25-Hydroxyvitamin D3 1alpha-hydroxylase splice variants in breast cell lines MCF-7 and MCF-10.
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.


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.


CONCLUSIONS
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.
Fischer
D
D
Klinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Lübeck, Germany. dorle.fischer@web.de
Seifert
M
M
Becker
S
S
Ludders
D
D
Cordes
T
T
Reichrath
J
J
Friedrich
M
M
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics*; D017398:Alternative Splicing; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005091:Exons; D005260:Female; D014644:Genetic Variation; D006801:Humans; D012334:RNA, Neoplasm / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012334:RNA, Neoplasm; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase