The mechanism of Tn antigen expression in human colon cancer HT-29 cells / 中华微生物学和免疫学杂志

ABSTRACT

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 im-mune magnetic beads.Total RNA, genomic DNA ( gDNA) and cytoplasmic proteins in these cells were ex-tracted 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 ana-lyzed 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 ( Wt-Cosmc) were transfected into tumor cell lines and normal cells to define the function of Cosmc.The expres-sions 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.