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.
(2004) In Carcinogenesis 25(8). p.1327-1333- Abstract
- phingomyelin (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... (More)
- phingomyelin (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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/121317
- author
- Wu, Jun LU ; Cheng, Yajun LU ; Nilsson, Åke LU and Duan, Rui-Dong LU
- organization
- publishing date
- 2004
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- sphingomyelin, phosphatidylcholine, PC, lyso-PC, lysophosphatidylcholine, SM, sphingomyelinase, alk-SMase
- in
- Carcinogenesis
- volume
- 25
- issue
- 8
- pages
- 1327 - 1333
- publisher
- Oxford University Press
- external identifiers
-
- wos:000223142700003
- pmid:15016655
- scopus:4344656249
- ISSN
- 0143-3334
- DOI
- 10.1093/carcin/bgh140
- language
- English
- LU publication?
- yes
- id
- 74a69fc7-5d58-4850-829a-65131d662457 (old id 121317)
- alternative location
- http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15016655&dopt=Abstract
- date added to LUP
- 2016-04-01 11:50:56
- date last changed
- 2024-02-23 10:02:00
@article{74a69fc7-5d58-4850-829a-65131d662457,
abstract = {{phingomyelin (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.}},
author = {{Wu, Jun and Cheng, Yajun and Nilsson, Åke and Duan, Rui-Dong}},
issn = {{0143-3334}},
keywords = {{sphingomyelin; phosphatidylcholine; PC; lyso-PC; lysophosphatidylcholine; SM; sphingomyelinase; alk-SMase}},
language = {{eng}},
number = {{8}},
pages = {{1327--1333}},
publisher = {{Oxford University Press}},
series = {{Carcinogenesis}},
title = {{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.}},
url = {{http://dx.doi.org/10.1093/carcin/bgh140}},
doi = {{10.1093/carcin/bgh140}},
volume = {{25}},
year = {{2004}},
}