Patent Application: US-76417601-A

Abstract:
the invention relates to the field of apoptosis . the invention provides novel therapeautic 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 nonfunctional .

Description:
we have used the yeast - 2 hybrid system ( durfee et al ., 1993 ) to identify apoptin - associating cellular compounds , which are essential in the induction of apoptosis . the used system is an in vivo strategy to identify human proteins capable of physically associating with apoptin . it has been used to screen cdna libraries for clones encoding proteins capable of binding to a protein of interest ( fields and song , 1989 ; yang et al ., 1992 ). the invention provides a , for example , novel apoptin - associating protein , which is named apoptin - associating protein 2 abbreviated as aap - 2 . the invention also provides a method for inducing apoptosis through interference with the function of this newly discovered aap - 2 protein or functional equivalents or fragments thereof and / or the induction of apoptosis by means of ( over ) expression of aap - 2 or related gene or functional equivalents or fragments thereof . in addition , the invention also provides another apoptin - associating protein , named aap - 3 . the invention also provides an antitumor therapy based on the interference with the function of aap - 2 - like proteins and / or its ( over ) expression . an aberrant high level of aap - 2 - like proteins will result in the induction of the opposite process of cell transformation , namely apoptosis . the invention furthermore provides a mediator of apoptin - induced apoptosis , which is tumorspecific . the invention provides a therapy for cancer , autoimmune diseases or related diseases which is based on aap - 2 - like proteins alone or in combination with apoptin and / or apoptin - like compounds . for the construction of the bait plasmid , which enables the identification of apoptin - associating proteins by means of a yeast - two - hybrid system , plasmid pet - 16b - vp3 ( noteborn , unpublished results ) was treated with ndel and bamhi . the 0 . 4 kb ndei - bamhi dna fragment was isolated from low - melting - point agarose . plasmid pgbt9 ( clontech laboratories , inc ., palo alto , usa ) was treated with the restriction enzymes ecori and bamhi . the about 5 . 4 - kb dna fragment was isolated and ligated to an ecori - ndei linker and the 0 . 4 - kb dna fragment containing the apoptin - encoding sequences starting from its own atg - initiation codon . the final construct containing a fusion gene of the gal4 - binding domain sequence and apoptin under the regulation of the yeast promoter adh was called pgbt - vp3 and was proven to be correct by restriction - enzyme analysis and dna - sequencing according to the sanger method ( 1977 ). all cloning steps were essentially carried out as described by maniatis et al . ( 1992 ). the plasmid pgbt - vp3 was purified by centrifugation in a cscl gradient and column chromatography in sephacryl s500 ( pharmacia ). the expression vector pact , containing the cdnas from epstein - barr - virus - transformed human b cells fused to the gal4 transcriptional activation domain , was used for detecting apoptin - associating proteins . the pact c - dna library is derived from the lambda - act cdna library , as described by durfee et al . 1993 . the e . coli strain jm109 was the transformation recipient for the plasmid pgbt9 and pgbt - vp3 . the bacterial strain electromax / dh10b was used for the transformation needed for the recovery of the apoptin - associating pact - cdnas and was obtained from gibco - brl , usa . the yeast strain y190 was used for screening the cdna library and all other transformations , which are part of the used yeast - two - hybrid system . for drug selections , luria broth ( lb ) plates for e . coli were supplemented with ampicillin ( 50 microgram per ml ). yeast ypd and sc media were prepared as described by rose et al . ( 1990 ). transformation of competent yeast strain y190 with plasmids pgbt - vp3 and pact - cdna and screening for beta - galactosidase activity the yeast strain y190 was made competent and transformed according to the methods described by klebe et al . ( 1983 ). the yeast cells were first transformed with pgbt - vp3 and subsequently transformed with pact - cdna , and these transformed yeast cells were grown on histidine - minus plates , also lacking leucine and tryptophan . hybond - n filters were layed on yeast colonies , which were histidine - positive and allowed to wet completely . the filters were lifted and submerged in liquid nitrogen to permeabilize the yeast cells . the filters were thawed and layed with the colony side up on whattman 3mm paper in a petridish with z - buffer ( per liter : 16 . 1 gr na 2 hpo 4 . 7h 2 o , 5 . 5 gr nah 2 po 4 . h 2 o , 0 . 75 gr kcl and 0 , 246 gr mgso 4 . 7h 2 o , ph 7 . 0 ) containing 0 . 27 % beta - mercapto - ethanol and 1 mg / ml x - gal . the filters were incubated for at least 15 minutes or over night . total dna from yeast cells , which were histidine - and beta - galactosidase - positive , was prepared by using the glusulase - alkaline lysis method as described by hoffman and winston ( 1987 ) and used to transform electromax / dh10b bacteria via electroporation using a bio - rad genepulser according the manufacturer &# 39 ; s specifications . transformants were plated on lb media containing the antibiotic agent ampicillin . by means of colony - filter assay , the colonies were lysed and hybridized to a radioactive - labeled 17 - mer oligomer , which is specific for pact ( see also section sequence analysis ). plasmid dna was isolated from the pact - clones and by means of xhoi digestion analysed for the presence of a cdna insert . the subclone containing the sequence encoding the apoptin - associating protein was partially sequenced using dideoxy ntps according to the sanger - method , which was performed by eurogentec , seraing , belgium the used sequencing primer was a pact - specific 17 - mer comprising the dna - sequence 5 ′- taccactacaatggatg - 3 ′ ( seq id no . 11 ). the sequences of the apoptin - associating cdnas were compared with known gene sequences from the embl / genbank . in order to generate polyclonal antisera against the aap - 2 and aap - 3 protein , we designed three peptides per protein . these peptides were for aap - 2 : the numbers in parenthesis correspond respectively to the amino acid sequences of fig3 ( seq . id no . 3 ) and fig6 ( seq . id no . 5 ). these peptides were synthesized at eurogentec ( belgium ) with the standard addition of a c - terminal or n - terminal cysteine residue and all subsequent antibody syntheses were also performed there . these peptides were coupled to keyhole limpet hemocyanin ( klh ) and injected as a cocktail into two separate specific pathogen free rabbits with an immunization schedule of one injection and three subsequent boosts . blood samples were taken before and after immunization . the sera were tested in - house for specific reactivity to the peptide cocktail by elisa . the titers from each rabbit were high (& gt ; 200 , 000 ). furthermore , for certain subsequent purposes , the aap - 2 and aap - 3 antibodies were immune - purified using peptide cocktail coupled to immobilized diaminodipropylamine agarose columns ( pierce ) according to the manufacturer &# 39 ; s protocol . the best aap - 2 and aap - 3 antibody preparation of the two generated was selected for further use . we tested the efficacy of this antibody by transfecting 6 cm plates of subconfluent primate cos - 7 and human u 2 os cells using the calcium phosphate coprecipitation method with 5 μg of the aap - 2 - myc or aap - 3 - myc plasmid dna construct and , as a control , untransfected cells . two days post - transfection , cells were washed briefly in pbs , lysed in ripa buffer ( 10 mm tris 7 . 5 , 150 mm nacl , 0 . 1 % sds , 1 . 0 % np - 49 and 1 . 0 % sodium deoxycholate ), clarified by centrifugation , and the supernatant fractionated on sds - denaturing polyacrylamide gel electrophoresis . proteins were western - transferred to pvdf membranes ( immobilon , millipore ) using standard methodology . membranes were blocked in 5 % nonfat dry milk in tris - buffered saline containing 0 . 1 % tween - 20 , then incubated in the unpurified aap - 2 or aap - 3 antisera at a concentration of 1 : 5000 . after a brief wash , membranes were further incubated in hrp - conjugated goat - anti - rabbit ig at a concentration of 1 : 2000 . after a thorough series of wash steps , proteins were detected using enhanced chemiluminescence ( amersham ) according to the manufacturer &# 39 ; s protocol and exposed to x - ray film and developed using standard automated machinery . in addition , we tested the purified aap - 2 and aap - 3 antibody using immunoprecipitation in a manner the same as above , except that after centrifugation , the supernatant was added to 10 ul of aap - 2 or aap - 3 antibody precoupled to protein - a - sepharose beads , incubated for 1 hour with tumbling , then washed before fractionation on sds - page gels and western analysis . detection in this case was performed with the anti - myc tag monoclonal antibody 9e10 ( evan et al . 1985 ). finally , the purified antibody was tested for utility in immunofluorescence by including glass coverslips in the above transfections . coverslips were fixed with 4 % paraformaldehyde , blocked with normal goat serum , incubated in aap - 2 or aap - 3 antibody diluted 1 : 5 , washed , incubated in fitc - conjugated goat - anti - rabbit ig , mounted and visualized under fluorescence microscopy . apoptin induces specifically apoptosis in transformed cells , such as cell lines derived from human tumors . to identify the essential compounds in this cell - transformation - specific and / or tumor - specific apoptosis pathway , a yeast genetic screen was carried out . we have used a human cdna library , which is based on the plasmid vector pact containing the complete cdna copies made from epstein - barr virus - transformed human b cells ( durfee et al ., 1993 ). construction of a bait plasmid expressing a fusion gene product of gal4 - dna - binding domain and apoptin to examine the existence of apoptin - associating proteins in the human transformed / tumorigenic cdna library , a so - called bait plasmid had to be constructed . to that end , the complete apoptin - encoding region , flanked by about 40 basepairs downstream from the apoptin gene , was cloned in the multiple cloning site of plasmid pgbt9 . the final construct , called pgbt - vp3 , was analyzed by restriction - enzyme analysis and sequencing of the fusion area between apoptin and the gal4 - dna - binding domain . a gene ( fragment ) encoding an apoptin - associating protein is determined by transactivation of a gal4 - responsive promoter in yeast the apoptin gene is fused to the gal4 - dna - binding domain of plasmid pgbt - vp3 , whereas all cdnas derived from the transformed human b cells are fused to the gal4 - activation domain of plasmid pact . if one of the proteinaceous substances encoded by said cdnas binds to apoptin , the gal4 - dna - binding domain will be in the vicinity of the gal4 - activation domain resulting in the activation of the gal4 - responsive promoter , which regulates the reporter genes his3 and lacz . the yeast clones containing plasmid expressing apoptin and a plasmid expressing an apoptin - associating protein fragment can grow on a histidine - minus medium and will stain blue in a beta - galactosidase assay . subsequently , the plasmid with the cdna insert encoding the apoptin - associating protein can be isolated and characterized . before we could do so , however , we have determined that transformation of yeast cells with pgbt - vp3 plasmid alone , or in combination with an empty pact vector , did not result in the activation of the gal4 - responsive promoter . identification of apoptin - associating protein encoded by cdna derived from a human transformed b cell line we have found two independent yeast colonies , which upon transformation with pgbt - vp3 and pact - cdna were able to grow on a histidine - minus medium ( also lacking leucine and tryptophan ) and stained blue in a beta - galactosidase assay . these results indicate that the observed yeast colonies contain , besides the bait plasmid pgbt - vp3 , a pact plasmid encoding a potential apoptin - associating protein . plasmid dna was isolated from the positive yeast colony , which was transformed in bacteria . by means of a filter - hybridization assay using a pact - specific labeled dna - probe , 2 independent clones containing pact plasmid could be determined . subsequently , pact dna was isolated and digested with restriction enzyme xhoi , which resulted in the presence of a 1 . 1 - kbp ( clone i ) and a 1 . 3 - kbp ( clone ii ) cdna insert , respectively . finally , the pact plasmids containing the two independent cdna inserts were partially sequenced by using the sanger method ( sanger et al ., 1977 ). the yeast genetic screen for apoptin - associating proteins resulted in the detection of two cdna clones comprising a single type of protein , namely a novel protein called apoptin - associating protein 2 , abbreviated as aap - 2 . the determined dna sequence part of the aap - 2 cdna clones aap - 2 - i and aap - 2 - ii are shown in fig1 ( seq . id no . 1 ) and 2 ( seq . id no . 2 ), respectively . the amino acid sequence , derived from the detected dna sequence of clone aap - 2 - ii is given in fig3 ( seq . id no . 3 ). below the experiments will be described for aap - 2 - ii , which will be referred as aap - 2 . construction of an expression vector for the identification of aap - 2 protein in mammalian cells to study whether the cloned cdna aap - 2 indeed encode ( apoptin - associating ) a protein product , we have carried out the following experiments . the dna plasmid pmt2sm contains the adenovirus 5 major late promoter ( mlp ) and the sv40 ori enabling high levels of expression of foreign genes in transformed mammalian cells , such as sv - 40 - transformed cos cells . furthermore , the pmt2sm vector contains a myc - tag ( amino acids : eqkliseedl ) ( seq . id no . 18 ) which is in frame with the foreign - gene product . this myc - tag enables the recognition of , e . g ., apoptin - associating proteins by means of the myc - tag - specific 9e10 antibody . the pmt2sm vector expressing myc - tagged aap - 2 cdna was constructed as follows . the pact - aap - 2 cdna clone was digested with the restriction enzyme xhoi and the cdna insert was isolated . the expression vector pmt2sm was digested with xhol and treated with calf intestine alkaline phosphatase and ligated to the isolated aap - 2 cdna inserts . by sequence analysis , the pmt2sm constructs containing the aap - 2 cdna in the correct orientation was identified . the synthesis of myc - tagged aap - 2 protein was analyzed by transfection of cos cells with plasmid pmt2sm - aap - 2 . 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 antibody 9e10 . the cos cells transfected with pmt2sm - aap - 2 were proven to synthesize a specific myc - tagged aap - 2 product with the size of approximately 70 kda 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 isolate a cdna that is able to produce a protein product with the ability to associate to the apoptosis - inducing protein apoptin . coimmunoprecipitation of myc - tagged aap - 2 protein with apoptin in a transformed mammalian cell system next , we have analyzed the association of apoptin and the aap - 2 protein by means of coimmunoprecipitations using the myc - tag - specific antibody 9e10 . the 9e10 antibodies were shown not to bind directly to apoptin , which enables the use of 9e10 for carrying out coimmunoprecipitations with ( myc - tagged ) apoptin - associating proteins and apoptin . to that end , cos cells were cotransfected with plasmid pcmv - vp3 encoding apoptin and with plasmid pmt2sm - aap - 2 . as a negative control , cells were transfected with pcmv - vp3 expressing apoptin and a plasmid pcdna3 . 1 . lacz - myc / his - lacz encoding the myc - tagged betagalactosidase , which does not associate with apoptin . two days after transfection , the cells were lysed in a buffer consisting of 50 mm tris ( 7 . 5 ), 250 mm nacl , 5 mm edta , 0 . 1 % triton × 100 , 1 mg / ml na 4 p 2 o 7 and freshly added protease inhibitors such as pmsf , trypsine - inhibitor , leupeptine and na 3 vo 4 . the specific proteins were immunoprecipitated as described by noteborn et al . ( 1998 ) using the myc - tag - specific antibodies 9e10 and analyzed by western blotting . staining of the western blot with 9e10 antibodies and 111 . 3 antibodies , which are specifically directed against myc - tag and apoptin , respectively , showed that the “ total ” cell lysates contained the 16 - kda apoptin product and the myc - tagged aap - 2 protein . by means of a specific lacz polyclonal antibody , the beta - galactosidase product could be visualized . immunoprecipitation of the myc - tagged aap - 2 products was accompanied by the immunoprecipitation of apoptin product of 16 kda . in contrast , immunoprecipitation of myc - tagged betagalactosidase did not result in a detectable coprecipitation of the apoptin protein . in addition , immunoprecipitation of the apoptin protein , by means of a polyclonal antibody directed against the c - terminal part of apoptin ( noteborn and danen , unpublished results ) was accompanied by the immunoprecipitation of the aap - 2 product of approximately 70 - kda , but not by beta - galactosidase protein . in total , three independent immunoprecipitation experiments were carried out , which all showed the specific associating ability of apoptin protein to the aap - 2 protein . these results indicate that the novel determined aap - 2 protein is able to specifically associate with apoptin not only in the yeast background , but also in a mammalian transformed cellular system . over - expression of the novel aap - 2 protein in human transformed cells induces the apoptotic process in addition , we have examined whether aap - 2 carries apoptotic activity . first , we have analyzed the cellular localization of the novel aap - 2 protein in human transformed cells . to that end , the human osteosarcoma - derived saos - 2 cells were transfected , as described by danen - van oorschot ( 1997 ), with plasmid pmt2sm - aap - 2 encoding the myc - tagged aap - 2 protein , respectively . by indirect immunofluorescence using the myc - tag - specific antibody 9e10 and dapi , which stains the nuclear dna , it was shown that aap - 2 protein was mainly present in the nucleus of most of the tumor cells and in a minor part of the cells both in the nucleus and cytoplasm or cytoplasm alone . these features suggest that , at least in human tumor cells , aap - 2 is involved in nuclear transport processes . already , three days after transfection , a significant amount of saos - 2 cells synthesizing aap - 2 underwent induction of apoptosis . these aap - 2 - positive cells were aberrantly stained with dapi , which is indicative for induction of apoptosis ( telford , 1992 , danen - van oorschot , 1997 ). cells expressing apoptin also underwent apoptosis , whereas as expected the cells synthesizing the nonapoptotic betagalactosidase ( lacz ) protein did not . coexpression of apoptin and aap - 2 protein in human tumor cells , such as saos - 2 cells , results in a slightly faster apoptotic process than as with the expression of apoptin or aap - 2 protein alone . the results are shown in fig4 . the fact that aap - 2 protein can induce apoptosis in p53 - minus saos - 2 cells indicates that aap - 2 can induce p53 - independent apoptosis . these results imply that aap - 2 can be used as an antitumor agent in cases where other ( chemo ) therapeutic agents will fail . furthermore , the finding that both apoptin and aap - 2 induce a p53 - independent pathway indicates that aap - 2 fits in the apoptin - induced apoptotic pathway . in conclusion , we have identified an apoptin - associating protein , namely the novel aap - 2 protein , which is mainly present in the nucleus and able to induce ( p53 - independent ) apoptosis in human tumor cells . next , we have examined whether aap - 2 behaves similar in normal human diploid nontransformed cells as has been found for aap - 2 in human tumor cells . to that end , human diploid vh10 fibroblasts ( danen - van oorschot , 1997 ) were transfected using fugene according the protocol of the supplier ( roche , almere , the netherlands ) with plasmid pmt2sm - aap - 2b encoding the myc - tagged aap protein . in parallel , human tumor - derived saos - 2 cells were also transfected with plasmid pmt2sm - aap - 2 . three days after transfection , the cells were harvested and analyzed by indirect immunofluorescence using the myc - tag - specific antibody 9e10 . within the majority of aap - 2 - positive human diploid cells , aap - 2 is located in the cytoplasm only or both in the nucleus and cytoplasm as expected , in most of the human tumor saos - 2 cells , aap - 2 is only located in the nucleus . furthermore , the aap - 2 - positive human normal diploid fibroblasts did not show a sign of aap - 2 - induced apoptosis , as was examined by dapi staining ( see above ). in conclusion , we have identified an apoptin - associating protein , namely aap - 2 , which has a tumor - specific preference for induction of apoptosis and nuclear accumulation . a further sequence analysis of the human aap - 2 nucleic acid sequence yielded the 5331 bp long nucleic acid sequence given in fig7 a - 7d ( seq . id no . 6 ). an open reading frame was found in this nucleic acid sequence at position 300 - 4499 . the deduced amino acid sequence is given in fig8 ( seq . id no . 7 ). a protein domain called phd - finger was found in the amino acid sequence of the human aap - 2 protein . it spans the region of amino acid 852 to amino acid 900 . the cys 4 - his - cys 3 zinc - finger - like motif which is characteristic for a phd - finger domain ( r . aasland et al ., 1995 ; tibs 20 , 56 - 59 ) is found in said region ( see , fig9 ). the phd - finger is found in nuclear proteins thought to be involved in chromatin - mediated transcriptional regulation . the phd - finger was originally identified in a set of proteins that includes members of the drosophila polycomb and trithorax group genes . these genes regulate the expression of the homeotic genes through a mechanism thought to involve some aspect of chromatin structure . other proteins which have this motif also have additional domains or characteristics that support that suggestion that the phd - finger is involved in chromatin - mediated gene regulation . phd - fingers are thought to be protein - protein interaction domains . such protein - protein interactions are important for , e . g ., the activity of multicomponent complexes involved in transcriptional activation or repression . phd - fingers may also recognize a family of related targets in the nucleus such as the nucleosomal histone tails ( r . aasland et al ., 1995 ; tibs 20 , 56 - 59 ). phd - finger domains are also found in a number of proteins closely associated with human tumorigenisis such as hrx / all1 / mll / htrx , cbp , moz , all of which are part of aberrant fusion proteins derived from chromosomal translocations found in a high percentage of human leukemias ( for review see jacobson and pillus , 1999 ; current opinion in genetics & amp ; dev . 9 , 175 - 184 ). other phd - finger domain - containing proteins are overexpressed in certain tumor types ( lu , p . j . et al , 1999 ; j . biol . chem . 274 , 15633 - 45 ). therefore , interfering with the functional activity of the phd - finger domain of aap - 2 should have therapeutic effects against human tumors . the phd - finger domain can be used to identify substances which bind to the phd - finger domain . this can be done by methods known to persons skilled in the art , e . g ., by binding studies , where an aap - 2 peptide comprising the phd - finger domain is bound to a matrix and it is tested whether test substances bind to the aap - 2 peptide or by coimmunoprecipitation of an aap - 2 peptide comprising the phd - finger domain with test substances using antibodies generated against the aap - 2 peptide comprising the phd - finger domain . test substances may be small organic compounds derived , e . g ., from a compound library or peptides or proteins derived , e . g ., from a peptide library or from a natural source like a cell extract . the test substances may be labeled for easier detection . the substances found to bind to the phd - finger domain may either enhance or inhibit one or more effects of aap - 2 . this can be tested by measuring the apoptotic activity of aap - 2 as described above in the presence of said substances and by determining the nuclear localization of aap - 2 as described above in the presence of said substances . the genetic yeast screen with pgbt - vp3 as bait plasmid and pact plasmid containing cdnas from transformed human b cells also delivered the novel gene apoptin - associating protein 3 ( aap - 3 ). the dna sequence of the aap - 3 is shown in fig5 whereas the aap - 3 cdna - encoded amino - acid sequence is shown in fig6 . to analyze into further detail the associating properties of apoptin and this aap - 3 protein , we have expressed a myc - tagged aap - 3 cdna by means of the psm2nt vector ( as described for aap - 2 ) in transformed mammalian cos cells . western blot analysis using the myc - tag - specific antibodies 9e10 showed a specific ( myc - tagged ) aap - 3 protein of approximately 22 - kda . this major 22 - kda aap - 3 product is accompanied by smaller and larger minor aap - 3 - specific products . these results indicate that the isolated cdna indeed encodes a protein of the expected size . next , immunoprecipitation assays were carried out with transiently transfected cos cells cosynthesizing myc - tagged aap - 3 and apoptin . the results clearly showed that both 9e10 antibodies and apoptin - specific 111 . 3 antibodies precipitate aap - 3 protein and apoptin , which indicates that apoptin associates with this new aap - 3 protein in a mammalian transformed background . in total , three independent immunoprecipitation experiments were carried out , which all showed the associating ability of apoptin to the aap - 3 protein . immunofluorescence assays of human transformed saos - 2 cells and normal diploid vh10 fibroblasts expressing aap - 3 revealed that aap - 3 is located in both cell types predominantly in the cytoplasm and nucleus , but in lower percentages also mainly in the nucleus or mainly in the cytoplasm . cosynthesis of aap - 3 and apoptin in both cell types showed a clear perinuclear colocalization of aap - 3 and apoptin . tumor cells that have become apoptotic showed a nuclear localization of apoptin and a perinuclear stainings pattern of aap - 3 . as expected , normal diploid vh10 cells synthesizing both apoptin and aap - 3 did not undergo apoptosis . these data indicate that aap - 3 will release apoptin when the cell has become tumorigenic and / or transformed , resulting in the nuclear localization of apoptin and induction of apoptosis . in summary , our findings prove that our newly discovered aap - 3 protein is able to associate to the tumor - specific apoptosis - inducing protein apoptin in both a yeast and mammalian cellular background . therefore , this aap - 3 protein plays an important role in the induction of ( apoptin - regulated ) tumors - specific apoptosis . the best aap - 2 and aap - 3 antibody preparations obtained from the two rabbit derived antisera were selected for further use . we tested the efficacy of these antibody preparations against aap - 2 and aap - 3 , respectively , by transfecting primate cos - 7 and human u 2 os cells with the aap - 2 - myc or aap - 3 - myc construct . western analysis showed that the approximately 70 kda aap - 2 - myc protein and the approximately 22 kda aap - 3 - myc were detected strongly only in samples where the dna was transfected . similarly , in immunoprecipitation experiments , aap - 2 - myc or aap - 3 - myc protein was also strongly detected . finally , localization of aap - 2 - myc or aap - 3 - myc protein in a cell using the aap - 2 or aap - 3 antibody could be determined by immunofluorescence analysis . the genetic yeast screen with pgbt - vp3 as bait plasmid and pact plasmid containing cdnas from transformed human b cells also delivered another gene , which also encodes an apoptin - associating protein . this apoptin - associating protein was called aap4 ( see copending application ep00204396 . 6 , which is incorporated herein by reference ). the nucleic acid sequence of aap - 4 is shown in fig1 a - 10d ( seq . id no . 9 ). an open reading frame was found in this nucleic acid sequence at position 236 to 2866 . the deduced amino acid sequence is given in fig1 ( seq . id no . 10 ). just like aap - 2 and aap - 3 , aap4 is able to associate with apoptin not only in the yeast background , but also in a mammalian transformed cellular system . furthermore , this protein is present in the nucleus and able to induce ( p53 - independent ) apoptosis in human tumor cells . a functional equivalent or a functional fragment of aap - 4 is herein also included . a functional equivalent or a functional fragment of aap - 4 is a derivative or a fragment having the same kind of activity possibly in different amounts . it is clear to a person skilled in the art that there are different ways of arriving at a functional equivalent or functional fragment . a functional equivalent can , for example , be a point mutant or a deletion mutant or a equivalent derived from another species . another way to arrive at a functional equivalent is a molecular evolution of equivalents and / or fragments having the same kind of activity possibly in different amounts . to study whether two separate apoptin - associating proteins can not only bind to apoptin but also to another apoptin - associating protein , we carried out the following experiment . immunoprecipitation assays were carried out with transiently transfected cos cells cosynthesizing myc - tagged aap - 3 and myc - tagged aap - 4 . the results clearly showed that antibodies directed against aap - 3 and antibodies directed against aap - 4 both precipitate aap - 3 and aap - 4 , which suggests that aap - 3 and aap - 4 associate in this mammalian transformed background . in total , three independent immunoprecipitation experiments were carried out , which all showed the associating ability of aap - 3 and aap - 4 . the fact that two proteins , which showed to be apoptin - associating proteins can independently coassociate in the absence of apoptin strengthens the idea that the aap - 3 / apoptin coassociation is physiologically relevant . based on the present report , we can conclude that the cellular localization of aap - 2 is different in tumorigenic / transformed human cells in comparison to normal human nontransformed cells . furthermore , accumulation of aap - 2 in the nucleus correlates with apoptosis induction , whereas cytoplasmic / nuclear localization correlates with cell viability and normal proliferative capacity . therefore , we are able to develop a diagnostic assay for the identification of ( human ) cancer cells versus normal “ healthy ” nontransformed cells . the assay consists of transfecting “ suspicious ” ( human ) cells , for instance from human origin , with a plasmid encoding aap - 2 or infecting the cells with viral vectors expressing aap - 2 . subsequently , the cells will be examined 1 ) for the ability to undergo apoptosis by the over - expressing aap - 2 gene and 2 ) for a main shift in the localization of aap - 2 from the cytoplasm to the nucleus . the intracellular localization of aap - 2 can be determined , using an immunofluorescence assay with monoclonal antibodies specific for aap - 2 and / or specific for a tag linked to aap - 2 such as the herein described nyc - tag . if the percentage of apoptosis and / or the nuclear localization of aap - 2 in the analyzed cells expressing aap - 2 is significantly higher than in aap - 2 - positive control “ healthy ” cells , one can conclude that the analyzed cells has become tumorgenic / transformed . as positive control known human tumorigenic cells will be used for expressing aap - 2 . coexpression of sv40 large t antigen and aap - 2 results in translocation of aap - 2 and induction of apoptosis we have examined the effect of expression of transforming genes on aap - 2 - induced apoptosis in normal human cells derived from healthy individuals . to that end , human vh10 diploid fibroblasts were transiently cotransfected with plasmid pmt2sm - aap - 2 encoding aap - 2 protein and either plasmid pr - s884 encoding sv40 large t antigen , or the negative - control plasmid pcmv - neo ( noteborn and zhang , 1998 ). by indirect immunofluorescence , the cells were analyzed for aap - 2 - induced apoptosis . the normal vh10 cells did not undergo apoptosis when aap - 2 was transfected with the negative - control plasmid . the results showed , as expected , that expression of aap - 2 is not able to induce apoptosis in normal human diploid cells , confirming the above mentioned data . however , normal diploid human fibroblasts expressing both aap - 2 and sv40 large t antigen underwent aap - 2 - induced apoptosis . the transition of normal human cells , from aap - 2 - resistance to aap - 2 - susceptibility , can probably be explained by the fact that the aap - 2 protein translocates from a cytoplasmic localization to a nuclear localization . this transition becomes apparent already 2 days after transfection of plasmids encoding the transforming protein sv40 large t antigen . one can conclude that an event takes place , in this example due to expression of a transforming product derived from a dna - tumor virus , which results in the translocation of over - expressed aap - 2 from the cytoplasm to the nucleus , which is followed by induction of apoptosis . diagnostic assay for cancer - inducing genes , agents and cancer - proneness based on aap - 2 - induced apoptosis based on the present report , we are able to develop a diagnostic assay for the identification of cancer - inducing and / or transforming agents or genes . a first type of assay consists of transfecting “ normal ” cells , for instance from human origin , with a plasmid encoding aap - 2 , or infecting the cells with viral vectors expressing aap - 2 , together with a plasmid encoding a putative transforming / cancer - inducing gene . subsequently , the cells will be examined 1 ) for the ability to undergo apoptosis by the over - expressing aap - 2 gene and 2 ) for a shift in the localization of aap - 2 from the cytoplasm to the nucleus . the intracellular localization of aap - 2 can be determined using an immunofluorescence assay with monoclonal antibodies specific for aap - 2 and / or specific for a tag linked to aap - 2 such as the herein described myc - tag . if the percentage of apoptosis and / or the nuclear localization of aap - 2 in normal cells coexpressing aap - 2 and the putative transforming / cancer - inducing gene is significantly higher than in aap - 2 - positive control cells expressing a control plasmid , one can conclude that the analyzed gene indeed has transforming / cancer - inducing activity . a second example of a diagnostic test is based on the treatment of cultured normal diploid cells with a putative carcinogenic agent . the agent can be added , for instance , to the culture medium for various lengths of time . subsequently , the cells are transfected with a plasmid encoding aap - 2 . this approach can also be carried out by first transfecting / infecting the normal diploid cells and then treating the cells with the agent to be tested . the subsequent steps of the assay are the same as the first type of diagnostic assay described in this section . if the percentage of apoptosis and / or the nuclear localization of aap - 2 in normal cells expressing aap - 2 and the putative carcinogenic agent is significantly higher than in aap - 2 - positive control cells expressing a control agent , one can conclude that the analyzed agent indeed has transforming / cancer - inducing activity . a third example of a diagnostic test is based on the treatment of cultured normal diploid cells derived from a skin biopsy of the potential cancer - prone individual to be tested and cultured in suitable medium . next , the cells are irradiated with uv and subsequently transfected with a plasmid encoding aap - 2 or infected with a viral vector expressing aap - 2 or the cells are first transfected and / or infected and then irradiated . in parallel , diploid cells from a normal healthy individual will be used as a control . the subsequent steps of the assay are the same as the first type of diagnostic assay described in this section . if after uv - treatment the percentage of apoptosis and / or the nuclear localization of aap - 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