[Preparation and identification of monoclonal antibody against human GCRG213].

AIM: To prepare and characterize the monoclonal antibody against human GCRG213. METHODS: The HIS-GCRG213 fusion protein was expressed in E.coli. Mice were immunized with the purified HIS-GCRG213 protein. Hybridoma cell lines secreting monoclonal antibodies against GCRG213 were screened by regular cell fusion and subcloning approach. The titer and specificity of the antibody was characterized by ELISA and Western blot, respectively. The expression of GCRG213 was determined using immunohistochemistry technique on paraffin-embedded tissue sections from normal gastric mucosal tissues and advanced gastric cancer. RESULTS: The HIS-GCRG213 fusion protein with relative molecular mass of 20 800 was over expressed in E.coli. Two hybridoma cell lines which secreted monoclonal antibody specifically against human GCRG213 fusion protein were successfully obtained. The ascite titers of this monoclonal antibody reached 1:10(6);. Western blot analysis showed that the monoclonal antibody could bind to the recombinant HIS-GCRG213 protein specifically.The immunohistochemistry showed that GCRG213 were expressed higher in gastric cancer tissues than in normal ones. CONCLUSION: The monoclonal antibody against human GCRG213 with high titer and specificity has been successfully prepared, which could be utilized as a useful reagent for further studying the biological function of the GCRG213.

[Preparation, identification and preliminary application of the polyclonal antibody against gastric cancer-related protein GCRG224].

AIM: To prepare the polyclonal antibody against gastric cancer-related protein GCRG224. METHODS: The thioredoxin/GCRG224 fusion protein was expressed in E.coli. The polyclonal antibody against GCRG224 was obtained by immunizing a rabbit with the purified GCRG224 protein. The titer and specificity of the antibody were determined by ELISA and Western blot, respectively. The expression of GCRG224 in paraffin-embedded tissue sections from normal gastric mucosal tissues and advanced gastric cancer was determined using immunohistochemistry technique. RESULTS: The thioredoxin/GCRG224 fusion protein with relative molecular mass of 16.8 kDa was over-expressed in E.coli. The purity of the expressed product directly purified from a denaturing polyacrylamide gel was about 100%. The polyclonal antibody against GCRG224 was prepared. ELISA detection proved the titer of antiserum against GCRG224 was about 1:256,000. Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically. GCRG224 was found to have higher expression in gastric cancer tissues than in normal ones by immunohistochemistry. CONCLUSION: The successful preparation of the polyclonal antibody against GCRG224 lays a foundation for further study of the biological function and the possible role of GCRG224 in the development of gastric carcinoma.

[Expression of S100A6 in primary and metastatic human gastric cancer].

OBJECTIVE: Some members of the S100 gene family have been suggested to be associated with cancer development and metastasis. Our previous cDNA micro-array studies have showed S100A6 expression is elevated in gastric cancer compared with that in paired normal mucosa. To validate our previous results and further investigate the possible role of S100A6 gene in gastric cancer, we carried out this detailed S100A6 expression analysis in more matched gastric cancer samples. METHODS: S100A6 expression was detected in 20 paired fresh surgical samples of gastric tumor tissue and matched non-cancerous mucosa by QRT-PCR. A gastric cancer tissue microarray (TMA) containing 1020 duplicate matched normal mucosa, gastric cancer tissue and metastatic lymph node tissue cores from 208 gastric cancer patients was constructed. S100A6 expression was detected by immunohistochemistry and the correlation between S100A6 expression with clinicopathological factors and survival was analyzed. RESULTS: As quantitated by QRT-PCR, S100A6 transcript level was elevated in 73.7% of the primary cancer lesions with an average 2.25-fold up-regulation than that in matched non-neoplastic mucosa. As displayed by immunohistochemistry, the positive rate of S100A6 in non-neoplastic mucosa, tumor lesions and metastatic lymph nodes was 34.3%, 84.1% and 90.9%, respectively. S100A6 expression level in cancer and metastatic lymph node was significantly higher than their matched non-neoplastic mucosa (P < 0.05). 65.5% of patients showed an increased S100A6 expression in cancer tissue compared with that in matched normal mucosa. S100A6 overexpression was associated with larger tumor size and deeper invasion (P = 0.022 and P = 0.009). No evidence was found for an association between S100A6 expression level and other variables, including tumor grade, nodal metastases, and TNM stage. There was no association between S100A6 expression level and survival. But compared with paired non-neoplastic mucosa, an increased S100A6 expression in tumor lesion predicated a decreasing suvival if compared with a decreased S100A6 expression, though the difference was statistically not significant. CONCLUSION: Elevated expression of S100A6 gene may be an early event in the development and progression of gastric cancer. Further study of this gene may be helpful for understanding the nature of gastric carcinoma.

LINE-1 family member GCRG123 gene is up-regulated in human gastric signet-ring cell carcinoma.

AIM: To analyze the expression profiles of a human gastric-cancer-related gene, GCRG123, in human gastric signet-ring cell carcinoma tissues, and to perform bioinformatics analysis on GCRG123. METHODS: In situ hybridization was used to explore the GCRG123 expression pattern in paraffin-embedded gastric tissues, including 15 cases of signet-ring cell carcinoma, 15 of intestinal-type adenocarcinoma, and 15 of normal gastric mucosa. Northern blotting was used to analyze the differences in GCRG123 expression between stomach signet-ring cell carcinoma and intestinal-type adenocarcinoma tissues. Online software, including BLAST, Multalin and BLAT, were applied for bioinformatics analysis. National Center for Biotechnology Information (NCBI) and the University of California Santa Cruz (UCSC) databases were used for the analyses. RESULTS: The in situ hybridization signal appeared as blue precipitates restricted to the cytoplasm. Ten out of 15 cases of gastric signet ring cell carcinoma, normal gastric mucosal epithelium and pyloric glands showed high GCRG123 expression. Low GCRG123 expression was observed in gastric intestinal-type adenocarcinoma and normal gastric glands. Northern blotting revealed that GCRG123 was up-regulated in signet-ring cell carcinoma tissue but down-regulated in intestinal-type adenocarcinoma tissue. BLAST and Multalin analyses revealed that the GCRG123 sequence had 92% similarity with the ORF2 sequence of human long interspersed nuclear element retrotransposons (LINE-1, L1). BLAT analysis indicated that GCRG123 mapped to all chromosomes. GCRG123 was found to integrate in the intron-17 and -23 of Rb, 5' flanking region of IL-2 and clotting factor IX genes. CONCLUSION: GCRG123, an active member of the L1 family, was up-regulated in human gastric signet-ring cell carcinoma.

[Construction of a gastric cancer related gene GCRG213-specific siRNA expression vector and its effect on gastric cancer cells in vitro].

OBJECTIVE: To investigate the effect of gene GCRG213 siRNA transfection into gastric cancer cell line MKN45 cells. METHODS: Two pairs of DNA sequences containing small hairpin structure to GCRG213 were designed and synthesized. The complement form was obtained by annealing and inserted into RNAi expression vector IMG-800. They are IMG-800-1 and IMG-800-2 correspondingly. The recombinant plasmid IMG-800-1, IMG-800-2 and the vector IMG-800 were separately transfected into MKN45 cells conducted by lipofectamine 2000. After G418 selecting, the cells were transfected steadily. Expression of GCRG213 was detected by semi-quantitative RT-PCR and Western Blot. The growth graph of six steady transfected cell cultures was protracted by cell counting. FACS was used to detect the cell cycle, and Annexin V FITC/PI double labeling were used to detect the effects on cell apoptosis in the above-mentioned cells. The clone formation rate in plate and in nude mice was tested to investigate the tumorigenic characteristics of the six steadily transfected cells in vitro and vivo. RESULTS: Through sequencing, two pairs of DNA sequences containing small hairpin structure to GCRG213 were proved to be successfully cloned into siRNA expression vector IMG-800, correspondingly called IMG-800-1 and IMG-800-2. The recombinant plasmid IMG-800-1, IMG-800-2 and vector IMG-800 were transfected separately into MKN45 cells conducted by lipofectamine 2000. After G418 selecting, the cells were transfected steadily. Transfecting the siRNA vector (IMG-800-1, IMG-800-2 ) into the MKN45 cells significantly decreased the expression of GCRG213, at both mRNA and protein levels. The growth graph showed that the growth of IMG-800-1 and IMG-800-2 transfected cells were slower than that of vector transfected cells. The proportion of cells in G2/M and/or S phase decreased in the cells transfected with IMG-800-1 and IMG-800-2 and cell apoptosis increased. The average clone formation rate in vitro decreased in the cells transfected with IMG-800-1 and IMG-800-2, compared with those transfected with vector. In vivo, the time of tumor formation of IMG-800-1 and IMG - 800-2 transducted cells in nude mice was prolonged and the tumor size was smaller. CONCLUSION: GCRG213 SiRNA transfection may induce inhibition of growth and proliferation of tumor cells, promote cell apoptosis, and inhibit the tumorigenicity in vitro and vivo.

[Preparation and characterization of rabbit antibody against gastric cancer-related protein GCRG213].

AIM: To prepare the rabbit antibody against gastric cancer-related protein GCRG213. METHODS: The thioredoxin/GCRG213 fusion protein was expressed in E. coli. The rabbit antibody against GCRG213 was obtained by immunizing a rabbit with the purified GCRG213 protein. The titer and specificity of the antibody was determined by ELISA and Western-blot, respectively. RESULTS: The thioredoxin/GCRG213 fusion protein with relative molecular mass (Mr) of 29,400 was overexpressed in E. coli. The purity of expressed product directly purified from a denaturing polyacrylamide gel was about 100%. The rabbit antibody against GCRG213 was obtained. The ELISA titer of antiserum against GCRG213 was about 1:256,000. Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically. CONCLUSION: The rabbit antibody against GCRG213 has been successfully prepared, which lays the foundation for further studying the biological function and the possible role of the GCRG213 in the development of gastric carcinoma.

[Gene expression profile of human adenocarcinoma by cDNA microarray and clustering].

OBJECTIVE: To investigate the gene expression profile of human gastric adenocarcinoma by means of cDNA microarray and to analyze its biological significance. METHODS: Paired tumor and non-tumor specimens from 18 cases of advanced gastric adenocarcinoma were studied. Total RNA was isolated and labeled by reverse transcription reaction with cy5 and cy3 for cDNA probe. cDNA microarrays containing 148 genes were hybridized with labeled cDNA probe. Data from cDNA microarray experiments were analyzed by average-linkage hierarchical clustering and significance analysis of microarrays (SAM). RESULTS: Eighteen tumor and non-tumor specimens were clearly divided by clustering analysis. Three features of gene expression profile were found in gastric adenocarcinoma and non-tumor tissues. The profile of differential gene expression in tumor and non-tumor tissues was mainly shown in feature B and feature C. In gastric adenocarcinoma tissues, the expression of genes in feature B was lower and that in feature C was higher. The profile of differential gene expression among gastric adenocarcinoma tissues was found in feature A. In feature A, the profile of similar gene expression was found in paired tumor and non-tumor tissues from 13 patients. SAM analysis showed that 19 genes in feature B and 12 genes in feature C were of significant difference between tumor and non-tumor specimens. The expression levels of genes related to cell cycle, growth factor, cell adhesion, and matrix remodeling were higher or lower in gastric adenocarcinoma tissues. CONCLUSION: Data from cDNA microarray experiments can clearly distinguish gastric adenocarcinoma from non-tumor tissues. The profiles show that gene expression in gastric adenocarcinomas is both homogeneous and heterogeneous. The homogeneous gene expression profile is found in both tumor and non-tumor tissues from 13 patients, suggesting that some gene aberrance is an early event of carcinogenesis of gastric adenocarcinoma. This study provides not only a new molecular basis for understanding biological properties of gastric adenocarcinoma, but also useful resources for future development of diagnostic and prognostic markers for gastric adenocarcinoma.

Detection of K-ras gene mutation in fecal samples from elderly large intestinal cancer patients and its diagnostic significance.

AIM: To study the diagnostic significance of K-ras gene mutations in fecal samples from elderly patients with large intestinal cancer. METHODS: DNA was extracted in the fecal and tissue samples from 23 large intestinal cancer patients, 20 colonic adenomatoid polypus patients and 20 healthy subjects. The K-ras gene mutations at the first and second bases of codon 12 were detected by the allele specific mismatch method. RESULTS: The K-ras gene mutation was 56.52%(13/23) in the large intestinal cancer patients, which was notably higher than that in the normal subjects whose K-ras gene mutation was 5%(1/20) (chi (2)=12.93, P<0.001). There was no significant difference in comparison with that of colonic adenomatoid polypus patients whose K-ras gene mutation was 30%(6/12) (chi (2)=3.05, P>0.05). The K-ras gene mutation at the second base of codon 12 was 92.13%(12/13) in the large intestinal cancer patients. There was no significant difference between the detection rate of K-ras gene mutation in the fecal and tissue samples (chi (2)=9.35, P<0.01). CONCLUSION: Our results indicate that detection of the K-ras gene mutations in fecal samples provides a non-invasive diagnostic method for the elderly large intestinal cancer patients. Its significance in the early diagnosis of large intestinal cancer awaits further studies.

[Influence of dendritic cell infiltration on prognosis and biologic characteristics of progressing gastric cancer].

OBJECTIVE: To study the relation between dendritic cell (DC) infiltration and clinicopathologic parameters, biologic characteristics and prognosis of progressing gastric cancer. METHODS: The development of apoptotic cell death (apoptotic index, AI) in 61 progressing gastric carcinoma tissues was analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method. The PCNA labeling index (PCNA-LI), density of dendritic cells in the tumor were detected by immunohistochemical method by the LSAB kit using antibody against S-100 protein and PC-10. RESULTS: DC infiltration was negatively correlated with lymph node metastasis, clinical stage and PCNA-LI, but positively with AI. The DCs in gastric cancer groups with and without lymph node metastasis were (5.63 +/- 4.37)/HPF and (8.51 +/- 5.57)/HPF with difference significant (P < 0.05). The DC infiltration in I, II, III stage lesions were (11.23 +/- 6.05)/HPF, (6.28 +/- 4.37)/HPF and (5.53 +/- 5.19)/HPF also with differences significant (P < 0.01). The PCNA-LI was significantly higher in the low DC group (57.10% +/- 14.18%) than that of high DC group (48.15% +/- 10.59%, P < 0.01). AI findings were 3.77% +/- 1.26% and 2.95% +/- 1.07% in the high and low DC groups (P < 0.01). A positive correlation was observed between DC infiltration and AI (r = 0.39, P < 0.01) whereas a negative correlation between DC infiltration and PCNA-LI (r = -0.47, P < 0.01). The prognosis of high DC infiltration patients was significantly better than those with low ones. CONCLUSION: The infiltrating dendritic cells in and around tumor, representing the local immune status of the host, may play an important role in immunological defense mechanism of host versus tumor. Dendritic cells may inhibit the proliferation and induce the apoptosis of the tumor cells, thus affecting the clinical features and improve the prognosis of gastric carcinoma.

A gene encoding an apurinic/apyrimidinic endonuclease-like protein is up-regulated in human gastric cancer.

AIM: To identify the gene that may predispose to human gastric cancer and to analyze its expression in gastric cancer and non-tumorous gastric mucosa. METHODS: Cancer, para-tumor, and non-tumor gastric tissues were studied for gene expression profile using fluorescent differential display reverse transcription polymerase chain reaction (DDRT-PCR). The differentially expressed bands of interest were analyzed by cloning, Northern blotting, and sequencing. The sequencing results were compared with the GenBank database for homology and conserved domain analysis. In situ hybridization with DIG-labeled cRNA probes was used to detect the expression of gene in paraffin embedded gastric adenocarcinoma and non-cancerous tissues. RESULTS: A gene expressed higher in tumor and para-tumor tissues than in their non-tumor counterparts of all 7 tested gastric adenocarcinoma patients was identified by means of DDRT-PCR analysis. It was named GCRG213 (gastric cancer related gene 213). Northern blot confirmed the differential expression. GCRG213 (GenBank No. AY053451) consisted of 1094 base pairs with an open reading frame (ORF) which encoded 142 amino acids. The deduced amino acid sequence contained a putative conserved domain, apurinic/apyrimidinic endonuclease (APE). In situ hybridization analysis showed that GCRG213 was expressed higher in gastric cancer tissues than in their corresponding non-tumor ones. Precancerous leisions of gastric adenocarcinoma showed a high GCRG213 expression, too. No difference of the expression patterns was found between the early and advanced gastric cancer. CONCLUSION: A gene named GCRG213 was identified in human gastric adenocarcinoma. It encoded an APE-like protein which was probably a new member of the APE family. GCRG213 was over-expressed not only in gastric cancer, but also in its precancerous leisions. The role of GCRG213 expression in carcinogenesis needs further study.

[Relationship between Helicobacter pylori infection and proliferation and apoptosis of gastric epithelial dysplasia cell].

BACKGROUND & OBJECTIVE: Gastric epithelial dysplasia is the precancerous lesion of gastric cancer. However, the mechanism that dysplasia evolves to malignancy is not clear. In order to clarify the relationship between Helicobacter pylori (HP) infection and its virulence factor and changes of cell kinetics of dysplasia, the authors measured the changes of proliferation and apoptosis and the status of HP infection. METHODS: A total of 117 gastric mucosal biopsy specimens were enrolled, including 12 of chronic superficial gastritis (CSG) and 105 of dysplasia. Dysplasia samples were divided into two groups: 35 of high-grade dysplasia [carcinogenesis group (n=30), regression group (n=5)], 70 of low-grade dysplasia [carcinogenesis group (n=18), regression group (n=52)]. The expression of proliferating cell nuclear antigen (PCNA) was measured by immunohistochemical staining; cell apoptosis was determined by terminal deoxynucleotidyl transferase mediated nick end labeling(TUNEL); the status of HP infection was detected by polymerase chain reaction(PCR) with specific primers of urea A and cagA gene. RESULTS: There was no significant difference of HP infection between dysplasia and CSG(84.76% vs. 83.33%), but CagA-positive strain infection rate in dysplasia was slightly higher than that in CSG (85.39% vs. 60.00%). Proliferation indexes(PI) in the patients with HP infection and CagA(+) strain infection were higher than that in the patients without HP infection and CagA(-)strain, respectively (P< 0.05). PI was positively associated with the status of HP and CagA(+) strains infection (P< 0.05). AI/PI ratio (AI: apoptosis index) was negatively associated with CagA-positive strain infection (P< 0.05). CONCLUSION: Gastric epithelial dysplasia cells have abnormal changes in PI and AI when it evolves to malignancy, and the abnormal cell kinetics is partly correlated with HP and CagA(+) strain infection. So treatment of HP infection may produce a good result for the evolution of dysplasia.

[Cloning and tissue expression analysis of up-regulated cDNA fragment in human gastric cancer].

OBJECTIVE: To identify novel human gastric cancer-associated susceptibility gene for early diagnosis and treatment of gastric cancer. METHODS: A primer was designed for 3'-rapid amplification of cDNA end(RACE) and amplified fragments were cloned, then they were analyzed by sequencing. Compared with ESTs in Genbank, the EST fragment represented a novel gene. Combination of Northern blot and virtual Northern and multiple tissues Northern blot, expression of the cDNA in multiple normal and carcinoma tissues were analyzed. RESULTS: One of the important cDNA bands with poly(A) tail was cloned. This band was named W41. Sequence analysis showed that W41 consists of 533 bp. Basic local alignment search tool analysis revealed that W41 has low identity with any genes from GenBank. This sequence data was submitted to GenBank with accession No. AF 325202. Northern blot revealed that W41 presented higher expression in gastric cancer tissue than in normal tissue. Multiple tissue Northern blot revealed that W41 presented higher expression in multiple cancers than in normal tissues. Virtual Northern revealed that the cDNA presented higher expression in tumor series analysis of gene expression libraries than in normal. CONCLUSION: A novel human gastric cancer-associated cDNA fragment was identified.

[Expression of human anti-apoptotic gene survivin and its splice in normal human gastric tissue and gastric cancer].

OBJECTIVE: To analyze the expression of human anti-apoptotic gene survivin (SVV) in normal human gastric tissues and gastric cancer. METHODS: SVV cDNA clone was obtained from human gastric cancer tissues by virtue of RT-PCR, using Dig-marked cRNA probe in situ hybridization to analyze its expression in normal human gastric tissues and gastric cancer. RESULTS: Two SVV cDNA clones, SVV-S4A and SVV-S1B were obtained. The sequence of the former is identical to that of the well-known SVV cDNA; however, in the sequence of the latter, the third exon was missed, i.e., there are only two exons in SVV-S1B. In situ hybridization showed that SVV-S4A is mainly expressed in gastric cancer tissues whereas SVV-S1B is mostly expressed in normal gastric tissues. CONCLUSION: There is difference between SVV-S4A and SVV-S1B in respect to their characteristics of expression in gastric cancer and normal gastric tissues.

A novel gene, GCRG224, is differentially expressed in human gastric mucosa.

AIM: To clone genes that may predispose us to human gastric cancer and to analyze it's expression in gastric tissues. METHODS: Specimens of paired tumor, paratumor and normal gastric mucosa tissues collected from fifteen patients who suffered from stomach antrum adenocarcinoma were used for analysis. Seven out of the fifteen cases were first studied by fluorescent differential display reverse transcription polymerase chain reaction (DDTR-PCR) analysis. The differentially expressed bands of interest were cloned, analyzed by Northern blot, sequencing and RT-PCR. Through BLAST, the sequencing results were compared with GenBank database for homology analysis. In situ hybridization with DIG-labeled cRNA probes was used to analyze the expression of interesting cDNA bands in paraffin embedded paired normal gastric mucosa and cancer tissues isolated from 30 gastric adenocarcinoma patients. RESULTS: DDRT-PCR showed that one of the interesting cDNA bands, which was named W2, expressed much higher in all seven tested tumor and paratumor samples than in their normal counterparts, it was sub-cloned into a pGEM-T Easy vector. Two subclones were subsequently obtained. One of the subclone, GCRG224, was studied further. The sequencing result showed that GCRG224 consisted of 1 159 base pairs and had one open reading frame (ORF). It located at human chromosome 11q14. No homologue was found in GenBank database with GCRG224-ORF. This nucleotide sequence data were submitted to GenBank with accession No. AF438406. RT-PCR showed that GCRG224 expressed higher in 11/15 gastric cancer tissues than in non-tumor tissues. However, the result of Northern blot analysis showed a higher GCRG224 expression in the non-tumor tissue than in the tumor one. Human multiple tissue Northern blot analysis revealed that GCRG224 also expressed in human normal colon tissue, and peripheral blood leukocyte. In situ hybridization analysis showed that only 5/30 adenocarcinoma, 3/18 dysplasia and 6/18 intestinal metaplasia showed higher GCRG224 expression level than the normal gastric glands. However, GCRG224 was over-expressed predominantly in 26/30 cases of normal mucosal epithelium. CONCLUSION: A novel gene named GCRG224 was identified from human gastric mucosal tissue. It overexpressed in almost all gastric mucosal epithelium but only a small portion of cancer and precancerous leisions. The role of GCRG224 expression in gastric epithelium needs further study.