Diffuse-type Gastric Cancer Cell Research Articles

Identification and characterization of human CKTSF1B2 and CKTSF1B3 genes in silico

Bone morphogenetic proteins (BMPs) are implicated in the regulation of morphogenesis and proliferation during embryogenesis and carcinogenesis. We have previously reported over-expression of BMP4 in diffuse-type gastric cancer cells. BMP signaling is regulated by tissue-specific expression of ligands and receptors as well as by secreted-type antagonists, such as CKTSF1B1 (Gremlin), CER1 (Cerberus 1), Noggin, SOSTDC1 (Ectodin), and Chordin. Here, we identified two novel genes related to CKTSF1B1 and CER1 by using bioinformatics. Two novel members of human CKTSF1B gene family were designated CKTSF1B2 (GREM2 or PRDC) and CKTSF1B3 (GREM3 or DANTE). FLJ21195 (BC046632.1) was the representative human CKTSF1B2 cDNA, and CKTSF1B2 gene was mapped to human chromosome 1q43. Human CKTSF1B2 showed 94.0% total amino-acid identity with mouse Cktsf1b2 (Prdc). FLJ38607 (AK095926.1) was the representative human CKTSF1B3 cDNA, and CKTSF1B3 gene was mapped to human chromosome 19p13.2. Human CKTSF1B3 showed 61.9% total amino-acid identity with mouse Cktsf1b2 (Dante). N-terminal signal peptide and DAN domain with nine cysteine residues were conserved among CKTSF1B1, CKTSF1B2, CKTSF1B3 and CER1. Phylogenetic analyses revealed that CKTSF1B2 was more related to CKTSF1B1, and that CKTSF1B3 was more related to CER1. CKTSF1B1, CKTSF1B2, CKTSF1B3 and CER1 constitute the CKTSF1B family among secreted-type cysteine knot superfamily proteins. This is the first report on identification and characterization of the human CKTSF1B2 and CKTSF1B3 genes.

Identification of NOL8, a nucleolar protein containing an RNA recognition motif (RRM), which was overexpressed in diffuse-type gastric cancer.

In an attempt to identify novel therapeutic targets for diffuse-type gastric cancer, we had previously compared expression profiles of 20 diffuse-type gastric-cancer tissues with corresponding non-cancerous mucosae by means of a cDNA microarray consisting of 23,040 genes. Among 153 genes whose expression levels were elevated in cancers compared to non-cancerous mucosae, we focused on a gene termed NOL8 that encodes a putative 150-kDa protein with an RNA-recognition motif (RRM) domain in its amino-acid terminal region. Comparison of expression profiles between diffuse-type and intestinal-type gastric cancers showed that NOL8 was specifically up-regulated in diffuse-type cancers. Northern blot analysis revealed that NOL8 was expressed in skeletal muscle, but not expressed or hardly detectable in 22 other tissues examined. Immunocytochemical staining of NOL8 showed specific localization in the nucleolus. Subsequent protein phosphatase analysis coupled with western analysis revealed the presence of the phosphorylated form. Furthermore, transfection of short-interfering RNA (siRNA) specific to NOL8 into three diffuse-type gastric cancer cells, St-4, MKN45 and TMK-1, effectively reduced expression of this gene and induced apoptosis in these cells. These findings provide a new insight into diffuse-type gastric carcinogenesis and may contribute to the development of new therapeutic strategies for diffuse-type gastric cancer.

Trefoil factors and human gastric cancer (Review)

TFF1/pS2, TFF2/SP and TFF3/ITF are soluble peptides with trefoil domain(s) and C-terminal dimerization domain, which are conserved among human, cow, mouse and rat. TFF1 mRNA is expressed in stomach (mucous cells in fundus and antrum), TFF2 mRNA in stomach (mucous neck cells in fundus and basal cells in antral and pyloric glands) and duodenum (Brunner's gland), TFF3 mRNA in small intestine and large intestine (goblet cells). Expression of TFF1, TFF2 and TFF3 mRNAs are differentially regulated by FGF2/bFGF, FGF7/KGF, estrogen, aspirin, arachidonic acid, X-ray irradiation, and hydrogen peroxide. Gastric cancer is classified into the intestinal type and the diffuse type. TFF mRNAs are preferentially expressed in diffuse-type gastric cancer cells. Custom-made microarray (TFF mRNAs) and ELISA (TFF proteins) might be applicable for screening methods of peritoneal and bone marrow dissemination from diffuse-type gastric cancer. TFF1 and TFF2 mRNAs are frequently down-regulated in intestinal-type gastric cancer. TFF1 gene, inactivated by deletion, missense mutation and promoter hypermethylation, is a tumor suppressor gene implicated in gastric cancer. TFF2 is a candidate tumor suppressor gene; however, genetic and epigenetic alterations of TFF2 gene in human gastric cancer remain unclear. TFF1, TFF2 and TFF3 play key roles in mucosal protection through mucous-barrier formation, and also in mucosal repair through promotion of restitution after injury. Patients with chronic atrophic gastritis and those with ulcerative colitis are at risk of gastric cancer and colorectal cancer, respectively. TFF1, TFF2 and TFF3 proteins might be applicable for chemoprevention of gastrointestinal cancer associated with chronic persistent inflammation.

Expression of TFF1, TFF2 and TFF3 in gastric cancer

Expression of some members of the trefoil factor (TFF) and the WNT gene families is regulated together by estrogen. We have cloned and characterized human WNT signaling molecules using bioinformatics, cDNA-library screening and cDNA-PCR to investigate expression profile of WNT signaling molecules in human gastric cancer. Here, we investigated expression profile of TFF1/pS2, TFF2/SP and TFF3/ITF in human gastric cancer. Among 7 gastric cancer cell lines, TFF1 was expressed in OKAJIMA, TMK1, MKN45, and KATO-III, TFF2 in KATO-III, and TFF3 in MKN45 and KATO-III. TFFs were preferentially expressed in diffuse-type gastric cancer cell lines. Expression of TFFs in primary gastric cancer was next investigated. TFF1 was down-regulated in 7 cases out of 12 cases (58.3%) of primary gastric cancer. TFF2 was down-regulated in 10 out of 12 cases (83.3%) of primary gastric cancer. TFF3 was down-regulated in 2 out of 12 cases (16.7%) of primary gastric cancer, and was up-regulated in 5 out of 12 cases (41.7%). TFF1 and TFF2 were frequently down-regulated in primary gastric cancer, while TFF3 was up-regulated in some cases of primary gastric cancer. This is the first report on comprehensive expression analyses on TFFs in gastric cancer.

Sequential Numerical Changes of Chromosomes 7 and 18 in Diffuse-Type Stomach Cancer Cell Lines: Combined Comparative Genomic Hybridization, Fluorescence In Situ Hybridization, and Ploidy Analyses

Sequential changes of chromosomal copy number were analyzed retrospectively in five diffuse-type gastric cancer cell lines by comparative genomic hybridization (CGH), DNA cytometry, and fluorescence in situ hybridization (FISH) with centromeric and painting probes. By CGH, we found loss of 18q21 in all of the cell lines and gains of 7p11–q31, 20q, and 22 in four of the five cell lines. Actual copy numbers of chromosomes 7 and 18 were determined by FISH: disomy 18 with (partial) loss of 18q in the two DNA-diploid cell lines (AGS and MKN-45), trisomy 7 in MKN-45, disomy 18 and tetrasomy 7 with one-copy loss of 7p and one-copy gain of 7q tip in DNA-triploid HSC-39/40A, and trisomy 18 and hexasomy 7 with one-copy loss of 7q in DNA-tetraploid KATO-III. Because the DNA aneuploidy is thought to result through tetraploidization, and the duplicated chromosomal changes in DNA aneuploid tumors seem to precede tetraploidization, the duplicated gain of chromosome 7 and one-copy loss of 7q in KATO-III were inferred to have occurred before and after tetraploidization, respectively. Similarly, HSC-39/40A were inferred to be preceded by the DNA-diploid stage with disomy 7 and monosomy 18. As the loss of 18q21 and the gain of 7p11–q31 were inferred to have occurred already in the DNA diploid stage in at least four and two of the cell lines, respectively, the 18q21 loss may be more important than the 7q gain as an earlier event in the genesis of diffuse-type stomach cancer. The combined CGH, FISH, and ploidy analyses thus give us a clue to extract important earlier events from the chromosomal changes that were screened by CGH alone.

Alterations and hypermethylation of thep14ARF gene in gastric cancer

p14(ARF), generated through an alternative splicing process that replaces the first exon, 1alpha, of p16(INK4a) with exon 1beta, located >15 kb upstream of exon 1alpha, has been shown to function as a growth suppressor. We examined 11 gastric cancer cell lines for mRNA expression, homozygous deletion, mutation, and promoter methylation of the p14(ARF) gene. No mRNA expression was detected in 5 of the 7 diffuse-type cell lines. All intestinal cell lines displayed normal levels of expression except for one with a low level of expression. Of the 5 cell lines without expression, 3 (MKN45, NUGC-2, and NUGC-4) and 1 (KATO III) displayed homozygous deletion and methylation of the p14(ARF) gene, respectively. No mutation was found in the whole coding region of the p14(ARF) gene in 8 cell lines without homozygous deletion. Our results indicate that the p14(ARF) gene is more frequently inactivated by homozygous deletion or methylation in diffuse-type gastric cancer cell lines (5/7, 71.4%) than in intestinal ones (0/4, P = 0.022). When we also analyzed 62 primary gastric cancers for the methylation status of the p14(ARF) promoter region, the methylation frequency tended to be higher in diffuse-type gastric cancers (15/33, 45.5%) than in intestinal ones (7/28, 25%). Thus, p14(ARF) alterations might be involved in diffuse-type gastric carcinogenesis.