Deletion of A-antigen in a human cancer cell line is associated with reduced promoter activity of CBF/NF-Y binding region, and possibly with enhanced DNA methylation of A transferase promoter
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Employing blood group A− and A+ clones derived from the same parental colonic cancer cell lines, we studied the molecular mechanism of deletion/ reduction vs. continuous expression of A antigen in A tumors, a crucial determinant of human tumor malignancy. A− transferase mRNA level in one of the A− clones (A− SW480) was undetectable, while that in A+ SW480 was strongly detectable by semiquantitative RT-PCR. Relatively lower (∼1/3) transcript level was detectable in another A− clone (A− HT29) in comparison to A+ HT29 by the same RT-PCR procedure, although none of these tumor cell lines showed detectable level of A transcript by Northern blotting or RNase protection methods. Therefore, subsequent studies were performed employing A− vs. A+ SW480 clones. Deletion of A transcript in A− cells was not due to gene deletion, since Southern blot analysis showed equal presence of genomic DNA regardless of A− vs. A+ (SW480 or HT29) or B+ (KATOIII) tumor cells. Two transcriptional control mechanisms leading to differences of A expression in SW480 cells are indicated.
i. Luciferase assay in A− and A+ SW480 cells showed that promoter activities of segments of 5′ flanking sequence of ABO gene reflected transcript levels in these cell lines. The enhancing activity of a 43bp tandem repeat unit located between −3899 to −3618 was reduced in A− compared to A+ cells.
ii. Distinct differences in the pattern of CpG dinucleotide methylation were found in A− vs. A+ cells. Therefore, the methylation process of A promoter DNA may be another important factor controlling A activity in SW480 tumor cells.
Since proliferation and motility of tumor cells are associated closely with A expression, transcription control mechanism for expression of A transferase as described above may be of crucial importance in defining human tumor malignancy.
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