One hundred seventy (n=170) formalin-fixed paraffin-embedded gastric cancer specimens were obtained from the College of Medicine, The Catholic University of Korea, between 2003 and 2004. For gastric cancer, 73 cases were the intestinal-type and 97 were the diffuse-type gastric cancer. The mean size of the tumors was 6.5 cm, and 154 cases were found located in the middle or lower section of the stomach. Two pathologists screened the histological sections and selected areas representative of the tumor cells. Two and one tissue core samples from each cancer and normal area were taken and placed in a new recipient paraffin block using a commercially available microarray instrument (Beecher Instruments, Micro-Array Technologies, Silver Spring, MD, USA), according to established methods.(19) The institutional review board of the Catholic University of Korea, College of Medicine (CUMC09U042) approved this study. There was no evidence of familial cancer in patients from which the specimens were obtained.

For the immunohistochemical analysis, 2 µm sections were cut the day before use and stained according to standard protocols. To maximize the signal on immunohistochemistry, two strategies were used in this study: antigen retrieval in citrate buffer and signal amplification with biotinylated tyramide. For the former, heat-induced epitope retrieval was conducted by immersing the slides in Coplin jars filled with 10 mmol/L citrate buffer (pH 6.0) and boiling the buffer for 30 min in a pressure cooker (Nordic Ware, Minneapolis, MN, USA) inside a microwave oven at 700 W. The jars were then cooled for 20 min. For the latter, the Renaissance TSA indirect kit (NEN Life Science, Boston, MA, USA), which included streptavidin-peroxidase and biotinylated tyramide, was used. After rinsing with PBS, the slides were treated with 1% H₂O₂ in PBS for 15 min at room temperature to abolish endogenous peroxidase activity. After washing with TNT buffer (0.1 mol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl and 0.05% Tween 20) for 20 min, the slides were treated with TNB buffer (0.1 mol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl and 0.5% blocking reagent). Sections were incubated overnight at 4℃ with antibodies (1/100 dilution) to SIRT1 protein (Abcam, Cambridge, UK). Detection was carried out using biotinylated rabbit anti-rabbit antibody (Sigma, St. Louis, MO, USA), followed by incubation with peroxidase-linked avidin-biotin complex. Diaminobenzidine was used as a chromogen, and the slides were counterstained with Mayer's hematoxylin. The specificity of anti-SIRT1 antibody was confirmed in 9 cancer cell lines by Western blot analysis (data not shown). Staining for SIRT1 antigen was determined as positive when more than 30% of nucleus was positively stained. Two pathologists reviewed the results independently. As negative controls, the slides were treated with primary antibody replacement by non-immune serum.

Since DNA extracted from formalin fixed paraffin embedded tissue is of low quality for mutational analysis, we extracted DNAs from 86 methacarn-fixed paraffin-embedded tissues, which were not included in our tissue microarray. These samples were also obtained from the College of Medicine, The Catholic University of Korea, between 2003 and 2004. Tumor cells were selectively procured from Hematoxylin & Eosin stained slides using a laser microdissection device (ION LMD, JungWoo International Co, Seoul, Korea). The surrounding normal gastric mucosal cells were also obtained to study the corresponding normal DNA from the same slides in all cases. DNA extraction was performed using a modified single step DNA extraction method, as described previously.(20)

Genomic DNAs from each cancer cell and the corresponding non-cancerous gastric mucosal tissues were amplified with primers covering the entire exons of the SIRT1 and DNA binding domain (exon 4~9) of the p53 gene. Primer sequences are shown in Table 1. Numbering of the sequences of SIRT1 was done with respect to the ATG start codon according to the genomic sequence obtained from Genbank accession no. NM_012238. All cases were screened by SSCP analysis of each exon for the presence of an aberrant band in the tumor DNA compared to normal DNA. Each polymerase chain reaction (PCR) procedure was performed under standard conditions in a 10 µl reaction mixture containing 1 µl of the template DNA, 0.5 µM of each primer, 0.2 µM of each deoxynucleotide triphosphate, 1.5 mM MgCl₂, 0.4 unit of Ampli Taq gold polymerase (Perkin-Elmer, Foster City, CA, USA), 0.5 µCi of [³²P]dCTP (Amersham, Buckinghamshire, UK), and 1 µl of 10X buffer. The reaction mixture was denatured at 94℃ for 12 min and then incubated for 35 cycles (denaturing at 94℃ for 40 s, annealing at 52~62℃ for 40s and extension at 72℃ for 40s). A final extension step at 72℃ was performed for 5 min. After amplification, the PCR products were denatured for 5 min at 95℃ in a 1 : 1 dilution of sample buffer containing 98% formamide/5 mmol/L NaOH. These products were loaded onto a SSCP gel (FMC Mutation Detection Enhancement system; Intermountain Scientific, Kaysville, UT) containing 10% glycerol. After electrophoresis, the gels were transferred to 3 MM Whatman paper and dried. Autoradiography was then performed using Kodak X-OMAT film (Eastman Kodak, Rochester, NY, USA). DNAs showing mobility shifts were cut out from the dried gels and amplified for 35 cycles using the same primer set. Sequencing of the PCR products was sequenced on the ABI prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

The Chi-square test for association was used to test the relationship between SIRT1 expression and the clinicopathologic parameters of gastric cancers. A P-value<0.05 was considered statistically significant.

Moderate to strong immunopositivity for SIRT1 was clearly marked on the nucleus of gastric cancer cells (Fig. 1). However, the corresponding normal gastric mucosa and surrounding stromal cells, including fibroblasts, were focal and weak positive for SIRT1 protein. Aberrant expression of SIRT1 protein was detected in 95 (55.9%) of 170 gastric cancers. SIRT1 expression was found in 47 (64.3%) and 48 (49.5%) of 73 intestinal-type and 97 diffuse-type gastric cancers, respectively. Statistically, there was no significant relationship between SIRT1 protein expression and the clinicopathologic parameters, including differentiation, tumor location, tumor size, lymph node metastasis, lymphatic invasion and vein invasion (Chi-Square test, P>0.05) (Table 2).

In our previous report, moderate to strong nuclear staining for p53 protein was found in 79 (42.5%) of 186 cancers.(21) Interestingly, 29 cases were immunopositive for both SIRT1 and p53 proteins. 50 cases with p53 nuclear staining were negative for SIRT1, and 45 cases demonstrated negative staining for both proteins. Statistically, SIRT1 protein expression was not significantly associated with p53 immunostaining (Chi-Square test, P=0.3366).

The presence of any mutations, possibly associated with aberrant SIRT1 expression, was examined using PCR-based SSCP and sequencing analysis. Expectedly, we found SIRT1 and p53 mutations in 2 and 12 cases, respectively. As shown in Fig. 2 and Table 3, one of the SIRT1 mutations was a missense mutation caused by a single nucleotide substitution, G to A transition at nucleotide 1258. Another SIRT1 mutation was a missense mutation caused by a single nucleotide substitution, C to T transition at nucleotide 599. Two cases showed aberrant bands of the mutant allele with that of the wild type in SSCP analysis, indicating a hemizygous mutation in 1 allele in the presence of the remaining allele. The corresponding normal samples showed no evidence of mutation by repeated SSCP, indicating that the mutations occurred somatically. In immunohistochemistry, two cases with SIRT1 mutation showed aberrant increased expression of SIRT1 in the cancer cells (Data not shown).

The experiments, including tissue microdissection, PCR, SSCP and sequencing analysis, were repeated three times to ensure the reliability of the results.