Gene expression levels of  4-galactosyltransferase 5 correlate with the tumorigenic potentials of B16-F10 mouse melanoma cells

Abstract

Our previous studies showed that mouse β4-galactosyltransferase 5 (β4GalT5) is a lactosylceramide (Lac-Cer) synthase, and that its gene expression increases by 2- to 3-fold upon malignant transformation of cells. In the present study, we examined whether or not the tumorigenic and metastatic potentials of B16-F10 mouse melanoma cells can be suppressed by reducing the expression of the β4GalT5 gene. We isolated a stable clone named E5 whose β4GalT5 gene expression level was reduced to 35% that of a control clone C1 by transfection of its antisense cDNA. Thin-layer chromatography analysis of glycosphingolipids showed that the amounts of Lac-Cer and ganglioside GM3 are significantly less in clone E5 than in clone C1. Clone C1 and E5 cells were each transplanted subcutaneously or injected intravenously into C57BL/6 mice, and the sizes of tumors and numbers of colonies formed in the lungs were determined. The average tumor size and average number of colonies formed with clone E5 were decreased to 44 and 49%, respectively, of those formed with clone C1. Furthermore, the numbers and sizes of colonies formed in the soft agarose gels, and the volumes of tumors formed in athymic mice with fibroblasts from wild type, heterozygous and homozygous β4GalT5-knockout mouse embryos upon transformation with the polyoma virus oncogene correlated with the β4GalT5 gene dosage. These results strongly indicate that the amounts of Lac-Cer synthesized by β4GalT5 correlate with the tumorigenic potentials of malignantly transformed cells.

Downregulation of Transcription Factor Sp1 Suppresses Malignant Properties of A549 Human Lung Cancer Cell Line with Decreased β4-Galactosylation of Highly Branched <i>N</i>-Glycans

“…16,17) After being cultured for 72 h, the plasmid-transfected cells were selected with DMEM containing 10% FCS and hygromycin B (500 µg/mL) for two weeks.…”

“…Our previous studies demonstrated that the malignant properties of cancer cells are suppressed by regulation of the expression levels of the β4GalT genes, 16,17) indicating that the changes of cell surface glycosylation contribute to the suppression of malignant properties of cancer cells. Therefore, if Sp1-expression is downregulated by RNA interference (RNAi), not only the expression of cancer-related molecules but also glycosylation patterns can be changed in accordance with the altered gene expression of glycosyltransferases, which may lead to the suppression of malignant properties of cancer cells.…”

Novel Screening Method for Anti-Colon Cancer Drugs Using Two Sensor Cell Lines with Human &beta;4-Galactosyltransferase 4 Gene Promoters

“…The cell lysates were prepared from the hGT4-0.3-sensor cells treated with 20 μM U0126 or DMSO for 24 h. Immunoblot analysis using the antibodies against p44/42 MAPK and phosphorylated p44/42 MAPK was conducted, and the band intensity was quantified as the method described previously [8,17].…”

“…There are seven members of the β4GalT family [7]. Previously, we showed that by regulating the expression level of the β4GalT5 gene, the malignant properties of cancer cells are suppressed [8]. The findings suggest that if the inhibitors for the expression of the β4GalT genes, which relate to the malignant potentials of specific cancer cell types, are discovered, the inhibitors are useful for cancer therapy.…”

Transcriptional Mechanism of the β4-Galactosyltransferase 4 Gene in SW480 Human Colon Cancer Cell Line

“…18) In brief, we designed the primer pairs for amplification of the β4GalT4 mRNA 5′-UTR 1, TS27-36 (5′-AGC TCG CCG CGG CCG CCT C-3′) and TS27-38 (5′-ATG AAG CCA CAA AGT GCC AC-3′), while for amplification of the β4GalT4 mRNA 5′-UTR 2, TS27-37 (5′-ACC TTT GAC CTT TCT CCA AA-3′) and TS27-38. The amplification efficiency of each primer pairs was calculated from the slope of the standard curve, and was 123.1 and 122.8% for the amplicons of the β4GalT4 mRNA 5′-UTR 1 and 5′-UTR 2, respectively.…”