Melanoma × Macrophage Fusion Hybrids Acquire Increased Melanogenesis and Metastatic Potential: Altered N‐Glycosylation as an Underlying Mechanism

Abstract

We recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential. With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content and increased responsiveness to MSH compared to parental cells. Here we investigated the hybrid melanotic phenotype in more detail, comparing the pigmentary systems of hybrids and parental Cloudman S91 cells by several techniques. Cells were studied by electron microscopy, cell lysates were analyzed for tyrosinase (E.C.1.14.18.1) activity, and melanosomal proteins were analyzed by gel electrophoresis and immunoblotting. Melanosomes in parental Cloudman melanoma cells were few in number and relatively amorphous, whereas those in the hybrids were numerous and heavily pigmented, containing highly organized lattice structures. Both basal and MSH-inducible tyrosinase activities were elevated several fold in hybrids compared to parental cells. Tyrosinase, TRP-2, and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages, which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. By using 3H-glucosamine as a marker of N-glycosylation, its incorporation into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors, and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.

GnT-V expression and metastatic phenotypes in macrophage–melanoma fusion hybrids is down-regulated by 5-Aza-dC: Evidence for methylation sensitive, extragenic regulation of GnT-V transcription

“…4B). These results on LAMP-1 gel migration were similar to previous studies on treatment of high metastatic hybrids with Swainsonine, a GnT-V inhibitor (Dennis et al, 1987;Sodi et al, 1998). Together the results indicated that 5-Aza-dC decreased LAMP-1 glycosylation through an inhibition GnT-V activity.…”

“…Besides, GnT-V-associated other phenotypes like increased potential to melanin synthesis and dendrite formation, as mentioned earlier (Rachkovsky et al, 1998;Sodi et al, 1998), was also found to be inhibited during this treatment (not shown). While these effects point to DNA methylation as a regulator of GnT-V expression, no methylation differences in the promoter region were detected between the high and low-metastatic hybrids before and after bisulfite treatment.…”

“…Molecular analyses revealed that these hybrids expressed macrophage-like glycosylation, especially upregulation of GnT-V and increased β1,6 branching of glycoproteins (Sodi et al, 1998;Pawelek et al, 1999;Chakraborty et al, 2001). GnT-V upregulation was observed at both the mRNA and protein expression levels (Chakraborty et al, 2001).…”

Leukocyte-Cancer Cell Fusion: Initiator of the Warburg Effect in Malignancy?

“…In summary, the host-tumor cell hybrid lung metastasis showed the same characteristics as macrophage-melanoma hybrids experimentally fused in vitro, regarding aneuploidy, chemotaxis, enhanced pigmentation, and high levels of autophagy. This was also a characteristic of another spontaneous melanoma-host hybrid described previously ("PADA") [18][19][20] Thus, experimental macrophage-melanoma hybrids in cell culture and spontaneous host-melanoma hybrids arising in vivo and adapted to culture all appeared to show constitutive autophagy without autophagic death. It is important to note that this occurred in cell culture under normoxic conditions and ample nutrients indicating that hybrids expressed the aerobic glycolysis pathway (the Warburg effect) that includes autophagy [42][43][44][45][46][47][48].…”

“…Curiously, in melanoma the most metastatic clones tended to be highly melanized compared to parental melanoma cells or weakly metastatic hybrids as described below ( Fig. 8.4) [19,20]. This was subsequently explained by the acquisition of macrophage glycosylation patterns involving β1,6-branched oligosaccharides that were associated with many phenotypic changes, including pigmentation [21] In two separate rounds of isolation, a total of 75 clones of PEG-fused macrophage-melanoma hybrids were isolated in vitro.…”