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

Abstract

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.