K. Kabayama et al., Suppression of integrin expression and tumorigenicity by sulfation of lactosylceramide in 3LL Lewis lung carcinoma cells, J BIOL CHEM, 276(29), 2001, pp. 26777-26783
To investigate the cellular functions of sulfated glycosphingolipids, we in
troduced the cerebroside sulfotransferase (CST) gene into J5 cells, a subcl
one of 3LL, Lewis lung carcinoma cells. The J5 cells lack acidic glycosphin
golipids but accumulate their common biosynthetic precursor, lactosylcerami
de. We established the stable CST transfectants, J5/CST-1 and J5/CST-2 clon
es, highly expressing sulfated lactosylceramide (SM3). Both clones exhibite
d more spherical morphology in comparison to mock transfectant, and their a
dhesiveness to fibronectin and laminin was significantly lower. The loss of
cell-substratum interactions in these SM3-expressing cells could be attrib
uted to decreased expression of integrins (alpha (5), alpha (6), and beta (
1)) on the cell surface and their whole cellular levels. However, the level
s of H-2K(b) and H-2D(b) antigens remained unchanged. Reverse transcriptase
-polymerase chain reaction and Northern blot analyses for these integrins e
xhibited 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) transcri
pts. Deglycosylation by endoglycosidase H treatment clearly demonstrated th
at the precursor form of beta (1) integrin, possessing high mannose oligosa
ccharide chains, was preferentially decreased in the CST transfectants. The
se results demonstrate that endogenous SM3 negatively regulates beta (1) in
tegrin expression at the transcriptional level, and the decrease of alpha i
ntegrin proteins in the CST transfectants was due to the posttranscriptiona
l modification. We suggest the putative importance of the intracellular pre
-beta (1) integrin pool for normal integrin maturation and subsequent funct
ion. Although the rates of cell proliferation in vitro for mock and CST tra
nsfectants were similar, tumorigenicity of J5/ CST-1 and -2 cells inoculate
d into syngeneic C57/BL6 mice was greatly decreased or even absent. This wa
s probably due to global loss of the efficient cell-matrix interactions, wh
ich are essential for the development of malignant tumors in vivo. Thus, we
showed the evidence that cellular SM3 negatively regulates the cell substr
atum interaction, resulting in the loss of tumorigenicity.