Infection by the bacterium Helicobacter pylori is a putative cause of various gastric disorders, including gastric adenocarcinoma. Incident rates are associated with variants of the H. pylori virulence factor cytotoxin-associated gene A protein (CagA), encoded by the gene cagA. However, these variants have not been characterized in China, where gastric cancer is common. We investigated the diversity of CagA variants in H. pylori strains isolated from a Chinese population. The 3' variable region of cagA genes from 66 clinical isolates in China were amplified by polymerase chain reaction, sequenced, aligned, and analyzed. All 66 H. pylori strains were CagA-positive, of which 93.9% were East Asian type and the tyrosine phosphorylation motifs (TPMs) were EPIYA-ABD. The remainder was Western type, in which TPMs were EPIYA-ABC. Interestingly, two of sixty-two strains (3.2%) of the East Asian type were mutated into ESIYA-B, whereas all four Western type (100%) strains were mutated into EPIYT-B. Both of the two strains with Western-type CagA obtained from gastric cancer patients contained a distinguished mutation on the first residue following the EPIYA site in the EPIYA-A motif. The predominant CagA type in these H. pylori strains isolated from Chinese patients in China was East Asian, with TPMs EPIYA-ABD, and there existed mutations in both the East Asian and Western type CagA.
Objective
To explore the mechanism of Tn antigen expression in colon cancer cells HT-29.
Methods
Tn(+ ) and Tn(-) cells were separated from human colon cancer cell line HT-29 by immune magnetic beads. Total RNA, genomic DNA (gDNA) and cytoplasmic proteins in these cells were extracted by using Trizol, DNA preparation kit and nuclear and cytoplasmic extraction reagents respectively. T-synthase activity was measured by a fluorescent assay. Cosmc and T-synthase transcriptional levels were analyzed by RT-PCR using mRNA as the templates. The coding sequence (CDS) and CpG islands of Cosmc were amplified by PCR using gDNA as the templates. Amplified products were analyzed on 1% agarose gel. The expected bands were purified, and then sequenced to examine Cosmc mutation. Wild type Cosmc (WtCosmc) were transfected into tumor cell lines and normal cells to define the function of Cosmc. The expressions of Cosmc protein in these cells were then examined by Western blot.
Results
Although no mutation appeared in HT-29-Tn(-) cells, the deletion of CDS and inactivity of T-synthase were observed in HT-29-Tn(+ ) cells. Additionally, transfected WtCosmc restored T-synthase activity and then decreased Tn antigen expression in Tn antigen positive cells.
Conclusion
The absence of CDS in Cosmc gene resulted in the loss of T-synthase activity and consequent Tn antigen expression in HT-29-Tn(+ ) cells.
Key words:
Tn antigen; HT-29 cells; Cosmc; Mutation
A novel Gram-stain-negative bacterium, designated strain BH-SD16T, was isolated from a marine sediment sample collected in the Bohai Sea. Cells of strain BH-SD16T are aerobic, non-flagellated oval-shaped rods, showing oxidase- and catalase-positive activities. Growth occurs between 15-45 °C (optimum, 30 °C), at pH 6.0-9.0 (pH 7.0-7.5) and with 1-10 % (w/v) NaCl (3.0 %). Strain BH-SD16T contains C18 : 1ω7c (49.2 %), C16 : 0 (17.7 %) and C18 : 1ω7c 11-methyl (16.6 %) as the predominant fatty acids and ubiquinone-10 as the major respiratory quinone. The major polar lipids comprise phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol and two glycolipids. The size of the draft genome is 3 442 538 bp, including 3213 protein-coding genes, 40 tRNA genes and three rRNA genes, and the DNA G+C content is 63.4 mol%. Strain BH-SD16T shows the highest 16S rRNA gene sequence similarity to Pseudooctadecabacter jejudonensis (95.7 %), strains of the genus Octadecabacter(95.4-95.6 %) and strains of the genus Loktanella(93.8-95.4 %). Phylogenetic trees based on 16S rRNA gene sequences show that strain BH-SD16T forms a distinct lineage within the family Hyphomonadaceae, which is also confirmed in the multigenic phylogenetic tree calculated by RAxML. Based on the results of phenotypic, chemotaxonomic and phylogenetic analysis, strain BH-SD16T is considered to represent a novel genus and species in the family Hyphomonadaceae, for which the name Thalassorhabdomicrobium marinisediminis gen. nov., sp. nov. is proposed. The type strain is BH-SD16T (=CCTCC AB 2017073T=KCTC 62201T).
The Tn antigen, which arises from mutation in the Cosmc gene is one of the most common tumor associated carbohydrate antigens. Cosmc resides in X24 encoded by a single gene and functions as a specific molecular chaperone for T-synthase. While the Tn antigen cannot be detected in normal cells, Cosmc mutations inactivate T-synthase and consequently result in Tn antigen expression within certain cancers. In addition to this Cosmc mutation-induced expression, the Tn antigen is also expressed in such cell lines as Jurkat T, LSC and LS174T. Whether the Cosmc mutation is present in the colon cancer cell line HT-29 is still unclear. Here, we isolate HT-29-Tn+ cells from HT-29 cells derived from a female colon cancer patient. These HT-29-Tn+ cells show a loss of the Cosmc gene coding sequence (CDS) leading to an absence of T-synthase activity and Tn antigen expression. Additionally, almost no methylation of Cosmc CpG islands was detected in HT-29-Tn+ as well as in HT-29-Tn- and Tn- tumor cells from male patients. In contrast, the methylation frequency of CpG island of Cosmc in normal female cells was ~50%. Only one active allele of Cosmc existed in HT-29-Tn+ and HT-29-Tn- cells as based upon detection of SNP sites. These results indicate that Tn antigens expression and T-synthase inactivity in HT-29-Tn+ cells can be related to the absence of CDS in Cosmc active alleles, while an inactive allele deletion of Cosmc in HT-29 cells has no influence on Cosmc function.
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