Subjects consisted of 16 patients (12 male, 4 female), aged 44.6 on the average, and the period ranged from 1 month to 27 years in this study. Patients showing endoscopic signs of inflammation were classified as normal (N=0), mildly active (N=6), moderlately active (N=2), severe (N=8) according to Sninsky et al⁸⁾. The 6 pieces of mucosal biopsy specimens in each patient were obtained from the rectal mucosa approximately 15 cm from the anal verge by sigmoidoscopy or colonoscopy. All the patients had no evidence of dysplasia-associated lesions or masses on endoscopic finding. Among the 6 specimens in each patient, 4 specimens were used for DNA extraction, 1 specimen for H&E stain and 1 specimen for immunohistochemical stainning of p53 protein. The normal mucosal specimens (control) for PCR-SSCP were obtained from the colonic mucosa which showed no evidence of inflammation on endoscopic examination.

The histopathoiogy of all biopsies taken from UC and normal subjects was blindly evaluated by two gastrointestinal pathologists. Each biopsy specimen was classified as negative for dysplasia, indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia according to the microscopic criteria of the Inflammatory Bowel Disease Dysplasia Morphology Study Group⁴⁾.

For analsysis of mutational assay of p53 locus, DNA was extracted from 4 specimens of normal and UC mucosa in each patient. Each specimen was put in a bath containing liquid nitrogen and homogenized finely. Powdered tissue product was added with 1 ml of STE (0.1 M Tris-HCL, 40 mM EDTA, 20% sodium dodecyl sulfate) and with proteinase K 10μl (50 mg/ml) in a microcentrifuge tube.

This mixture was reacted in a 55°C tremulous cistern for 10 to 12 hours for digesting the protein. The same volume of phenol/chloroform/isoamyl alcohol (Sigma Chemical Co, Saint Louis Mo. USA) was added to it and centrifuged at 12,000 rpm for 15 minutes. After the centrifuging, the supernatant was taken. This process was repeated 3 times. The supernatant was mixed with 3 M sodium acetate (pH 5.2, 1/10 of the supernatant) and then 100% ethanol were added with the mixture. Finally, genomic DNA was picked up and cleaned with 75% ethanol, dried and dissolved in TE (10 mM Tris pH 8.0, 1 mM EDTA pH 7.2) solution. The DNA content was measured by a spectrophotometer at 260 nm and 280 nm and then used after the confirmation of the absorbancy ratio.

0.5 μg genomic DNA was used for the PCR amplification. The used primer sequence was displayed in Table 1. 10 pmol of primer with a 50 μl capacity was used. The Taq DNA polymerase, 10× PCR buffer (Mg²⁺, 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl₂), and dNTP mixture (2.5 mM) was used. The PCR products were pre-denaturated at 94°C for 5 minutes, annealed at 57°C for 1 minute, at 72°C for 1 minute and 30 seconds, and then at 72°C for 7 minutes. This process was repeated for 35 cycles. This PCR process was done by mastercycler 5330 (Eppedorf, Germany), and PCR products were confirmed with 2% agarose gel (Sigma, Chemical Co.). Gels were stained with ethidium bromide, photographed under UV light and we evaluated the differences in band intensities between normal and tumor DNAs.

Single-strand conformational polymorphism (SSCP) analysis was used to detect for mutations with exons 4–8 of the p53 gene. We used modified cold SSCP method which did not require radioisotope and the adjustment of temperature.

15 μl of loading mixture was composed of PCR product (50 ng DNA) with 0.2% glycerol, 1 N NaOH, TBE (4°C) buffer.

The mixture was kept at 75°C for 4 minutes and we put it into ice and rapidly performed electrophoresis in the gel. A 4–20% gradient polyacrylamide gel (8.0 × 8.0 × 0.1cm, 39°C)(Novex, San Dieg, CA) was used. Electrophoresis lasted at 280 volts 25 amphere for 1 hour 45 minutes. The staining of gel was carried out by ethidium bromide (0.5 μg/ml) for 10 minutes. Destaining with the third distilled water was done for 20 minutes, and we observed the band in 340 nm UV box.

The 16 μl of the mixture composed of 6% polyacrylamide gel (7 M urea), 30 ng template DNA (PCR products), and DNA sequencing buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDT pH 8.0) was put into the tubes containing GATC annealing buffers and then sequencing of the PCR products was done. Initially, the mixture was predenaturated at 95°C for 2 minutes, denaturated at 94°C for 10 seconds, annealed at 42°C for 30 seconds, at 72°C for 55 seconds, and amplified at 72°C for 55 cycles. After this process, addition of 3 μl of DNA sequencing solution (10 mM NaOH, 95% formide, 0.05% bromophenol blue, 0.05% xylene cyanol) was done. This solution was kept at 70°C for 3 minutes, stuck into ice for 3 minutes. It was electrophoresised in 6% urea gel, with 60 W/1500 V for 1½ hour until bromophenol blue perished. Then, the eletrophoresised gel was kept in stop solution (10% glacial acetic acid) for 20 minutes and cleansed with the third distilled water for 4 minutes. This cleansing process was done 3 times. After cleansing, gel was put in a flask shaker with staining soution (1 g/L silver nitrate, 0.056% formaldehyde) and was shaken slowly for 30 minutes. It was kept in distilled water for 5 seconds and then kept in developing solution (30 g/L sodium carbonate, 0.056% formaldehyde, 2 mg/L sodium thiosulfate) until the band was visible. After visuallization of the band, gel was kept in stop soultion for 10 minutes, and dried at 55°C for 2 to 3 hours. We observed the sequence.

Eight patients (50%) had proctosigmoiditis, 6 patients had proctitis (37%) and the other 2 had pancolitis (12%). The p53 mutated patients were composed of 3 proctosigmoiditis and 1 pancolitis. The sex (M:F) ratio in p53 mutated patients was 3:1. Non-mutated group showed 9:3 (M:F). The mean age of patients group of positive for p53 gene mutation was 44±11.5 years, the patients of negative for p53 gene mutation was 44 ± 16.4 years. The mean duration of colitis in p53 mutated group was 6 ± 1.7 years and non-mutated group was 8 ± 7 years. All patients who showed p53 gene mutation were poor response to conventional medical treatment such as 5-ASA and/or steroids. But all patients belonging to non-mutated group were good responders except one patient. All the characteristics of patients are displayed in Table 2.

Among 16 UC patients, two patients (2%) with mild inflammation on colonoscopy showed low grade dysplasia on H&E stain. In addition, there were 4 patients who showed p53 overexpression in immunohistochemical stain (Fig. 1) and all of them were also shown to be positive for p53 mutation.

Positive results of SSCP assays in 4 patients performed on exon 4, 5, 6, 7, 8 of p53 are displayed in Table 3. Examples are shown in Figure 2. In the other 12 patients and in normal looking mucosal specimens, no mutations were found by SSCP. Point mutations were observed in 2 histologically normal tissues and 2 patients who showed dysplastic mucosal change.

Findings of SSCP were confirmed and further characterized by DNA sequencing in the four SSCP positive patients. All samples had been sequenced repeatedly. Sequence abnormalities included single-base mis-sense mutation in codons 249 (AGG to AGT), 260 (TCC:arginine to TAC:methionine) and 123 (ACT:stop codon to ACC⁺T) and non-sense mutation in codon 121 (TCT: serine to TGAT: stop codon) (Table 3), (Fig. 3).