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J. Biol. Chem., Vol. 276, Issue 29, 26777-26783, July 20, 2001

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Suppression of Integrin Expression and Tumorigenicity by Sulfation of Lactosylceramide in 3LL Lewis Lung Carcinoma Cells^*

Kazuya Kabayama, Nozomi Ito, Koichi Honke^§, Yasuyuki Igarashi, and Jin-ichi Inokuchi^¶

From the  Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-chome, Kita-ku, Sapporo 060-0812 and the ^§ Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

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 (₅, ₆, and 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 ₁ gene expression in J5/CST-1 and 2, but there was no change in the levels of ₅ and ₆ transcripts. Deglycosylation by endoglycosidase H treatment clearly demonstrated that the precursor form of ₁ integrin, possessing high mannose oligosaccharide chains, was preferentially decreased in the CST transfectants. These results demonstrate that endogenous SM3 negatively regulates ₁ integrin expression at the transcriptional level, and the decrease of  integrin proteins in the CST transfectants was due to the post-transcriptional modification. We suggest the putative importance of the intracellular pre-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.

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