Glycosphingolipids of Metastatic Variant RNA Virus-Transformed Nonproducer Balb/3T3 Cell Lines: Altered Metabolism and Cell Surface Exposure23

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Abstract
The composition, metabolism, and cell surface exposure of neutral glycolipids and gangliosides were studied to correlate the biologic properties of several metastatic variant lines of RNA virus-transformed nonproducer Balb/3T3 cells, of control 3T3 cells, and of flat revertants of a metastatic line. Chemical and radiochemical analyses of neutral glycolipid and ganglioside composition revealed differences between Moloney (M) murine sarcoma virus (MuSV) transformants and Kirsten (Ki) MuSV transformants and nontransformed 3T3. A quantitative increase of asialo GM2 (Cer-Glc-Gal-GalNAc) and simultaneous decrease of GM3 (Cer-Glc-Gal-NAN) was seen in all Ki-MuSV transformants relative to M-MuSV transformants and control 3T3 cells. The disialo ganglioside GD1b [Cer-Glc-Gal-(NAN-NAN)-GalNAc-Gal] was low in all transformants. Reaction sequences for the synthesis of glycolipids of representative intermediately and highly metastatic Ki-MuSV-transformed cells were determined with [14C]galactose as labeled precursor. These studies revealed significant incorporation first in CD (Cer-Glc-Gal) and later in asialo GM2 and GM2 (Cer-Glc-Gal-(NAN)-GalNAc) for the highly metastatic line KA31, whereas the intermediately metastatic line KB521 first showed high levels of incorporation in CD and later in GM3, asialo GM2, GM2, and GM1 [Cer-Glc-Gal-(NAN)-GalNAc-Gal]. The kinetics of radioactive incorporation of galactose in variant lines and their chemical composition suggested different biosynthetic pathways among them. Gangliosides might be synthesized via the CD→GM3→GM2→GM1 pathway in 3T3 and MuSV85 Cl 3 lines, whereas in KB521 ganglioside synthesis might also proceed via the alternate CD→asialo GM2→GM2→GM1 pathway. Highly metastatic Ki-MuSV transformants that lack GM3 showed a concomitant increase of asialo GM2, which suggested that the alternate pathway was predominant in this line. Cell surface exposure of gangliosides and neutral glycolipids was detected with the galactose oxidase:NaB3H4-labeling method. Cell surface labeling of individual ganglioside components by this method revealed some differences, but none could be correlated with metastatic activity. However, labeling of asialo GM2 was positively correlated with metastatic activity of the variant lines examined. Study of glycolipids of 2 flat revertant lines of the highly metastatic K234 line showed no reversion of the ganglioside pattern to that of control 3T3, but the ganglioside profile was similar to that of the highly metastatic parental line. However, cell surface exposure of asailo GM2 of the flat revertant line caused a reversion to that of the control 3T3. In BALB/c inbred mice, qualitative composition of neutral glycolipids and gangliosides from subcutaneous tumors of 3 metastatic variant lines, MuSV85 CI 3, KB521, and KA31, did not differ despite their remarkable differences in composition in vitro and between in vivo and in vitro patterns. The results demonstrate altered metabolism of glycosphingolipids that resulted in increased cell surface exposure of asialo GM2 in highly metastatic Ki-MuSV transformants in culture relative to poorly metastatic M-MuSV transformants.

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