Patent Application: US-4308308-A

Abstract:
the present invention relates to methods for identifying pre - neoplastic and neoplastic states in mammals and in particular to a method for identifying pre - neoplasia and neoplasia in cells and tissues based on differential expression of p2y purineric receptors .

Description:
the present invention is based on the finding that pre - neoplastic and - neoplastic cells differentially express p2y purinergic receptors . this differential expression can therefore be used to identify pre - neoplastic or neoplastic cells . further , because the p2y purinergic receptors are distributed differently in cells at different stages of pre - neoplasia and neoplasia , the expression profile of the p2y purinergic receptors can be used to stage pre - neoplastic and neoplastic states . the differential expression patterns of p2y can also be used to determine the aetiology of carcinogenesis . further , the invention includes a kit for diagnosing a pre - neoplastic and / or neoplastic state comprising means for detecting a p2y purinergic receptor expression profile ; a kit for staging a pre - neoplastic and / or neoplastic state comprising means for detecting a p2y purinergic receptor expression profile ; and a kit for determining the aetiology of carcinogenesis comprising means for detecting a p2y purinergic receptor expression profile . preferred embodiments of the invention will now be described by way of example only . the consensus sequences of the human p2y 2 [ 12 ], human p2y 4 [ 13 ], and human p2y 6 [ 14 ] cloned receptors were examined for suitable epitopes following the approach adopted in hansen et al . [ 15 ]. the non - homologous epitope corresponding to the segment leu226 - lys242 in p2y 2 was used , with the comparable sequences in p2y 4 ( arg226 - arg242 ) and p2y 6 ( cys220 - lys236 ). each was attached to diphtheria toxoid via maleimidocaproyl - n - hydroxysuccinimide crosslinker through adding an n - terminal cys to p2y 2 and p2y 4 epitopes . all syntheses were carried out using standard t - boc chemistry on an abi synthesiser [ 16 ]. the peptide - antigen conjugates were suspended in water at 5 mg / ml and aliquots emulsified by mixing with complete freund &# 39 ; s adjuvant . emulsion volumes of 1 ml containing 2 mg of peptide were injected intramuscularly with second , third , fourth and fifth immunisations followed at 2 week intervals using incomplete freund &# 39 ; s adjuvant . final bleeds via venepuncture were obtained at 10 - 12 weeks , after it was established that adequate antibody titres had been obtained in the rabbits or sheep used for each epitope . the blood was incubated at 37 ° c . for 30 min , and stored at 4 ° c . for 15 h after which the serum was collected following centrifugation and stored at − 20 ° c . in small aliquots . sera were tested with an elisa assay for antibodies specific for each peptide . the antibody titre , defined as the reciprocal of the serum dilution resulting in an absorbance of 1 . 0 above background in the elisa assay , was in the range 75 , 000 +− 4 , 000 compared with 225 +− 25 for the pre - immune samples . each antibody was found to be specific against the particular subtype . affinity purification of each of the antibodies against the specific epitope for that antibody resulted in reduced background but identical labelling trends . a monoclonal antibody raised against the same p2y 2 epitope in mice yielded the same results . sections with a thickness of 8 μm were cut from unfixed , frozen tissue using a reichert jung 2800 frigocut cryotome or else were sectioned from paraffin - embedded tissue . sections were air dried at room temperature for 1 hour , fixed for 12 hours in acetone at − 20 ° c . and air dried at room temperature for 1 hour prior to antibody labelling . they were then incubated at room temperature with either a monoclonal mouse , rabbit or sheep anti - p2y 2 primary antibody . after washing , sections were then incubated for 30 min in the secondary antibody : a 1 : 30 dilution of hrp - conjugated goat anti - mouse secondary antibody ( dako ) for mouse primary , brp - conjugated anti - rabbit secondary antibody for rabbit primary , or hrp - conjugated goat anti - sheep secondary antibody . slides were again rinsed and then immersed in 15 % diaminobenzidine tetrahydrochloride ( dab - sigma ) for 10 minutes . sections were rinsed , air dried and mounted in dpx ( merck ). control slides were incubated in diluent buffer during the first incubation and then treated in the same manner as the experimental slides . negative control slides were treated in the same manner as the experimental slides except that the primary antibody was replaced with non - immune serum . in a study of 40 normal and 40 human prostate cancer cases , p2y 2 subtypes were markedly increased in human prostate cancer tissue . there was no observable labelling of normal tissue / benign prostatic hyperplasia . the labelling pattern for p2y 2 in the cancerous tissue was particularly informative in that there was a greater proportion of labelled acinar epithelial cells with each stage of prostate disease , showing a direct correlation between neoplastic transformation and the extent of p2y 2 acinar labelling commencing with the nuclei ( stage 1 ; fig1 ), progressing to extra - nuclear or cytoplasmic punctate labelling ( stage 2 ; fig2 ) and progressing to deposition on the apical epithelium and lateral epithelium ( stage 3 ; fig3 ) in advanced cases where stage 3 is also accompanied by changes in morphology . p2x are fast acting ligand ( atp )- gated ion channels that open the cell to ions , particularly calcium . p2y receptors are much slower acting metabotropic receptors that act on internal cellular calcium stores following activation by atp or utp or even adp by triggering a g - protein coupled cascade of activity and so function entirely differently to p2x receptors . when p2x receptor antibodies were used in combination with p2y receptor antibodies , a similar pattern to that described using p2y receptor antibodies alone was obtained . in particular , labelling of prostate cells / tissues using antibodies against p2y 2 and either or both p2x 1 and p2x 2 receptor subtypes enhanced results obtained using antibodies against the p2y 2 receptor alone . the consensus sequences of the human p2x 1 [ 17 ], human p2x 2 [ 18 ], human p2x 3 [ 19 ], human p2x 4 [ 20 ], human p2x 5 [ 21 ], human p2x 6 [ 22 ] and human p2x 7 [ 23 ] were examined for suitable epitopes following the approach used for the rat antibodies [ 15 , 24 ]. the non - homologous epitopes corresponding to the segment lys68 - val84 used in p2x 1 with an added n - terminal cys . his209 - cys226 was selected from p2x 2 . asn185 - cys203 was selected in p2x 3 . cys270 - glu285 was selected from p2x 4 . cys272 - ser288 was selected from p2x 5 . asn200 - cys218 was selected from p2x 6 . val65 - lys81 to which was added a c - terminal cys was selected from p2x 7 . all epitopes were conjugated to diptheria toxin via maleimidocaproyl - n - hydroxysuccinimide for crosslinking to diphtheria toxin . antibodies were raised in rabbits and sheep and purified as described above for the p2y antibodies . fig4 shows the use of a combination of p2y 2 , p2x 1 and p2x 2 antibody labelling of prostate tissue from a stage 3 case ( obvious cancer ). the use of other p2x subtypes such as p2x 3 and p2x 7 yielded similar results in prostate samples . the labelling patterns were reversed in other cancers such as breast cancers ( single and multi - foci ) and skin cancers including malignant melanoma . in these tissues , p2y 2 antibody labelling of normal tissue was intense ( implying that the expression of p2y 2 in normal tissue was relatively high ) but p2y 2 antibody labelling was not observed in cancer tissue . results were enhanced by the use of antibodies against p2x receptors in combination with antibodies against p2y 2 receptor , in particular antibodies against p2x 1 and p2x 2 subtypes ( fig5 to 10 ). it is clear that the p2y ( and p2x ) receptor antibody diagnosis described above can be combined with complementary tests . such tests may provide further information as to the nature of the cancer eg . a test for malignancy such as the anti - telomerase antibody test , and / or anti - tenascin may be useful in devising treatment regimes . although the invention has been described with reference to specific examples , it will be appreciated by those skilled in the art that the invention may be embodied in many other forms . 1 . lian f r , bhuiyan m , li y w , wall n , kraut m , and sarkar f h , 1998 genistein - induced g ( 2 )- m arrest , p21 ( waf1 ) upregulation , and apoptosis in a non - small - cell lung cancer cell line . nutr . & amp ; 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