce apoptosis in human breast - tumor - derived mcf - 7 cells lacking the essential execution caspase - 3 ( danen - van oorschot et al ., 2000 ). to test if this is also the case for tap , we have transfected human mcf - 7 cells with plasmid pcmv - tap encoding myc - tagged tap protein . as control , the cells were transfected with the plasmid pcmv - lacz encoding the myc - tagged lacz , as was the case for all above described experiments . four or five days after transfection , the cells were fixed and analyzed for myc - tagged tap expression by means of immunofluorescence using the myc - tag specific is antibody 9e10 and apoptotic activity by dapi staining . a significant amount of mcf - 7 cells were stained aberrantly with dapi whereas the cells containing lacz were not , which is indicative for induction of tap - specific apoptosis . the level of apoptosis was to a comparable level as inp53 - minus saos - 2 cells or p53 - positive u20s cells , which indicates that despite the fact that functional caspase - 3 is absent in these human tumor cells , tap is able to force these cells undergoing apoptosis . tap induces apoptosis in cell lines derived from human lung - tumor and squamous cell carcinomas . next , we have examined whether cell lines derived from human lung tumors and squamous cell carcinoma were sensitive to tap - induced apoptosis . to that end , we have transfected human lung - tumor h1299 cells ( friedlander et al ., 1996 ) and human squamous cell carcinoma u8 cells ( van damme et al ., 1997 ) with plasmid pcmv - tap encoding myc - tagged tap protein . as control , the cells were transfected with the plasmid pcaw - lacz expressing the myc - tagged lacz . four or five days after transfection , the cells were fixed and analyzed for myc - tagged tap expression by means of immunofluorescence using the myc - tag specific antibody 9e10 and apoptotic activity by dapi staining . a significant amount of h1299 and u8 cells were stained aberrantly with dapi whereas the cells containing lacz were not , which is indicative for induction of tap - specific apoptosis . in conclusion , we have provided evidence that expression of tap proteins in various human tumorigenic cells results in induction of apoptosis . therapies based on induction of ( p53 - independent and / or bcl - 2 insensitive and / or caspase - 3 independent ) apoptosis are possible utilizing the tap proteins . combined therapies can be carried out using tap protein and other apoptosis - inducing proteins such as apoptin . another cav - derived protein , which is known to induce apoptosis and also known to enhance apoptin activity is vp2 ( noteborn et al ., 1997 ). an efficient anti - tumor therapy without obvious side effects is by far the preferable one . therefore , we have examined whether tap can induce apoptosis in normal human fibroblasts . in addition , we have also analyzed the tap apoptosis activity in rat primary hepatocytes . to that end , we have transfected human vh10 fibroblasts ( danen - van oorschot et al . 1997 ) with plasmid pcmv - tap encoding myc - tagged tap protein . as control , the cells were transfected with the plasmid pcmv - lacz encoding myc - tagged lacz or with pcmv - apoptin encoding apoptin ( negative controls ). four or five days after transfection , the cells were fixed as described above . in parallel , human transformed nw18 fibroblasts ( danen - van oorschot al ., 1997 ) were examined for tap - induced apoptosis as described for the human normal fibroblasts . by means of micro - injection of rat primary hepatocytes with plasmids pcmv - tap , pcmv - apoptin or pcmv - lacz , we examined whether tap was able to induce apoptosis in these normal rat hepatocytes . in parallel , human transformed hepg2 cells were microinjected with the above described43plasmids encoding myc - tagged tap , apoptin or myc - tagged lacz protein , respectively . six and 24 hours after micro - injection the cells were fixed . the cells were analyzed for myc - tagged tap or myc - tagged lacz expression by means of immunofluorescence using the myc - tag specific antibody 9e10 or the apoptin expression by using the apoptin - specific antibody 111 . 3 and apoptotic activity by dapi staining . the human vh10 fibroblasts and rat hepatocytes containing tap protein were stained aberrantly with dapi to a background level as the cells containing lacz or apoptin , whereas the transformed / tumorigenic controls samples nw18 and hepg2 cells clearly showed high levels of apoptosis induction when synthesizing tap or apoptin but not when produce / contain lacz . this data are indicating that tap does not or at least not significantly induce apoptosis in the human and rodent normal cells . for the production of polyclonal antibodies against tap proteins three putative immunogenic peptides were synthesized . n - terminus - thplpvpsetcpsdg - c - terminus ; n - terminus - crlsrlrqrrpeieh - c - terminus ; n - terminus - ctqtmeapledpktqt - c - terminus ; ( eurogentec sa , seraing , belgium ). subsequently , co - workers of eurogentec injected two rabbits with the tap - specific peptides according to standard procedures . the serum derived from the rabbits injected with the tap peptides was shown to recognize specifically for in this report described tap products by means of immunofluorescence and western - blot assays . these results imply that we have generated specific antibodies , which can be used for detecting our novel tap protein . furthermore , this antibody forms the basis for a diagnostic test in detecting tap in sera of ttv - infected individuals , which is indicative for a ttv strain with apoptotic activity . functional domains of tap although , tap does not share overall sequence similarities with any known protein sequence and in particular with the cav - derived apoptosis - inducing protein apoptin , it also has apoptotic activity in human tumor cells . tap harbors putative sequences resembling a nuclear location signal or a nuclear export signal as indicated in fig2 . furthermore , tap has a relatively high percentage (& gt ; 8 %) of proline residues . computer analysis revealed a significant structural homology among tap and apoptin , which might explain the similarity in their activity . 1 . bellamy , c . o . c ., malcomson , r . d . g ., harrison , d . j ., and wyllie , h . 1995 . cell death and disease : the biology and regulation of apoptosis . seminars in cancer biology 6 , 3 2 . charlton , m ., adjei , p ., poterucha , j ., zein , n ., moore , b ., therneau t ., krom , r ., and wiesner , r . 1998 . tt - 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