Sixty-eight cancer and corresponding normal tissues were obtained from surgically resected gastric carcinoma in our hospital. Each specimen was frozen immediately and stored at -80 °C until analyzed. A 5 µm section was cut from each tissue and stained with hematoxylin/eosin in order to ascertain whether the cancer cells in the tissues were predominant or not. Genomic DNA was isolated by standard proteinase-K digestion and phenol-chloroform extraction protocols. Of the 68 patients with gastric cancer, 45 were men and 23 were women with an age range of 30-76 years (a mean of 56.2 years at diagnosis). None of the patients included in the present series had a family history suggestive of HNPCC or had received chemotherapy or radiation therapy.

PCR and heteroduplexing Primer pairs for long-chain and short-chain PCR and GC-clamped primers used were shown in Table 1[17]. PCR reactions were carried out in 50 µl reactions in thin-walled tubes in a Perkin-Elmer 2400 thermocycler. A total of 200-400 ng of genomic DNA, varying concentrations of each primer, and the LA PCR kit (TaKaRa, Otsu, Shiga, Japan) were used for long PCR. Final concentrations of each LA PCR primer pair were as follows: hMLH1-4F and hMLH1-4R, 0.16 µM each; hMLH5-10F and hMLH5-10R, 0.125 µM each; hMLH11-13F and hMLH11-13R, 0.094 µM each; and hMLH14-19F and hMLH14-19R, 0.125 µM each. The reactions were carried out according to the manufacturer’s instructions. In brief, the conditions were as follows: a hot start of 94 °C for 2 min, with the addition of Taq Pol in between, followed by eight cycles of 98 °C × 20 sec, 69 °C × 1 min (with decrements of 0.5 °C/cycle), and 68 °C × 12 min; six cycles of 96 °C × 20 sec, and 68 °C × 12 min; 16 cycles of 96 °C × 20 sec, and 68 °C × 12 min (with increments of 15 sec/cycle), and finally a chain extension of 72 °C for 10 min.

After checking and visualizing the long PCR products on a 0.8% agarose gel, 1µl of the long PCR amplicons was used as template for subsequent extensive multiplex short PCR. The short PCR was performed in two multiplex groups of 11 and 10 amplicons, respectively. The final concentrations of each primer were shown in Table 2. The final concentrations of the PCR mix included 1 × PCR buffer, 7 mM MgCl₂, and 0.25 mM each dNTP. Cycling conditions included a hot start of 3 min at 94 °C with the Taq Pol added after 2 min, followed by five cycles of 94 °C × 1 min, 52 °C × 45 sec (decrements of 1 °C/cycle), and 72 °C × 1 min; 15 cycles of 94 °C × 1 min, 48 °C × 45 sec, and 72 °C × 1 min, 30 sec (with increments of 2 sec/cycle); 15 cycles of 94 °C × 1 min, 38 °C × 45 sec, and 72 °C × 1 min 30 sec. Each PCR reaction was teminated with a round of heteroduplexing: 72 °C × 10 min, 98 °C × 10 min, 45 °C × 30 min, and finally 37 °C × 30 min. Each tube reaction was directly mixed with 1/10 volume of 10 × loading buffer, 6.5 µl of multiplex group I and 8.5 µl of multiplex group II were loaded onto a slab gel for size separation.

Two-dimensional electrophoresis For two-dimensional electrophoresis, the DGGE instrument was from CBS Scientific Co. (Solana Beach, CA. Amplicons from each of the two mu ltiplex reactio ns were mixed and sub jected to electrophoresis in 0.5 × TAE (Tris-Acetate EDTA). A 10% polyacrylamide, 0.75 mm thick slab gel was used, the amplicons were fractionated at 140 V for 7.5 hr at 50 °C. The separation pattern was detected by SYBR green staining and UV-transillumination of the slab gel. The 120-to 420-bp region in the middle of each lane was quickly cut out and applied to a 1 mm thick DGGE gel.

The DGGE gel was prepared as a 1 mm thick slab gel with a 10%-6.5% reverse polyacrylamide gradient containing 25%-70% urea/formamide (UF) and 3%-9% glycerol gradients. Electrophoresis was carried out for 16 hr at 56 °C and 90-110 V. After electrophoresis, the gels were stained with SYBR-green I and II for 30 min. The DGGE band patterns were detected and documented with a gel documentation system (Oncor, Gaithersburg, MD).

Sequence analysis Amplicons were prepared by PCR such that a standard M13 primer site was incorporated at one end. These products were sequenced on an ABI 377 sequencer (Foster City, CA) with kits containing Taq FS DNA polymerase and dye primer technology, as recommended by the manufacturer.

DNA methylation patterns in the CpG islands of hMLH1 gene were determined by Methylation-specific PCR (MSP) as described[18]. The primer sequences of hMLH1 for unmethylated reaction were 5’-TTTTGATGTAGATGTTTTATTAGGGTTGT-3’ (sense) and 5’-ACCACCTCATCATAACTACCCACA-3’ (antisense), and for methylated reaction were 5’-ACGTAGACGTTTTATTAGGGTCGC-3’ (sense) and 5’-CCTCATCGTAACTACCCGCG-3’ (antisense).

MSI analyses included five microsatellite markers: BAT25, BAT26, BAT40, D2S123, and D5S346. PCR was performed as previous described[1]. MSI was defined as the presence of band shift in the tumor DNA that was not present in the corresponding normal DNA. Based on the number of mutated MSI markers in each tumor, carcinomas were characterized as MSI-H if they manifested instability in two or more markers, MSI-L if unstable in only one marker, and MSS if they showed no instability in any marker[19,20].

Chi-square test with Yates’ correction were used. A P value < 0.05 was considered significant.

Alterations of electrophoretic patterns of PCR products of five microsatellite markers were compared between the tumor and the normal DNA in each patient (Figure 1). MSI affecting at least one locus was observed in 17 (25%) of 68 tumors, among which eight (11.8%) were MSI-H, nine (13.2%) were MSI-L, and fifty-one (75%) were MSS.

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Figure 1 MSI in gastric cancer using 5 microsatellite loci (BAT-25, BAT-26, BAT-40, D2S123, and D5S346). Arrows indicate variant conformers. N: normal DNA pattern; T: tumor speci-mens containing variant conformers representing MSI.

Mutations and sequence alteration in various exons manifested as the four-spot pattern denoting a heterozygous variant (Figure 2). We found three mutations in 68 (4.4%) gastric cancer by DNA sequencing. Two mutations were caused by a single bp substitution (exon 8 at codon 226, CGG→CTG, Arg→Leu; exon 9 at codon 252, TCA→TTA, Ser→Phe);. One identical change was caused by a 2 bp substitution (exon 12 at codon 409, CAG→CGT, Gln→Arg), which displayed similar DGGE band pattern. A comparison of MSI status with hMLH1 mutation was shown in Table 3. Three hMLH1 mutations were all detected in MSI-H, but no mutation was found in MSI-L or MSS.

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Figure 2 Detection of mismatching repair gene hMLH1 muta-tion in gastric cancer by two-dimentional DNA electrophoresis. Four-band pattern was observed at exon 8.

To examine methylation of promoter region of hMLH1, we adapted MSP for the 5’ CpG islands in this gene. The region chosen spaned the area of greatest CpG density immediately 5’ to the transcription starting site, in an area previously studied for methylation changes[15]. In gastric mucosal samples without cancer, only unmethylated hMLH1 genes were present. Eleven of 68 (16.2%) gastric cancers exhibited prominent methylation, which always had both methylated and nonmethylated hMLH1 genes (Figure 3). A comparison of MSI status with hMLH1 methylation status was shown in Table 4. Seven of 8 (87.5%) cancers with MSI-H exhibited prominent methylation, whereas methylated hMLH1 gene was only found in 1/9 (11.1%) gastric cancer with MSI-L and 3/51 (5.9%) with MSS, suggesting that hMLH1 methylation was more correlated with gastric cancers with MSI-H than that with MSI-L or with MSS (P < 0.01-0.001).

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Figure 3 Methylation of hMLH1 in gastric cancer. A: No me-thylation was found in normal mucosa; B: Methylation of hMLH1 in MSI-H gastric cancer. Cases 1, 3 showed both hypermethylation and unmethylation; C: Methylation of hMLH1 in MSI-L gastric cancer. Case 3 showed both hypermethylation and unmethylation; U: Unmethylation; M: methylation.