Document Detail


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.
MedLine Citation:
PMID:  15016655     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Jun Wu; Yajun Cheng; Ake Nilsson; Rui-Dong Duan
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2004-03-11
Journal Detail:
Title:  Carcinogenesis     Volume:  25     ISSN:  0143-3334     ISO Abbreviation:  Carcinogenesis     Publication Date:  2004 Aug 
Date Detail:
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    
Other Details:
Languages:  eng     Pagination:  1327-33     Citation Subset:  IM    
Affiliation:
Gastroenterology Lab, Biomedical Center B11, Lund University, S-221 84 Lund, Sweden.
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MeSH Terms
Descriptor/Qualifier:
Alkaline Phosphatase / metabolism
Amino Acid Sequence
Animals
Blotting, Western
COS Cells
Caco-2 Cells
Cell Differentiation
Chromosome Mapping
Chromosomes, Human, Pair 17 / ultrastructure
Cloning, Molecular
Colonic Neoplasms / enzymology*,  genetics*
DNA / metabolism
DNA, Complementary / metabolism
Exons*
Gene Deletion*
Gene Expression Regulation, Enzymologic*
Gene Expression Regulation, Neoplastic*
Humans
Intestines / enzymology*
Introns
Kinetics
Lysophosphatidylcholines / metabolism
Models, Genetic
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Phosphatidylcholines / metabolism
RNA / chemistry,  metabolism
Reverse Transcriptase Polymerase Chain Reaction
Sphingomyelin Phosphodiesterase / genetics*
Time Factors
beta-Galactosidase / metabolism
Chemical
Reg. No./Substance:
0/DNA, Complementary; 0/Lysophosphatidylcholines; 0/Phosphatidylcholines; 63231-63-0/RNA; 9007-49-2/DNA; EC 3.1.3.1/Alkaline Phosphatase; EC 3.1.4.12/Sphingomyelin Phosphodiesterase; EC 3.2.1.23/beta-Galactosidase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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