Patent Application: US-21452208-A

Abstract:
a method for the analysis of differential expression in colorectal cancer based on the variation in the expression levels of genes encoding for proteins forming part of the condensin complex or associated proteins that occurs in patients with the disease and that can be used as markers for the diagnosis of the cancers , as well as for the prevention and treatment thereof .

Description:
the present invention is based on the over - expression of proteins of the condensin complex and associated proteins observed in colorectal cancer patients . the data showed an over - expression of hcap - e in the western blot analysis using specific anti - hcap - e antibodies in colorectal cancer samples in comparison with samples of normal tissue , regardless of its tumor stage , with a 90 % incidence of tumor over - expression ( 18 / 20 ) ( fig4 ). this over - expression was also observed in all immunohistochemical analyses of colorectal cancer tumor tissue ( fig5 ). likewise it was observed that the expression of hcap - e is also specific for pluripotent ( stem ) cells of the colon crypt ( fig6 ), which are the undifferentiated cells from which colorectal tumors originate . their expression pattern indicates that the latter virtually disappears as the pluripotent cells are transformed into epithelial cells ( goblet cells ) in a normal crypt to form the epithelium , so that it could be regarded as a marker of cell differentiation . table 3 shows the levels of over - expression of hcap - e in colorectal cancer according to the staging . the incidence of over - expression (+) was 90 % ( 18 out of 20 tumors ). only one case was observed in which expression was below normal (−) and one case in which normal tissue and tumor tissue showed similar expression levels (=). the expression levels of other proteins forming the condensin complex , such as hcap - c , hcap - d2 , hcap - d3 , hcap - g , hcap - g2 and hcap - h , were likewise analyzed by real - time pcr , as were other associated proteins that interact with the complex , such as kif4a , in certain tumors in which over - expression of hcap - e had been observed . the results indicated that all the analyzed proteins showed increased expression levels in tumor tissue in comparison with the levels in normal tissue ( fig7 ). it is , therefore , concluded that all the proteins that make up the condensin complex and other proteins interacting with the complex are over - expressed in tumors . accordingly , the proteins forming the condensin complex and other associated proteins that interact with it may be used as markers of colorectal cancer or of a premalignant condition thereof , potentially acting as a diagnostic marker and / or a marker for recommending colonoscopy . these proteins can also act both as markers of pluripotent stem cells of the colon crypt and as markers of cell differentiation . moreover , these proteins can be histological markers of cancer and / or be useful in imaging analysis systems . in addition , these proteins can constitute direct or indirect therapeutic targets , enabling tumor - targeted anticancer treatments to hit the tumor through interactions with any of these proteins or by modulation of their expression levels . interestingly , it has recently been reported that the expression levels of the proteins in the condensin complex do not vary throughout the cell cycle and , therefore , do not vary during mitosis ( takemoto et al ., 2004 ). so , the fact that high levels of these proteins are found in cancer cells , as described in the present invention , cannot be attributed simply to an increase in replication activity ( mitosis ) of the tumor cell , but rather to actual relative over - expression due to the development of the disease . therefore , the present invention shows that there is a complete association between the expression levels of the proteins that make up the condensin complex and other proteins associated with the complex and the presence of colorectal cancer , whatever its stage of development , which means that there is now a new molecular tool available that enables the disease to be diagnosed even in its earliest stages , something that is not possible using methods currently available . the practice of the present invention employs , unless otherwise indicated , conventional techniques of chemistry , molecular biology , microbiology , recombinant dna , genetics , immunology , cell biology , cell culture and transgenic biology , which are within the skill of the art . see , e . g ., t . maniatis et al . ( 1982 ), molecular cloning : a laboratory manual ( cold spring harbor laboratory , cold spring harbor , n . y . ); j . sambrook et al . ( 1989 ), molecular cloning : a laboratory manual , 2 nd ed . ( cold spring harbor laboratory , cold spring harbor , n . y . ); f . m . ausubel et al . ( 1992 ), current protocols in molecular biology ( j . wiley and sons , ny ); d . glover ( 1985 ), dna cloning , i and ii ( oxford press ); r . anand ( 1992 ), techniques for the analysis of complex genomes ( academic press ); g . guthrie and g . r . fink ( 1991 ), guide to yeast genetics and molecular biology ( academic press ); harlow and lane ( 1988 ), antibodies : a laboratory manual ( cold spring harbor laboratory , cold spring harbor , n . y . ; w . b . jakoby and i . h . pastan ( eds .) ( 1979 ), cell culture : methods in enzymology , vol . 58 ( academic press , inc ., harcourt brace jovanovich ( ny )); nucleic acid hybridization ( b . d . hames and s . j . higgins eds . 1984 ); transcription and translation ( b . d . hames and s . j . higgins eds . 1984 ); culture of animal cells ( r . i . freshney , alan r . liss , inc ., 1987 ); immobilized cells and enzymes ( irl press , 1986 ); b . perbal ( 1984 ), a practical guide to molecular cloning ; the treatise , methods in enzymology ( academic press , inc ., n . y . ); gene transfer vectors for mammalian cells ( j . h . miller and m . p . calos eds ., 1987 , cold spring harbor laboratory ); methods in enzymology , vols . 154 and 155 ( wu et al . eds . ); immunochemical methods in cell and molecular biology ( mayer and walker , eds ., academic press , london , 1987 ). below is described a preferred , though not exclusive , embodiment of the invention . biopsies of normal and cancerous tissue were obtained from 20 patients diagnosed with colorectal cancer . the surgically obtained samples were immediately frozen in liquid nitrogen and kept at − 80 ° c . for later extraction of proteins and rna . in addition , histological sections were prepared for immunohistochemical testing . the clinicopathological characteristics were recorded , including the stage and differentiation grade of the tumors , as well as at least a three - year follow - up to detect any early recurrence . western blot : the proteins were extracted from the samples of normal and cancerous colorectal tissue by standard methods , using ripa lysis buffer . ninety μg of protein were fractionated in 10 % sds - page gel , transferred to a nitrocellulose membrane ( biorad , usa ), blocked with 5 % milk in tbs - t , and hybridized with a primary anti - hcap - e antibody ( abcam , uk ) diluted 1 : 2500 in blocking solution , and then with a secondary anti - rabbit antibody ( dako cytomation , denmark ) in a 1 : 500 dilution . the chemiluminescent signal was detected using the ecl kit ( amersham , usa ) and the expression levels of the tumor samples were compared with their normal counterparts . finally , the membrane was rehybridized with anti - actin antibody ( invitrogen , usa ), which was used as load control . as can be seen from fig4 , all the tumor samples exhibited increased hcap - e expression levels . immunohistochemistry : the histological sections taken from the tissues of patients with colorectal cancer were deparaffinized with xylene and rinsed in decreasing series of ethanol and distilled water . the sections were treated with citrate buffer at ph 6 ( five minutes at 800 w and ten minutes at 450 w in a microwave ) the endogenous peroxidase was blocked with h 2 o 2 , and they were then hybridized with the primary anti - hcap - e antibody ( abcam , uk ). for the immunohistochemical analysis , the envision + dual link system kit ( dako cytomation , denmark ) was used in accordance with the manufacturer &# 39 ; s recommendations . finally , the hcap - e expression levels in areas of normal and cancerous tissue were compared . fig5 shows that hcap - e expression is greater in cancerous than in normal tissue . analysis of the expression levels of other proteins of the condensin complex and associated proteins real - time pcr : the mrna levels corresponding to other proteins of the condensin complex and associated proteins were quantified by real - time pcr , for which purpose rna was extracted from samples of normal and cancerous colorectal tissue kept at − 80 ° c ., using trizol ( invitrogen , usa ). ten jig of rna was retro - transcribed using the high capacity cdna archive kit ( applied biosystems , usa ) and amplified with taqman ® gene expression assays ( applied biosystems , usa ) for hcap - c , hcap - d2 , hcap - d3 , hcap - g , hcap - g2 , hcap - h , and kif4a , respectively . the amplification reaction was carried out using taqman universal pcr master mix in the 7500 real - time pcr system ( both from applied biosystems , usa ). the relative mrna levels of each gene were quantified using the δδc t method and the program associated with the system . the test was carried out in triplicate and 18s rrna was used as the endogenous control , and the expression of normal tissue and of cancerous tissue from the same patient was compared . as fig7 shows , for all the genes analyzed , the cancerous samples exhibited substantially elevated mrna levels when compared to the control samples . hagstrom k . and b . j . meyer . condensin and cohesin : more than chromosome compactor and glue . nat . rev . genet . 2003 july 4 : 520 - 534 . hirano t . the abc of smc proteins : two - armed atpases for chromosome condensation , cohesion and repair . genes & amp ; 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