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 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
Published Version
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