Expression of PNA‐binding sites on specific glycoproteins by human melanoma cells is associated with a high metastatic potential

Abstract

Lectin-binding patterns of seven human melanoma clones and variants selected from the same parental cell line and differing in their spontaneous metastatic potential in an animal model were compared by flow cytometry and Scatchard analysis. Human melanoma clones and variants with high and low metastatic potential could be distinguished by their peanut agglutinin (PNA)-binding patterns, but not by their wheat germ agglutinin (WGA)-, Ulex europaeus agglutinin I (UEA I)-, and soybean agglutinin (SBA)-binding patterns. Low metastatic clones and variants proved to be made up of single poorly peanut agglutinin-binding cell population (2.20-3.52 x 10(6) sites/cell, Ka = 2.48-2.75 x 10(6) M-1). By contrast, highly metastatic variants were found to be constituted by two cellular subpopulations, exhibiting respectively a moderate 2.62-3.72 x 10(6) sites/cell) and a high peanut agglutinin staining (17.68-18.76 x 10(6) sites/cell). One highly metastatic clone was found to be homogeneously constituted by a single population of cells strongly binding this lectin (18.86 x 10(6) sites/cell) with an association constant of 4.06 +/- 10(6) M-1. Using an EPICS V cytometer, these two subpopulations were sorted from a highly metastatic variant and tested for their metastatic abilities: cells with high PNA binding generated a higher frequency of metastases than did moderately PNA-binding cells. Following treatment with Vibrio cholerae neuraminidase, all cells from all variants and clones were brightly labeled by PNA, collecting in a single peak with similar fluorescence intensities. Electrophoresis of total cellular proteins and subsequent detection with labeled PNA om Western blots show two major PNA-reactive glycoproteins with apparent molecular weights of 140 and 110 kDa (MAGP1 and MAGP2), expressed only in highly metastatic cells, but which can be strongly labeled by PNA in slightly metastatic cells following a treatment with neuraminidase. These results provide evidence that the expression of terminal galactose (beta 1-3)N-acetyl galactosamine structure, positioned on MAGP1 and MAGP2 glycoproteins, is associated with the metastatic potential of human melanoma cells.

The gangliosides as a possible molecular coupling factor between the proportion of radiosensitive cells in vitro and the metastatic potential in vivo within a human melanoma cell line

“…The variant Tlp26 was established in culture after two direct successive transplantations of the M4Be tumours into immunosuppressed newborn rats (Bailly et al, 1993). The variant Tlp26 is itself heterogeneous, containing two populations of cells that were separated by flow cytometry for their ability to bind the PNA lectin (peanut agglutinin lectin); the PNA recognizes specific glycoproteins at the cell surface, and the binding is higher for the subvariant Tlp26R than the subvariant Tlp26L; the metastatic potential of the subvariant Tlp26R is significantly higher than that of the subvariant T1p26L (Zebda et al, 1994; Table 1). The clones TlCll, T1C6 and T1C3 were obtained by limiting dilution from the Tlp26 variant.…”

“…The clones TlCll, T1C6 and T1C3 were obtained by limiting dilution from the Tlp26 variant. With the exception of the T1C6 and TICI1 clones, the derivation of the variants and clones from the M4Be cell line has been previously described (Zebda et al, 1994). The common origin of all these cell sublines is attested by their karyotype and by their sharing common marker chromosomes with the parental cell line M4Be; the cells appeared hypertriploid, showing quite similar modal numbers (around 70) (Bailly and Dore, 1992).…”

“…A significant correlation exists between X and the metastatic potential: the higher the value of X, the lower the metastatic potential (P < 0.006, ANOVA analysis) with M4Be and clone 4 ( Figure 3B). Figure 3C shows the survival curves obtained from variant 1 and the two subvariants 1+ and 1-; the two subvariants were separated by flow cytometry for their ability to bind PNA, a lectin recognizing specific glycoproteins at the cell surface (Zebda et al, 1994). The subvariant 1+, which binds more PNA than the subvariant 1-, is much more radiosensitive, while an intermediate response to radiation is observed for variant 1.…”

Deficiency of ganglioside biosynthesis in metastatic human melanoma cells: relevance of CMP‐NeuAc: LacCer α2‐3 sialyltransferase (GM3 synthase)

“…The procedures for selection and cloning and the culture conditions have been described elsewhere [18,20]. Cell cultures were regularly tested for absence of mycoplasma and preserved metastatic capability.…”

“…In a spontaneous metastasis model of human melanoma recently established in our laboratory [18,19], PNA-binding glycoproteins are expressed in human melanoma cells able to give lung metastases whereas these cell surface glycoproteins in non-metastatic cells are sialylated and do not bind PNA lectin [20]. Sialyltransferases being also essential enzymes in the biosynthesis of gangliosides, we determined the patterns of gangliosides of metastatic (T 1 C3) and non-metastatic (IC8) clones of our melanoma model, as well as those of the corresponding tumors grown in immunosuppressed newborn rats.…”