Patent Abstract:
the present invention relates to the treatment of epidermal growth factor - family receptor - dependent tumors . more specifically , the present invention relates to the use of abin for the preparation of a medicament to inhibit epidermal growth factor - induced proliferation , and to treat erbb - dependent tumors .

Detailed Description:
embryonic kidney epithelial hek293t cells were grown in 24 - well plates ( 50 , 000 cells / well ) in dmem supplemented with 10 % fetal calf serum , 2 mm l - glutamine , 0 . 4 mm sodium pyruvate and antibiotics , and transiently transfected by dna calcium phosphate coprecipitation with the following plasmids : an egfr - encoding plasmid ( pmt2 - egfr obtained from dr . roovers ( university of utrecht ), transfected at 200 ng / μg total dna ), pnfconluc ( 100 ng / μg ), encoding the luciferase reporter gene driven by a minimal nf - κb - responsive promoter ( kimura et al ., 1986 ), and put651 ( 100 ng / μg ) encoding β - galactosidase driven by the constitutively active cmv promoter ( eurogentec , seraing , belgium ). the day after transfection , cells were either non - stimulated or stimulated for 24 hours with 100 ng / ml egf . cells were subsequently lysed in lysis buffer ( 25 mm tris - phosphate ph 7 . 8 , 2 mm dithiothreitol , 2 mm 1 , 2 - cyclohexaminediaminetetraacetic acid , 10 % glycerol and 1 % triton x - 100 ). inducible promoter activity was measured by measuring the luciferase and β - galactosidase activity present in cell extracts . luciferase values were normalized for β - galactosidase values in order to correct differences in transfection efficiency . as shown in fig1 , egf addition to egfr - transfected hek293t cells induced nf - κb activation . in a similar way , the effect of her - 2 expression on nf - κb activation was measured . hek293t cells were transfected with increasing amounts of her2 dna . as can be seen in fig2 , the presence of her2 is sufficient to obtain egf - induced nf - κb activation , but the signal is strongly increased when both egfr , as well as her2 , are present . full - length abins , as well as their minimal active domain ( mad ), inhibit egf - mediated nf - κb activation to analyze the nf - κb - inhibiting effects of abins and their minimal active domain ( mad ), egfr - expressing hek293t cells were transiently transfected in 24 - well plates ( 50 , 000 cells / well ) with expression plasmids encoding murine abin - 1 ( 100 ng / μg ), mabin1 - mad ( aa 444 - 601 ) ( 100 ng / μg ), human abin - 1 ( 100 ng / μg ), habin - 1 - mad ( 100 ng / μg ), habin - 2 ( 100 ng / μg ) or habin - 3 ( 100 ng / μg ). after transfection , cell were serum - starved for 24 hours in serum - free medium supplemented with its ( insulin - transferrin - selenium supplement obtained from invitrogen ), after which they were either non - stimulated or stimulated for six hours with 100 ng / ml egf . the effect of abin on egf - induced activation of nf - κb was studied by luciferase reporter tests . as shown in fig3 , all full - length abins inhibit egf - mediated nf - κb activation . a dominant - negative kinase - defective mutant of ikkβ , ikkb - dn , thereby strongly repressing nf - κb activation , was used as a positive control ( 100 ng / μg ). overexpression of the minimal active domains of mabin - 1 ( 50 ng / μg ) and habin - 1 ( 100 ng / μg ) resulted in a similar inhibition of egf - mediated nf - κb activation as that provided by their full - length counterparts ( fig4 ). in a similar way , the different abins , as well as their minimal active domain , strongly repressed the egf - induced activation of nf - κb when both egfr and her - 2 were present as receptors ( fig5 ). abins and their mads inhibit egf - mediated nf - κb activation in a431 human carcinoma cells a431 carcinoma cells were grown in dmem supplemented with 10 % fetal calf serum , 2 mm l - glutamine , 0 . 4 mm sodium pyruvate , 1 mm non - essential amino acids , 100 iu / ml penicillin and 0 . 1 mg / ml streptomycin . to determine whether or not abins and their mad domains can block nf - κb activity following egf treatment of human epidermoid carcinoma a431 cells , which overexpress egf receptors ( 2 × 10 6 receptors per cell ), a431 cells were seeded in six - well plates ( 150 , 000 cells / well ). the following day , cells were transiently transfected with expression vectors encoding the different abin constructs ( 2 μg / well ) and their effect on egf - induced nf - κb activation was studied by luciferase reporter assays ( both pnfconluc and put651 were applied at 2 μg / well ). forty - eight hours after transfection , cells were serum starved overnight ( serum - free medium supplemented in its ). cells were then either stimulated with 1 ng / ml egf for six hours or left untreated . overexpression of both habin - 1 and habin - 2 clearly inhibited egf - mediated nf - κb activation in a431 cells ( fig6 ). habin1 - mad was even more potent in inhibiting nf - κb activity in a431 cells upon egf stimulation , as compared to full - length habin - 1 . as expected , ikkb dn strongly inhibited nf - κb activity . adenoviral expression of abin inhibits the proliferative capacity of a431 human vulvar squamous carcinoma and human androgen - insensitive du145 prostate cancer cells recombinant adenoviruses for mabin - 1 were prepared as described previously ( el bakkouri et al ., 2005 ). briefly , the murine abin - 1 cdna , n - terminally fused to an e - tag , was amplified via pcr with a forward 5 ′- cgggatccgccatgggtgcgccggtgcc - 3 ′ primer and reverse 5 ′- ccccaagcttaaatgacccactgcagcc - 3 ′ primer . a recombinant adenoviral vector adabin - 1 was generated by cloning the abin - 1 pcr fragment into a bamhi and hindiii opened pacplpa . cmv shuttle vector ( gomez - foix et al ., 1992 ) and co - transfected with the rescue plasmid pjm17 ( mcgrory et al ., 1988 ) ( which encodes the adenovirus dl309 genome , lacking e1 and e3 functions ) into hek293 cells via calcium phosphate coprecipitation . recombinant plaques were isolated , extracted dna was verified via pcr , and expression of the correct transgene from the ubiquitously active cytomegalovirus ( cmv ) promoter was confirmed by means of western blotting . recombinant adenoviruses for the other abins were prepared in a similar way . control viruses without transgene ( adrr5 ) or expressing the β - galactosidase gene ( adlacz ), and a virus expressing the iκbα super - repressor ( adiκbα s ) were generated with the same pjm17 adenoviral backbone vector . the iκbα super - repressor means a nondegradable mutant form of iκb - α with s32a and s36a mutations ( grempler et al ., 2004 ). the latter locks nf - κb in a cytosolic protein complex , preventing its nuclear action . high titer virus stocks were prepared in hek293 cells and purified via single cscl banding . titers were determined via plaque assay in hek293 cells and calculated as plaque - forming units ( pfu ) per ml virus stock . a431 and du145 cells were maintained in dmem supplemented with 10 % fetal calf serum , 2 mm l - glutamine , 0 . 4 mm sodium pyruvate , 1 mm nonessential amino acids , 100 iu / ml penicillin and 0 . 1 mg / ml streptomycin . du145 cells ( 2 × 10 5 egf receptors per cell ; macdonald et al ., 1990 ) and a431 cells ( 2 × 10 6 egf receptors per cell ; haigler et al ., 1978 ) were adenovirally transfected with 200 moi of mabin - 1 , habin - 1 , habin - 3 , the iκbα super - repressor ( iκb - sr ), lacz , or the empty vector rr5 , and their effects on the proliferation of the cancer cells was monitored via incorporation of 3 h - 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