The Changes Of Molecular Genetics In Ulcerative Colitis Associated Colorctal Cancer

AIMTo assess the possible roles of p53 , K - ras, hMSH2 genes in ulcerative colitis associated colorectal cancers(UCACRC). To study microsatellite instability (MSI) status during neoplastic and inflammatory changes in ulcerative colitis (UC). To explore methylation of hMSH2 promoter in UCACRC and UC with dysplasia.METHODSSerial paraffin sections of 5 jim thickness were cut and used for hematoxylin and eosin(HE)and immunohistochemical stains. Immunohistochenical SP method was used to detect p53, K - ras, hMSH2 genes in paraffin - embedded tissues by immunohistochemical (S -P technique) in 87 cases (examined group: 25 cases of ulerative colitis, 7 cases of ulcerative colitis with dysplasia, 8 cases of UCACRC. control group:30 cases of sporadic colon cancer(SCRC) ,14 cases of chronic colitis). Anti - p53 , anti - K - ras and anti - hMSH2 monoclonal antibodies wers purchased from Boster Co. Analysis of immunohistovhemical staining : Cells with brown granules under microscope were regarded as positive. p53 and hMSH2 protein expression scattered in nucleu, K - ras protein expression scattered in cell - plasma. The intensity of staining for p53, K - ras and hMSH2 in tumorous cells and nontumorous cells was scored in each specimen on a scale of 0 to 3,in witch 0 = negative staining, 1 = weakly positive staining,2 = moderately positive staining, and 3 = strongly positive staining. The percentage of positive cells in each soecimen was estimated and scored on a scale of 0 to 4, in which 0 = negative,1 = positive staining in 1% to 25% of cells counted,2,in 26% to 50% ; in 51% to 75% ;and 76% to 100%. Each section was evaluatedfor the sum of these two parameters.Genomic DNA extracted from the tissues was amplified by the polymerase chain reaction (PCR) at 6 loci containing polymorphic microsateUite repeate;LN-SCA,MFD41,D18S47,D2S123,D3S1317 and D13S158. PCR was performed in a total volume of 25 l in 70 cases (examined group; 25 cases of UC,7 cases of UC with dysplasia, 8 cases of UCACRC. control group:30 cases of SCRC). MSI was detected by PCR - SSCP. MSI was dedined by the presence of novel alleles in DNA extracted from regenerative or neoplastic tissue when co - pared with DNA extracted from normal tissue in the same patient. MSI was classified into MSI low( MSI - L) if only 1 loci showed instability ,they were classified into MSI high (MSI - H) if 2 or more loci were unstable. MSI was classified into MSI negertive (MSS) if only no loci showed instability.Methylation of hMSH2 promoter was measured with methylation - specific PCR in 45 cases ( examined group: 7 cases of UC with dysplasia, 8 cases of UCACRC. control group:30 cases of SCRC) ,a PCR - based Hpa II restriction enzyme assay was used. Both tumor and normal DNA were digestived with the methylation sensitive restriction endonuclease Hpa II prior to the PCR. Hie restriction enzyme digest contained 20 units of Hpan and 1.0 pug of DNA on a 20 l reaction. A "mock" digest without enzyme was also performed on normal and tumor DNA samples. All sampled were invubated 4 hours at 37t.RESULTSThe positive expressions of p53 and K - ras in UC with dysplasia or UCACRC were significantly higher than that in UC ( p < 0.05 and p < 0. 01) , however, no significan difference was found between UC with dysplasia and UCACRC ( P > 0.05 ). The loss expression rates of hMSH2 in UC, UC with dys-plsia and UCACRC were not different in statistical analysis( p >0.05). Hie positive rate of MSI in UC with dysplasia is significantly higher than that in UC ( p <0.01) ,however,no difference was found between UCACRC and SCRC (P > 0.05 ) , so does it between UC with dysplasia and UCACRC in statistical analysis ( p > 0.05 ). The loss of hMSH2 protein expression is not found in UC with dys-plasia and UCACRC. Methylation of HMSH2 promoter was detected in 1 (12. 5% ) UC with dysplasia with loss expression of hMSH2 protein.CONCLUTION1. The mutations of p53 gene and K - ras gene are early events in UCACRC.2. Microsatellite instability is an early event in UCACR...