Suppression of integrin expression and tumorigenicity by sulfation of lactosylceramide in 3LL Lewis lung carcinoma cells.

Abstract

To investigate the cellular functions of sulfated glycosphingolipids, we introduced the cerebroside sulfotransferase (CST) gene into J5 cells, a subclone of 3LL Lewis lung carcinoma cells. The J5 cells lack acidic glycosphingolipids but accumulate their common biosynthetic precursor, lactosylceramide. We established the stable CST transfectants, J5/CST-1 and J5/CST-2 clones, highly expressing sulfated lactosylceramide (SM3). Both clones exhibited more spherical morphology in comparison to mock transfectant, and their adhesiveness to fibronectin and laminin was significantly lower. The loss of cell-substratum interactions in these SM3-expressing cells could be attributed to decreased expression of integrins (alpha(5), alpha(6), and beta(1)) on the cell surface and their whole cellular levels. However, the levels of H-2K(b) and H-2D(b) antigens remained unchanged. Reverse transcriptase-polymerase chain reaction and Northern blot analyses for these integrins exhibited significant decrease of beta(1) gene expression in J5/CST-1 and 2, but there was no change in the levels of alpha(5) and alpha(6) transcripts. Deglycosylation by endoglycosidase H treatment clearly demonstrated that the precursor form of beta(1) integrin, possessing high mannose oligosaccharide chains, was preferentially decreased in the CST transfectants. These results demonstrate that endogenous SM3 negatively regulates beta(1) integrin expression at the transcriptional level, and the decrease of alpha integrin proteins in the CST transfectants was due to the post-transcriptional modification. We suggest the putative importance of the intracellular pre-beta(1) integrin pool for normal integrin maturation and subsequent function. Although the rates of cell proliferation in vitro for mock and CST transfectants were similar, tumorigenicity of J5/CST-1 and -2 cells inoculated into syngeneic C57/BL6 mice was greatly decreased or even absent. This was probably due to global loss of the efficient cell-matrix interactions, which are essential for the development of malignant tumors in vivo. Thus, we showed the evidence that cellular SM3 negatively regulates the cell-substratum interaction, resulting in the loss of tumorigenicity.

• Authors: K Kabayama, N Ito, K Honke, Y Igarashi, J Inokuchi
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 276 Issue 29 Pg. 26777-83 (Jul 20 2001) ISSN: 0021-9258 [Print] United States
• PMID: 11352905 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antigens, CD
• DNA Primers
• Integrins
• Lactosylceramides
• Sulfuric Acids
• CDw17 antigen

Topics

• Animals
• Antigens, CD
• Base Sequence
• Carcinoma, Lewis Lung (metabolism, pathology)
• Cell Adhesion
• Cell Division
• DNA Primers
• Integrins (metabolism)
• Lactosylceramides (physiology)
• Mice
• Reverse Transcriptase Polymerase Chain Reaction
• Sulfuric Acids (metabolism)
• Transfection
• Tumor Cells, Cultured