25-Hydroxyvitamin D3 1α-Hydroxylase Splice Variants in Breast Cell Lines MCF-7 and MCF-10

Cancer Genomics - Proteomics

S. BECKER²  T. CORDES²  D. LÜDDERS²  D. FISCHER²  J. REICHRATH¹  M. SEIFERT¹  M. FRIEDRICH² 

[1] linik für Dermatologie, Allergologie und Venerologie, Universitätsklinik des Saarlandes, Homburg, Germanylinik für Dermatologie, Allergologie und Venerologie, Universitätsklinik des Saarlandes, Homburg, Germanylinik für Dermatologie, Allergologie und Venerologie, Universitätsklinik des Saarlandes, Homburg, Germany;linik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Lübecklinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Lübecklinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Lübeck

关键词: Vitamin D3;    1α-hydroxylase;    splice variants;    MCF-7;    MCF-10;   

DOI  :  

来源: Delinasios GJ CO

【 摘 要 】

Background: It is known that 25(OH)D3 can be metabolized to 1,25(OH)2D3 by 1α-OHase in breast tissue. This tissue-specific expression of 1α-OHase may act as the pivotal link between vitamin D status (25(OH)D3 levels) and the anticancer effects of 1,25(OH)2D3. 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 1α-OHase in the human breast cancer cell line MCF-7 and thebenign breast cell line MCF-10A are described. Materials and Methods: Expression of 1α-OHase RNA and protein was assessed using a real-time polymerase chain reaction (RT-PCR). The expression of 1α-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 1α-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 1α-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 1α-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.

【 授权许可】

Unknown