Challenge to the suppression of tumor growth by the β4-galactosyltransferase genes

Abstract

It has been well established that structural changes in glycans attached to proteins and lipids are associated with malignant transformation of cells. We focused on galactose residues among the sugars since they are involved in the galectin-mediated biology, and many carbohydrate antigens are frequently expressed on this sugar. We found changes in the expression of the β4-galactosyltransferase (β4GalT) 2 and 5 genes in cancer cells: decreased expression of the β4GalT2 gene and increased expression of the β4GalT5 gene. The growth of mouse melanoma cells showing enhanced expression of the β4GalT2 gene or reduced expression of the β4GalT5 gene is inhibited remarkably in syngeneic mice. Tumor growth inhibition is probably caused by the induction of apoptosis, inhibition of angiogenesis, and/or reduced MAPK signals. Direct transduction of human β4GalT2 cDNA together with the adenovirus vector into human hepatocellular carcinoma cells grown in SCID mice results in marked growth retardation of the tumors. β4GalT gene-transfer appears to be a potential tool for cancer therapy.

Figure 1.

A variety of carbohydrate antigens expressed on the Galβ1→4GlcNAc groups of N- and O-glycans and a neolacto-series of GSLs. a, polysialic acid; b, HNK-1 carbohydrate; c, poly-N-acetyllactosamine; d, blood group O-antigen; e, blood group A-antigen; f, blood group B-antigen; g, Lewis X; h, sialyl Lewis X; and i, Lewis Y.

Figure 2.

Human β4GalT family proteins. For each transferase, the closed box indicates a putative transmembrane domain (TM), and other boxes in blue, green and red indicate amino acid sequences conserved among β4GalTs 1, 2, 3, 4, 5 and 6 but not β4GalT7. The positions of Cys residues (C) are also indicated. The total number of amino acids is shown at the right.

Figure 3.

Comparison of the expression of β4GalT genes between NIH3T3 and MTAg cells. Blots containing poly (A)⁺ RNA preparations from NIH3T3 (normal) and MTAg (transformed) cells were probed with [³²P]-labeled mouse β4GalT1–6 or β-actin cDNA fragment. The sizes of the transcripts are shown to the right of lane MTAg.

Figure 4.

Growth curves of clone C2 and clone D4 cells (panel a) and their morphological appearances at the sub-confluence in culture (× 80) (panel b). Open and closed circles in panel a indicate clone C2 and clone D4 cells, respectively. (Modified from ref. with permission from Nature Publishing Group.)

Figure 5.

Immunohistochemical stainings of clone C2- and clone D4-derived tumors. Tumor cryosections were stained for TUNEL assay (bar: 70 µm) or incubated with anti-CD31 antibody (bar: 200 µm) and anti-mouse Ki-67 antibody (bar: 70 µm), and visualized. Upper panels indicate clone C2-derived tumors and lower panels indicate clone D4-derived tumors. (Reproduced from ref. with permission from Nature Publishing Group.)

Figure 6.

Growth curves of clone C1 and clone E5 cells (panel a) and their morphological appearances at the sub-confluence in culture (× 100) (panel b). Open and closed circles in panel a indicate clones C1 and E5 cells, respectively. (Modified from ref. with permission from Oxford University Press.)

Figure 7.

Tumors and their GSL compositions. Tumors formed with clone C1 and clone E5 cells in mice two weeks after subcutaneous transplantation are shown in panel a. Panel b shows the GSL components of tumors formed with clone C1 and clone E5 cells. Neutral (left lanes) and acidic (right lanes) GSLs separated on TLC plates are shown. (Reproduced from ref. with permission from Oxford University Press.)

Figure 8.

Tumorigenic potentials of oncogene-transformed MEF cells. Panel a: Numbers of colonies formed from transformed MEF cells in soft agarose gels. Panel b: Sizes of colonies formed from transformed MEF cells in soft agarose gels. Panel c: Volumes of tumors formed from transformed MEF cells in athymic mice three weeks after transplantation. Panel d: Representative tumors formed from transformed MEF cells. +/+, +/−, and −/− indicate MEF cells isolated from β4galt5^+/+-, β4galt5^+/−-, and β4galt5^−/−-mice, respectively, and transformed with the viral oncogene. *p < 0.05 compared with β4galt5^+/+-MEF cells. (Modified from ref. with permission from Oxford University Press.)

Figure 9.

Immunoblot of proteins involved in the MAPK pathway. Blots incubated with antibody against Ras to detect Ras (a), GTP-bound Ras (b), and GDP-bound Ras (b′), with anti-c-Raf antibody (c), anti-phospho-c-Raf antibody (d), anti-MEK antibody (e), anti-phospho-MEK antibody (f), anti-ERK antibody (g), anti-phospho-ERK antibody (h) and anti-G3PDH antibody (i), are shown. (Reproduced from ref. with permission from Oxford University Press.)

Figure 10.

Tumor growth of HuH-7 hepatocellular carcinoma cells injected with Ad/β4GalT2. Panel a: tumors formed from HuH-7 cells transplanted subcutaneously into SCID mice were injected with Ad/β4GalT2 (closed circles) or Ad/mock (open circles), and tumor volumes are shown. Panel b: representative SCID mouse carrying tumors that were injected intratumorally with Ad/mock as indicated by the red arrow (left flank) or Ad/β4GalT2 indicated by the yellow arrow (right flank). Panel c: RT-PCR analysis of the β4GalT2 transcript in tumors injected with Ad/mock (lane C) and Ad/β4GalT2 (lane E). Panel d: β4GalT activities toward GlcNAcβ-S-pNP in tumors injected with Ad/mock (open circles with C) and Ad/β4GalT2 (closed circles with E). Panel e: Lectin blot analysis of membrane proteins from tumors injected with Ad/mock (lanes C) and Ad/β4GalT2 (lanes E). Blots were stained with CBB or incubated with Con A or RCA-I followed by visualization. (Reproduced from ref. with permission from Nature Publishing Group.)