The Mechanism Of Tn Antigen Expression In Human Colon Cancer Cell Line HT-29

Objective:Tn~+ cells were separated from human colon cancer cell line HT-29, which derived from a female patient, to investigate the mechanism of Tn antigen expression in these cells, and then provide new target points or theoretical basis for the diagnosis and therapy of human colorectal carcinoma.Methods:Human tumor cell lines including colon cancer cells HT-29, gastric carcinoma cells BGC823 and lymphoma cells Jurkat T were cultured in RPMI1640 or DMEM plus 10% FBS at 37℃ and 5% CO2. Peripheral blood mononuclear cells (PBMC) were separated from a female’s whole blood cells with the method of density gradient centrifugation. HT-29-Tn~+ and HT-29-Tn- cells were sorted from HT-29 cells using immune microbeads based on their Tn antigen expression or not. The status of Tn antigen expression were detected by flow cytometry (FCM). Total RNA, genome DNA (gDNA) and cytoplasmic protein were extracted from tumor and normal cells respectively. Cosmc cDNA were amplified using RNA as template by RT-PCR. Coding sequence (CDS), non-coding sequence (NCDS) and CpG islands of Cosmc DNA were amplified by PCR using gDNA as templates. All amplified products were analyzed on 1% or 1.5% TAE agrose gel. The expected bands were excised, purified and then sequenced to detect whether the gene mutation or SNP sites exsit in Cosmc. Methylated frequency of Cosmc CpG islands were detected by Bisufite Sequenced PCR (BSP). Wild type Cosmc (WtCosmc) were transfected into Tn antigen positive and Tn antigen negative cells, and the expression of Cosmc protein were then analyzed by Western blot. T-synthase activity was measured using florescence assay before and after WtCosmc transfection.Results:The deletion of Cosmc CDS and loss of T-synthse activity were detected in HT-29-Tn~+ cells, but there was no mutation of Cosmc exsit in HT-29-Tn- cells. Furthermore, while the SNP sites of NCDS and ～50% methylation frequency of CpG islands of Cosmc were examined in female normal cells, none of them were present in HT-29-Tn~+ and HT-29-Tn- cells. Additionally, after WtCosmc transfection, Tn antigen expression decreased but T-synthase activity increased significantly in Tn antigen positive cells.Conclusion: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 has been deleted in HT-29 cells, which has no influence on Cosmc function. WtCosmc transfection can restore T-synthase activity and decrease Tn antigen expression in Tn positive cells.