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.
Sphingomyelin (SM) metabolism in the gut has been implicated in colonic tumorigenesis. Intestinal alkaline sphingomyelinase (alk-SMase) hydrolyses SM 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 RT-PCR. 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 polarizing conditions increased alk-SMase activity and reduced SM level in Caco-2 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 differentiation-related 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.
Wu
Jun
J
Gastroenterology Lab, Biomedical Center B11, Lund University, S-221 84 Lund, Sweden.
Cheng
Yajun
Y
Nilsson
Ake
A
Duan
Rui-Dong
RD
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D018938:Caco-2 Cells; D002454:Cell Differentiation; D002874:Chromosome Mapping; D002886:Chromosomes, Human, Pair 17 / Q000648:ultrastructure; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007422:Intestines / Q000201:enzymology*; D007438:Introns; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010713:Phosphatidylcholines / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics*; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D018076:DNA, Complementary; D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; D012313:RNA; D004247:DNA; D000469:Alkaline Phosphatase; D013108:Sphingomyelin Phosphodiesterase; D001616:beta-Galactosidase