Sialylation and glycosylation modulate cell adhesion and invasion to extracellular matrix in human malignant lymphoma: Dependency on integrin and the Rho GTPase family

Abstract

To determine the biological roles of cell surface glycosylation, we modified the surface glycosylation of human malignant lymphoma cell lines using glycosylation inhibitors. The O-glycosylation inhibitor, benzyl-α-GalNAc (BZ) enhanced the fibronectin adhesion of HBL-8 cells, a human Burkitt's lymphoma cell line, and of H-ALCL cells, a human anaplastic large cell lymphoma cell line, both of which were established in our laboratory. The N-glycosylation inhibitor, tunicamycin (TM) inhibited the surface expression of Phaseolus vulgaris leukoagglutinating (L-PHA) lectin- and Canavalia ensiformis (ConA) lectin-reactive oligosaccharides in the HBL-8 cell line. Assay of the adhesion of HBL-8 cells to fibronectin showed that fibronectin adhesion is mediated by the integrin very late antigen (VLA)-4 and that not only BZ but also TM treatment enhanced HBL-8 cell adhesion to fibronectin. Furthermore, although BZ treatment also enhanced H-ALCL cell adhesion to fibronectin, this effect was not mediated by VLA-5 or the RGD sequence of fibronectin. We also showed that H-ALCL cell adhesion to galectin-3 was enhanced by pre-treatment with neuraminidase, which cleaves cell surface sialic acid. Additionally, H-ALCL cell adhesion to galectin-3 was inhibited by pre‑treatment with the RGD peptide suggesting that cell adhesion to galectin-3 is mediated by integrin (VLA-5). Furthermore, H-ALCL cell invasion of galectin-1 and galectin-3 was inhibited by pre-treatment with the RGD peptide. Therefore, cell adhesion to and invasion of galectin-1 and galectin-3 are integrin-dependent. In addition to these findings, cell adhesion to galectin-3 was markedly inhibited by treatment with β-lactose compared to treatment with sucrose. Therefore, interactions between integrins and galectin-3 may be mediated through β-galactose that is linked to glycans of integrins. AZA1, an inhibitor of Ras homolog oncoprotein (Rho) GTPase family proteins, RAS-related C3 botulinus toxin substrate 1 (Rac 1) and Cell division control protein 42 homolog (Cdc42) markedly inhibited cell invasion of galectin-1 and galectin-3 suggesting that Rac 1 and Cdc42 may be involved in the regulation of H-ALCL cell invasion of galectins. In conclusion, artificial modification of cell surface glycosylation revealed the biological roles of glycosylation in the adhesion to and invasion of the extracellular matrix (ECM) by human malignant lymphoma cell lines. These findings will provide new insight into the glycobiology of human malignant lymphoma.

Figure 1

Cell surface expression of VLA integrins. The expression of the integrins VLA-1 to VLA-5 on the surface of HBL-8 3G3 cells was analyzed using flow cytometry. The filled area is a control experiment, and the thick line indicates VLA expression. The data shown are representative of two independent experiments.

Figure 2

Effect of alteration of cell surface O- or N-linked oligosaccharides on lectin reactivity. Effect of inhibition of glycosylation in the HBL-8 3G3 cloned cells on their surface lectin reactivity was analyzed using FACS analysis. (A) HPA lectin reactivity without (thick line) or with (dotted line) BZ treatment. (B) L-PHA or ConA reactivity without (thick dotted line) or with (thin dotted line) SW treatment. (C) L-PHA or ConA reactivity without (thick dotted line) or with (thin dotted line) TM treatment. The filled area is the control experiment with avidin-FITC only. Data shown are representative of two independent experiments.

Figure 3

Effect of alteration of cell surface glycosylation on cell adhesion to fibronectin. The adhesion of cells to fibronectin, and the effect of anti-VLA-4 antibodies or alteration of cell surface glycosylation on this adhesion were assayed using fibronectin coated culture plates. Adhesion was monitored by measurement of absorption at 570–655 nm. (A) Effect of anti-VLA-4 or isotype control (cont) antibodies on HBL-8 3G3 cloned cell adhesion to plates coated with the indicated concentrations of fibronectin (fibro) (^*p=0.0003, ^**p=0.0035). (B) Effect of BZ or control (cont) treatment on adhesion to fibronectin (fibro) of HBL-8 3G3 cells (B-1) (^*p=0.0327, ^**p=0.0194) or of H-ALCL cells (B-2) (^*p=0.004). Data shown are representative of two independent experiments performed in triplicate. (C) Effect of SW or control (cont) treatment on adhesion to fibronectin (fibro) of HBL-8 3G3 cells (NS, not significant). Data shown are representative of three independent experiments performed in triplicate. (D) Effect of TM or control (cont) on HBL-8 3G3 cloned cell adhesion to fibronectin (^*p=0.0136, ^**p=0.0010, ^***p=0.0126). Data shown are representative of two independent experiments performed in triplicate. p-values were calculated based on Student's t-test.

Figure 4

BZ enhancement of H-ALCL cell adhesion to fibronectin is not mediated by VLA-5, or by CS-1 or RGD sequences. H-ALCL cells that were treated with BZ or with ethanol control were incubated with anti-VLA-5 or control antibodies (Ab) or with the CS-1 or RGD peptides and fibronectin adhesion was then assayed as in Fig. 3 (^*p=0.001, ^**p=0.00006, ^***p=0.00001, ^****p=0.0001). The data shown are representative of two independent experiments performed in triplicate. p-values were calculated based on Student's t-test.

Figure 5

Effect of neuraminidase treatment and the RGD sequence on galectin binding of H-ALCL cells. Cell adhesion to galectin-coated plates was analyzed by determination of the OD at 570 nm in an adhesion assay. (A) The effect of pre-treatment of H-ALCL cells with neuraminidase on cell adhesion to galectin-3 was analyzed (^*p=0.039). (B) The effect of pre-treatment of cells in serum-free medium with the RGD peptide (RGD) or with PBS control (cont) on H-ALCL cell adhesion to galectin-3 was analyzed (^*p=0.02). Data are representative of two independent experiments performed in triplicate.

Figure 6

Further analysis of the mechanism of H-ALCL interaction with galectin. (A) Effect of pre-treatment with the RGD peptide (RGD) or PBS control (cont) for 48 h on H-ALCL cell invasion of galectin-1 and galectin-3 (^*p=0.00002, ^**p=0.008). Data are representative of two independent experiments performed in triplicate. (B) Effect of pre-incubation with β-lactose, sucrose or control (cont) on H-ALCL cell adhesion to galectin-3 (^***p=0.001).

Figure 7

Immunohistochemical analysis of the expression of Rac 1 and Cdc42 proteins in the cytoplasm of H-ALCL cells. The H-ALCL cells were cytospun onto glass slides and were then fixed by 100% ethanol. Subsequently, the immunohistochemical staining was performed using standard methods.

Figure 8

Effect of an inhibitor of Rac 1 and Cdc42 on H-ALCL cell invasion of galectin-1 and galectin-3. H-ALCL cells were incubated with PBS control (cont) or with the Rac 1 and Cdc42 inhibitor, AZA1, following which cell adhesion to galectin-1 (GAL-1) or galectin-3 (GAL-3) was assayed (^*p=0.004, ^**p=0.008). Data are representative of two independent experiments that were performed in triplicate.

Figure 9

Schematic representation of the biological roles of cell surface glycosylation in human malignant lymphoma cell adhesion to and invasion of the extracellular matrix.