Fifty-three gastric cancer and corresponding paracan-cerous normal tissue samples were obtained at surgery. All the samples were formalin fixed, paraffin embedded, and pathologically confirmed. Of the 53 patients, 41 were males and 12 were females with a mean age of 65.6 years, ranging 39-81 years. The tumor diameter was greater than 3 cm in 37 patients. The tumor was located in gastric antrum of 30 patients, in gastric body of 16 patients, and in gastric cardia of 7 patients, respectively. Invasion was restricted in mucosa and submucosa of 2 patients (I), in muscular layer of 12 patients (II), in chorion and subchorion of 15 patients (III), in neighboring organs of 24 patients through chorion (IV). Lymph node metastasis was found in 32 patients, distant metastasis in 8 patients, embolization in 45 patients. Well-differentiated tumor was found in 1 patient, moderately-differentiated tumor in 35 patients, and poorly-differentiated tumor in 27 patients. pTMN stage I was identified in 13 patients, stage II in 6 patients, stage III in 26 patients, and stage IV in 8 patients.

Tris base, EDTA, 2H₂O-Na₂, Taq DNA polymerase and Taq I were purchased from Shanghai Sangon Biological Engineering Technology and Service Co. Ltd. SDS was purchased from AMRESCO Inc. Protein enzyme was purchased from Jingmei Biotechnology Co. Ltd. dNTPs was purchased from Pharmacia Inc. Polyclonal rabbit anti-PTEN antibody and immunohistochemistry staining reagent kit (Rabbit SP Kit) were purchased from Zymed Laboratories Inc. DAAB kit and citrate buffer were purchased from Beijing Zhongshan Biotechnology Co. Ltd. Phosphate-buffered saline (PBS) was purchased from Fuzhou Maixin Biotechnology Co. Ltd.

Three paraffin slices (7 &mgr;m) were dried in a galvano-thermy box at 60°C for 30 min, hydrated in gradient ethanol after deparaffinized in dimethylbenzenel, adequately rinsed with tap water and naturally dried. Necrotic tissues were removed under inverted microscope and no carcinoma cells were found in para-cancerous tissues. Gastric cancer and paracancerous tissues were put into a 1.5 mL eppendorf tube into which 50 &mgr;L digest buffer solution was added. The tube was overturned several times to blend it adequately, bathed in water for 8 h at 65°C and shaken several times. Protein enzyme k was deactivated at 95°C for 8 min and then centrifuged at 10 000 r/min for 10-15 min. Transfer supernatant, namely genome DNA, was transferred to another antisepsis tube and stored at 4°C for application.

PCR system is composed of 5 &mgr;L PCR buffer solution,5 &mgr;L dNTP (2.5 mmol/L), 2 &mgr;L primer (F) (10 pmol/&mgr;L), 2 &mgr;L primer (R) (10 mmol/L), 2 &mgr;L DNA template, 1 &mgr;L Taq DNA polymerase (5 units/&mgr;L), 33 &mgr;L ddH₂O. PCR conditions were at 94°C for 4 min × 1 cycle, at 94°C for 30 s, at 52°C (fifth, sixth and eighth exons) at 58°C (seventh exon) for 30 s, at 72°C for 30 s × 30 cycles, at 72°C for 7 min × 1 cycle. Five &mgr;L of the PCR amplified product was put on a 2% agarose gel containing 0.5 g/L EB, 100 bp DNA ladder as a standard reference, electrophoresed for 45 min at 100 V. The results were observed with an ultraviolet transmission reflect analysis instrument and photo was taken with an automatic gel documentation system. Primers used for detecting the mutation of exons 5-8 in the PTEN gene are listed in Table 1.

Enzyme cut reaction is composed of 3.2 &mgr;L ddH₂O, 1.5 &mgr;L buffer Taq I, 10.0 &mgr;L PCR, 0.3 &mgr;L Taq I (10 unit/&mgr;L), and a total volume of 15.0 &mgr;L. The mixture was centrifuged for 15 s and heated for 3.5 h at 65°C. Ten &mgr;L enzyme cut product was put on a 2% agarose gel containing 0.5 g/L EB, 100 bp DNA ladder as a standard reference, electrophoresed for 45 min at 100 V. The results were observed with an ultraviolet transmission reflect analysis instrument and photo was taken with an automatic gel documentation system to evaluate the enzyme cut reaction.

Eight percent neutral polyacrylamide gel electrophoresis was performed as previously described[16]. In brief, 3 mL 40% acrylamide solution, 3 mL 5 × TBE solution, 3 mL 50% glycerin, 6 mL ddH₂O, 75 &mgr;L 10% ammonium persulfate, 7 &mgr;L TEMED, were blended adequately and poured into the gel, then concreted for 1 h at room temperature. Four &mgr;L PCR product (eighth exon enzyme cut product of exon 8) and 6 &mgr;L formamide sample were mixed. The mixture was centrifuged for 15 s, denatured at 95°C for 10 min, bathed in ice for 10 min, put on an 8% neutral polyacrylamide gel, and electrophoresed with 1 × TBE buffer for 8 h at 14°C and 300 V. The fixation solution was infused into a flat utensil, into which gel was immerged, vibrated for 10 min, and washed 3 times (2 min each time) with ddH₂O. The gel was immerged into a staining solution, vibrated for 10 min, washed 3 times (20 s each time) with ddH₂O. The gel was then immerged into a display solution, vibrated until the sample signal became brown and the background became transparent yellow, and rinsed with tap water to stop display. The staining results were observed and photographs were taken.

According to the PCR-SSCP results of genome DNA, the difference in the single strand strip number and electrophoresis transference location, also known as the mobility shift, was considered PCR-SSCP positive.

Genome DNA from positive PCR-SSCP samples was amplified again in 80 &mgr;L reaction system. The product was identified by electrophoresis for bidirectional DNA sequencing.

PBS was used instead of the primary antibody for blank and normal non-immunized rabbit serum was used instead of the primary antibody for negative. Following the specifications provided with the SP staining reagent box, the deparaffinized tissues were cut into 5 &mgr;m thick sections, washed 3 times (5 min each time) with PBS, incubated at room temperature in 3% H₂O₂ to eliminate the endogenous peroxidase activity, then wash additional 3 times (3 min each time) with PBS. Antigens were repaired with microwave (citrate buffer pH 6.0), naturally refrigerated to room temperature, washed 3 times (3 min each time) with PBS, incubated at room temperature with normal non-immunized serum solution for 15 min to indicate the non-specific sites, then incubated at room temperature with the primary antibody solution and horse radish peroxidase (HRP) tagged streptavidin for 15 min respectively, washed 3 times (3 min each time) with PBS. DAE stain was rinsed with PBS for 3 min, counter stained with hematoxylin for 1 min, rinsed with tap water for 2 min, differentiated with 1% hydrochloric ethanol, rinsed with tap water for 5 min, dehydrated with gradient alcohol, transparentized with dimethylbenzene. The sections were coated with neutral balata.

Ten high power fields (50-300 cells/HP) were randomly selected for each section to measure histology (H) scores according to the percentage (P) and intensity (I) scores of positive cells ( H = P × I, P: percentage lower than 10% for score 0, 11%-40% for score 1, 41%-70% for score 2, and higher than 71% for score 3. I: intensity null for score 0, weak (faint yellow) for score 1, moderate (yellow) for score 2, strong (brown) for score 3. H measurement: score 0 or 1 for negative, score 2 or more for positive).

Fisher’s exact probability and chi-square test were used in statistical analysis. P < 0.05 was considered statistically significant.

Detection of the PTEN gene exons 5-8 of genome DNA in 53 paired gastric cancer and paracancerous tissue samples indicated that the amplified PCR product had no gene homozygous alteration and no large and/or alteration in the alleles.

Ten &mgr;L reaction product of PCR amplified exon 8 and Taq I enzyme cut reaction on a 2% agarose gel containing 0.5 g/L EB, 100 bp DNA ladder were used as a standard reference. The results indicated that the number and size were in accordance with the theory. The 281 bp, 247 bp, 30 bp segments were relatively justified as the complete enzyme cut reaction.

In terms of mutation of exons-5-8 in the PTEN gene, positive PCR-SSCP was considered abnormal single strand number and mobility location. Of the 53 gastric cancer tissue samples, mutation occurred in 5 samples, the mutation rate was 9.4%. A surplus shift strip of exon 5 was found in 4 samples, the mutation rate was 7.5% (Figure 1A). Abnormal motility velocity (single strand strip mobility location) was observed in 1 sample at exon 7, the mutation rate was 1.9% (Figure 1B). There was no abnormal SSCP strip in exons 6 and 8.

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Figure 1 PCR-SSCP showing exons 5 (A) and 7 (B) in PTEN gene. T7, T16, T19 and T37: Positive SSCP; T1: Negative SSCP; T32: Positive SSCP; T31 and T33: Negative SSCP. N: Paracancerous tissue samples; T: Gastric cancer tissue samples.

Genome DNA from positive PCR-SSCP samples was amplified for bidirectional DNA sequencing. The results indicated that only one mutation was found in exons. As in the sample, AA-TCC mutation was located at 40 bp upstream of 3’ lateral in exon 7 (Figure 2A). Such mutations led to terminator formation in codon 297 of the PTEN gene. The other 3 kinds of mutation were found in introns, including a G-C point mutation at 91bp upstream of 5’ lateral in exon 7 (Figure 2B), a T-G point mutation at 24 bp upstream of 5’ lateral in exon 5 (Figure 2C), single base A mutation was deleted at 7 bp upstream of 5’ lateral in exon 5 ( Figure 2D).

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Figure 2 AA-TCC mutation at 40 bp upstream of 3’ lateral in exon 7 (A), G-C point mutation at 91 bp upstream of 5’ lateral exon 5 (B), T-G point mutation at 24 bp upstream (C) and single base A mutation at 7 bp upstream (D) of 5’ lateral exon 5 in paired paracancerous and gastric cancer tissue samples.

The PTEN protein was expressed in gastric cancer and paracancerous tissue samples. The expression of PTEN protein in gastric cancer samples was 66.0% and 100% in gastric cancer and paracancerous tissue samples, respectively (P < 0.005, Table 2).

PTEN expression was not significantly correlated with the clinicopathological parameters in gastric cancer patients, such as gender and age of the patients, location and size of the carcinoma, distant metastasis, and embolization (P > 0.05), but was significantly correlated with infiltrating depth, lymph node metastasis, and pTMN staging (P < 0.05, Table 3). There was also a significant difference between the moderate and high differentiation groups (P < 0.005, Table 1).

The occurrence and development of gastric cancer, like other malignant tumors, are a complicated process involving participation of polygene and many factors. It is generally considered that protein tyrosine phosphatase level plays an important role in the process. Mutation of the PTEN gene encoding for protein tyrosine phosphatase and abnormal expression of the PTEN protein are significantly correlated with the occurrence and development of malignant tumors, such as glioblastoma, prostate cancer, malignant melanoma, and breast cancer.

Discovery of the PTEN gene is another important landmark in the field of anti-oncogenes. The relationship between PTEN gene and gastric carcinoma was analyzed for the genetic structure, expression and interaction with other genes in this study.

Few studies on PTEN gene mutation and protein expression in gastric cancer are available. However, cancer cells have not been isolated from normal cells that may lead to undetectable PTEN gene mutations because of plenty of normal genome DNAs.

PTEN protein phenotype can be used as an object index to judge the action of gastric carcinoma based on the cancer cells isolated from normal cells. In this study, we researched the PTEN gene mutations using PCR-SSCP-DNA sequencing technology, which can increase the detection rate of PTEN gene mutation, suggesting that it can be extended to research other tumor-related genes.

This article describes mutation of the PTEN gene in patients with gastric carcinoma. The results indicate that PTEN gene plays an important role in the occurrence and development of gastric cancer.