|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.|
|PMID: 15016655 Owner: NLM Status: MEDLINE|
|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.|
|Jun Wu; Yajun Cheng; Ake Nilsson; Rui-Dong Duan|
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|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2004-03-11|
|Title: Carcinogenesis Volume: 25 ISSN: 0143-3334 ISO Abbreviation: Carcinogenesis Publication Date: 2004 Aug|
|Created Date: 2004-07-23 Completed Date: 2004-09-10 Revised Date: 2006-11-15|
Medline Journal Info:
|Nlm Unique ID: 8008055 Medline TA: Carcinogenesis Country: England|
|Languages: eng Pagination: 1327-33 Citation Subset: IM|
|Gastroenterology Lab, Biomedical Center B11, Lund University, S-221 84 Lund, Sweden.|
|APA/MLA Format Download EndNote Download BibTex|
Amino Acid Sequence
Chromosomes, Human, Pair 17 / ultrastructure
Colonic Neoplasms / enzymology*, genetics*
DNA / metabolism
DNA, Complementary / metabolism
Gene Expression Regulation, Enzymologic*
Gene Expression Regulation, Neoplastic*
Intestines / enzymology*
Lysophosphatidylcholines / metabolism
Molecular Sequence Data
Phosphatidylcholines / metabolism
RNA / chemistry, metabolism
Reverse Transcriptase Polymerase Chain Reaction
Sphingomyelin Phosphodiesterase / genetics*
beta-Galactosidase / metabolism
|0/DNA, Complementary; 0/Lysophosphatidylcholines; 0/Phosphatidylcholines; 63231-63-0/RNA; 9007-49-2/DNA; EC 184.108.40.206/Alkaline Phosphatase; EC 220.127.116.11/Sphingomyelin Phosphodiesterase; EC 18.104.22.168/beta-Galactosidase|
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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