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.
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 (approximately 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 43 bp 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.
Iwamoto
S
S
Pacific Northwest Research Institute, and Dept. of Pathobiology, Univ. of Washington, Seattle 98122-4327, USA.
Withers
D A
DA
Handa
K
K
Hakomori
S
S
D000017:ABO Blood-Group System / Q000235:genetics* / Q000276:immunology; D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D022762:CCAAT-Enhancer-Binding Proteins; D002999:Clone Cells / Q000276:immunology / Q000378:metabolism; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020080:Tandem Repeat Sequences / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000017:ABO Blood-Group System; D000951:Antigens, Neoplasm; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase