Carcinogenesis Advance Access published online on March 11, 2004
Carcinogenesis, doi:10.1093/carcin/bgh140
© 2004 by Oxford University Press
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CANCER BIOLOGY
1 Gastroenterology Lab, Biomedical Center B11, Lund University, S-221 84 Lund, Sweden
* Corresponding author. E-mail: Rui-dong.duan{at}med.lu.se.
Received 4 November 2003
; revised 27 January 2004
; accepted 2 March 2004
Sphingomyelin metabolism in the gut has been implicated in colonic tumorigenesis. Intestinal alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin in the intestinal content and at the brush border. The enzyme activity is decreased in the tissues of human colorectal tumours. This study examines whether site or chain-mutation of alk-SMase occurs in colon cancer HT-29 cells and Caco-2 cells. Total RNA was isolated and the cDNA of alk-SMase was amplified by RTPCR. The size of the cDNA from HT-29 cells was smaller than that of the wild type cDNA. DNA sequencing identified a deletion of exon 4 in alk-SMase cDNA in HT-29 cells. No mutation in genomic alk-SMase DNA from exon 3 to 5 was identified. The exon 4 deletion was caused by a shift of RNA splice site in chromosome 17q25. In Caco-2 cells, no mutation of alk-SMase cDNA was identified. Transient expression in COS-7 cells showed that the enzyme from the cDNA in HT-29 cells had little alk-SMase activity whereas that in Caco-2 cells was as active as the wild type alk-SMase. The deleted region included residue His353, which is predicted to form a substrate binding site of alk-SMase. H353A substitution resulted in a protein with no alk-SMase activity. In monolayer cultured Caco-2 cells and HT-29 cells the alk-SMase activities were low. However, to culture the cells under polarising conditions increased alk-SMase activity and reduced SM level in Caco-2 but not HT29 cells. The alk-SMase activity varied in parallel with alkaline phosphatase activity. In conclusion, we identified an inactive deletion in alk-SMase in HT-29 cells, and a differentiationrelated expression of the enzyme in Caco-2 cells. The results provide a molecular mechanism related to previous findings of reduced alk-SMase activity in human colon cancers.
Identification of one exon deletion of intestinal alkaline sphingomyelinase in colon cancer HT-29 cells and a differentiation-related expression of the wild type enzyme in Caco-2 cells
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