Document Detail


25-Hydroxyvitamin D3 1alpha-hydroxylase splice variants in breast cell lines MCF-7 and MCF-10.
MedLine Citation:
PMID:  17878529     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
BACKGROUND: It is known that 25(OH)D3 can be metabolized to 1,25(OH)2 D3 by 1alpha-OHase in breast tissue. This tissue-specific expression of 1alpha-OHase may act as the pivotal link between vitamin D status (25(OH)D3 levels) and the anticancer effects of 1,25(OH)2 D3. Alternative splicing frequently occurs in breast cancer cells; different splice variants of a given protein can display different biological functions and may cause tissue-specific variations. With this study it is the first time that expression and alternative splicing of 1alpha-OHase in the human breast cancer cell line MCF-7 and thebenign breast cell line MCF-10A are described.
MATERIALS AND METHODS: Expression of 1alpha-OHase RNA and protein was assessed using a real-time polymerase chain reaction (RT-PCR). The expression of 1alpha-OHase splice variants was detected by a highly specific PCR that combines nested and touchdown PCR. To determine which variants are translated in protein western blot analysis was carried out.
RESULTS: The expression of 1alpha-OHase was found to be 1.25-fold higher in MCF-7 compared to MCF-10A cells. In MCF-10A cells, at least 6 splice variants were detected whereas MCF-7 showed no or marginal expression levels of these variants. In MCF-7 cells the antibody detected a signal at 56 kDa corresponding to the size of normal 1alpha-OHase protein. In MCF-10A cells this signal was weaker. In western blot analysis at least two smaller variants at 45 kDa were found in MCF-7 cells. In MCF-10A cells at least 6 proteins between 37 and 56 kDa were detected with an only faint signal.
CONCLUSION: We propose that alternative splicing of 1alpha-OHase can regulate the level of active enzyme. Splice variants may lead to a reduction of the protein. The significance of the smaller variants in MCF-7 cells has not been clarified either, but it is known that they are not able to use 25(OH)D3 as a substrate to generate 1,25(OH)D3. In MCF10A cells, more splice variants were identified, it may be that malignant cells contain inactive variants. How far they show a reduced activity remains unclear as no activity measurements were performed.
Authors:
D Fischer; M Seifert; S Becker; D Ludders; T Cordes; J Reichrath; M Friedrich
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Cancer genomics & proteomics     Volume:  4     ISSN:  1109-6535     ISO Abbreviation:  Cancer Genomics Proteomics     Publication Date:    2007 Jul-Aug
Date Detail:
Created Date:  2007-09-19     Completed Date:  2007-10-12     Revised Date:  2012-01-18    
Medline Journal Info:
Nlm Unique ID:  101188791     Medline TA:  Cancer Genomics Proteomics     Country:  Greece    
Other Details:
Languages:  eng     Pagination:  295-300     Citation Subset:  IM    
Affiliation:
Klinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Lübeck, Germany. dorle.fischer@web.de
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MeSH Terms
Descriptor/Qualifier:
25-Hydroxyvitamin D3 1-alpha-Hydroxylase / genetics*
Alternative Splicing*
Breast Neoplasms / genetics*
Cell Line, Tumor
Cloning, Molecular
Exons
Female
Genetic Variation*
Humans
RNA, Neoplasm / genetics
Reverse Transcriptase Polymerase Chain Reaction
Chemical
Reg. No./Substance:
0/RNA, Neoplasm; EC 1.14.-/25-Hydroxyvitamin D3 1-alpha-Hydroxylase

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


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