Patent Application: US-87886510-A

Abstract:
the present invention relates to methods of identifying pre - neoplastic and / or neoplastic states in mammals and in particular to a method for identifying pre - neoplastic and neoplastic cells in tissues and body fluids , based on differential expression of purinergic receptors in these cells .

Description:
a preferred embodiment of the invention will now be described by way of example only and with reference to the accompanying figures . the immunohistochemical method used in this study was adapted from barclay [ 31 ]. sections with a thickness of 8 μm were cut from unfixed , frozen tissue using a reichert jung 2800 frigocut cryotome . 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 one of either rabbit or sheep anti - p2x 1 , p2x 2 , p2x 3 , p2x 4 , p2x 5 , p2x 6 or p2x 7 antibody . after washing , sections were then incubated in the secondary antibody ; a 1 : 30 dilution of hrp - conjugated goat anti - rabbit secondary antibody ( dako ) for 30 mins for rabbit primaries and hrp - conjugated goat anti - sheep secondary antibody ( dako ) for sheep primaries . 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 . the consensus sequences of the rat p2x 1 [ 32 ], p2x 2 [ 33 ], p2x 3 [ 34 ], rat p2x 4 [ 35 ], rat p2x 5 [ 36 ], rat p2x 6 [ 36 ], rat p2x 7 [ 37 ], human p2x 7 [ 38 ], human p2x 1 [ 39 ], human p2x 3 , [ 40 ], human p2x 4 [ 41 ] and human p2x 5 [ 42 ] cloned receptors were examined for suitable epitopes following the approach adopted in hansen et al . [ 15 ]. the non - homologous epitopes corresponding to the segment lys199 - cys217 used in rat p2x 1 were utilised in rat p2x 3 , rat p2x 6 and rat p2x 7 . variations were applied to rat p2x 4 which used the sequence ile235 - gly251 to which was attached a c - terminal cys residue for cross - linking to a 6 kda diphtheria toxin domain . the p2x 2 epitope was selected from a region within the c1 domain [ 15 ], cys130 - gly153 . the rat p2x 5 epitope was selected from a region closer to the second transmembrane domain but still extracellular ( lys314 - ile333 to which was added a c - terminal cys also for conjugation ). although largely homologous with rat p2x 4 , cross - labelling of p2x 4 and p2x 5 did not occur . all antibodies against rat sequences were able to label corresponding human receptors . a separate epitope was used for the human p2x 1 and p2x 7 sequences . this was taken just c - terminal to the first transmembrane domain from lys68 - val84 with an n - terminal cys added for conjugation via a diphtheria toxin domain using maleimidocaproyl - n - hydroxysuccinimide . the epitope for human p2x 3 antibody was the equivalent sequence used for rat , while the epitopes for human p2x 4 and human p2x 5 were cys270 - asn287 and cys272 - ser288 respectively . all syntheses were carried out using standard t - boc chemistry on an abi synthesiser [ 43 ]. 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 [ 15 ]. 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 . affinity purification of each of the antibodies against the specific epitope for that antibody resulted in reduced background but identical labelling trends . each of the p2x antisera used has been shown to possess similar distributions in many cases but with distinctly different distributions in other cases indicating that the antisera do not lack specificity . specificity was demonstrated by affinity purification of the sera against the cognate peptides . to further verify antibody specificity , individual antibody such as the antibody to p2x 1 was added to cells transfected with the corresponding p2x 1 cdna in the presence and absence of a 10 mm concentration of the p2x 1 epitope . immunolabelling and confocal imaging of the tranfected xenopus oocytes demonstrated that the expressed p2x 1 is located , as expected , within the cell membrane and the presence of a 10 mm concentration of the cognate peptide as an absorption control resulted in the blocking of p2x 1 staining [ 18 ]. tissue was processed for morphological examination as follows : sections of approximately 3 mm × 3 mm in size were fixed in 2 . 5 % glutaraldehyde in 0 . 1m tris buffer ph 7 . 2 for 1 hour . they were then washed and post fixed in 2 % aqueous osmium tetroxide for 2 hours . after further washing , the tissue was dehydrated in a graded series of alcohols and embedded in spurr &# 39 ; s resin . curing was carried out at 50 ° c . for 18 hours . 100 nm sections were then cut with a diamond knife , stained with uranyl acetate and reynolds lead citrate in the usual manner and examined in a phillips 400 transmission electron microscope . the method of slater [ 44 ] was used . in short , thin sections ( 100 nm ) were cut and retrieved on 300 mesh nickel grids . after incubation in blocking solution ( 1 % bsa in pbs ) for 30 min , the sections were placed on the surface of a drop of the blocking solution ( with the addition of 0 . 05 % tween 20 ) containing hrp - conjugated goat anti - rabbit secondary antibody or hrp - conjugated goat anti - sheep secondary antibody ( diluted 1 : 100 ) for 1 h at room temperature . grids were then rinsed three times for 10 min in pbs and placed on drops of goat anti - rabbit secondary antibody conjugated to 10 nm gold ( nanoprobe ) for 1 h at room temperature . the grids were then washed twice with pbs followed by one wash with distilled water , for 10 min each and then placed in the vapour of 2 % aqueous osmium tetroxide for 1 minute . sections were then stained with aqueous uranyl acetate solution for 20 min , lead citrate for 10 min , rinsed twice for 10 min in distilled water and examined with a phillips 400 electron microscope at 80 kv . in a study of 4 normal and 6 human prostate cancer cases , p2x 1 , p2x 3 , and p2x 4 subtypes were markedly increased in human prostate cancer tissue . there was no labelling at all for these subtypes in normal tissue . the labelling patterns for p2x 1 ( fig1 ) in the cancerous tissue were particularly interesting in that there was a greater proportion of labelled acinar epithelial cells with each stage of prostate disease , suggesting a direct correlation between neoplastic transformation and the extent of p2x 1 acinar labelling . p2x 5 was also increased in some prostate cancer cells ( results not shown ). there was very little or no labelling for p2x 5 in normal tissue . p2x receptors , growth , innervation , and metabolic factors , ionic calcium modulation in young vs aged wistar rats studies comparing prostates from four 12 week - old rats and four 1 . 5 year - old rats resulted in the detection of a marked increase in epithelial hyperplasia in the aged rats , resembling bph in humans ( fig2 ). as with the human cancer tissue , p2x 1 , p2x 3 , and p2x 4 receptors and tyrosine kinase a receptor antibody were up - regulated in the prostatic epithelium of aged rats , when compared with that of young rats . as previously discussed , this indicates an increase in protein phosphorylation ( activation ), dna synthesis , intracellular microtubule expression ( organelle transport ), up - regulation of adjacent receptors for other neurotransmitters , cell proliferation and an influx of ions ( primarily ionic calcium ) into the epithelial cells indicating apoptosis . an increase in alpha ( 1b ) ( voltage - gated calcium channel ), and a reduction in the calcium - regulating hormone stanniocalcin was also observed in the aged rat prostates . pdgf and igf - 1 both inhibit apoptosis and were decreased in the aged rats [ 45 ]. thus , the aged rat prostate undergoes apoptosis and similar changes in p2x receptor expression as human prostate cancer tissue , and therefore may be used to investigate prostate cancer aetiology . in the aged rats , there was an increase in microtubular structures in the fibromuscular septa subjacent to the prostatic epithelium . these structures appeared similar in micrographs depicting the apoptosis - associated purinergic receptors p2x 1 , p2x 7 , ionic calcium , and the innervation factors vamp , muscarinic receptor ( m2 ), sv - 2 , snap - 25 , s100 , and transferrin receptor , all of which were up - regulated in the aged rats . alpha ( 1b ) voltage - gated calcium channels and tyrosine kinase a receptors were also up - regulated in the aged rats . stanniocalcin was down - regulated while the p2x 1 and p2x 7 apoptotic calcium channel receptors were up - regulated . these data indicate an increase of calcium ion inflow , metabolic rate , microtubule transport and innervation of the prostatic epithelium in the aged rats , and also suggest that this model could be used to investigate human prostate cancer . in 6 breast cancer cell lines supplied as frozen sections . p2x 1 , p2x 3 , and p2x 4 purinergic subtypes were labelled using the same techniques employed in the labelling of prostate tissues . the labelling pattern ( fig3 ) was suggestive of the labelling patterns seen in both human prostate cancer tissue ( fig1 ) and the prostate of the male aged wistar rat ( fig2 ). prostate cancer diagnoses ( fig4 a - f and 5 a - f ) the expression characteristics of the purinergic receptor calcium channels ( p2x 1 - 7 ) were examined in normal and pathological prostate tissue from 65 cases representing each stage of prostate disease : normal , bph , preneoplastic and cancerous ( gleason &# 39 ; s grade 5 - 9 ). clear translocation features were noted in tissue labelled with p2x 1 , p2x 2 , p2x 3 and p2x 7 . after a lengthy process of optimisation and standardisation of p2x antibody production and labelling protocols , a standardised protocol was developed . a mixture of p2x 1 , p2x 2 , p2x 3 and px2 7 subtypes at a concentration of 0 . 5 μg / ml igg each , diluted 1 : 100 with pbs , proved to be the best reagent for demonstrating the translocation features described . p2x 4 , p2x 5 or p2x 6 labelling was of lesser significance . using this reagent to label tissue sections from each category of prostate cancer it was found that there was a sequential expression and translocation of p2x labelling from the nuclei to the cytoplasm and lateral plasma membranes , ultimately expressing primarily in the apical epithelium , as cancer progressed ( fig4 f , 5 c , 5 f ). p2x labelling was completely de - expressed in bph tissue ( fig4 b , 4 e ). preneoplastic p2x translocation occurred in three distinct stages . stage 1 was characterised by dense , prominent p2x - labelled epithelial nuclei ( pen ) on a pale background ( fig4 c , 4 f ). stage 2 featured a progressive de - expression of pen and the appearance of dense and markedly punctate cytoplasmic labelling , nuclear membrane and lateral plasma membrane labelling , and an increasing signal on the apical epithelium ( fig5 b , 5 c ). stage 3 was represented by nuclei labelled only on the nuclear membrane ( no ), no cytoplasmic signal , homogeneous rather than punctate labelling , and a dense label in the apical epithelium ( fig5 e , 5 f ). in the present study , 56 % of cases diagnosed as normal or bph by haematoxylin and eosin ( h & amp ; e ) staining , showed stage 1 or stage 2 p2x labelling . the remaining cases , ranging from gleason score g5 to g9 , had p2x stage 2 or 3 labelling features . stage 3 labelling was always accompanied by the histological features of cancer ( fig5 e ). true non - neoplastic bph tissue was easily distinguished by the complete de - expression of all p2x subtypes in the epithelium and stroma . we propose that biopsy tissue that has been histologically diagnosed as normal but displays p2x labelling features , may be in the process of early ( preneoplastic ) transformation at a metabolic level . the demonstration of stage 2 features in ‘ normal ’ tissue suggests that the preneoplastic process is more advanced in that tissue . the p2x labelling features described are stage - specific and uniform throughout the entire area of cells representative of each histological classification . in cores that contained both bph and cancer areas , p2x labelling was clearly and uniformly demarcated into either bph or one of the cancer labelling patterns . it is proposed that this technique can be used to exclude ( and reassure ) patients with non - neoplastic prostatic conditions from those with early cancer and also identify rapidly - developing preneoplasia , that may lead to malignancy . this information may permit earlier and more accurate treatment decisions . subtypes p2x 2 , p2x 3 , and p2x 7 are significantly down - 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