Fischer D, Seifert M, Becker S, Ludders D, Cordes T, Reichrath J, Friedrich M
Klinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Schleswig-Holstein, Campus Lübeck, Lübeck, Germany.
Cancer Genomics Proteomics. 2007 Jul-Aug;4(4):295-300.
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.
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.
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.
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.
已知25(OH)D3可在乳腺组织中被1α-羟化酶代谢为1,25(OH)2D3。1α-羟化酶的这种组织特异性表达可能是维生素D状态（25(OH)D3水平）与1,25(OH)2D3抗癌作用之间的关键环节。替代剪接在乳腺癌细胞中经常发生；给定蛋白质的不同剪接变体可表现出不同的生物学功能，并可能导致组织特异性差异。本研究首次描述了人乳腺癌细胞系MCF-7和良性乳腺细胞系MCF-10A中1α-羟化酶的表达和替代剪接。
使用实时聚合酶链反应（RT-PCR）评估1α-羟化酶RNA和蛋白质的表达。通过结合巢式PCR和降落PCR的高度特异性PCR检测1α-羟化酶剪接变体的表达。为了确定哪些变体被翻译为蛋白质，进行了蛋白质免疫印迹分析。
发现MCF-7中1α-羟化酶的表达比MCF-10A细胞高1.25倍。在MCF-10A细胞中，检测到至少6种剪接变体，而MCF-7中这些变体的表达水平无或极低。在MCF-7细胞中，抗体在56 kDa处检测到一个信号，对应于正常1α-羟化酶蛋白的大小。在MCF-10A细胞中，该信号较弱。在蛋白质免疫印迹分析中，在MCF-7细胞中发现了至少两种45 kDa的较小变体。在MCF-10A细胞中，检测到至少6种37至56 kDa之间的蛋白质，信号较弱。
我们认为1α-羟化酶的替代剪接可调节活性酶的水平。剪接变体可能导致蛋白质减少。MCF-7细胞中较小变体的意义也尚未阐明，但已知它们不能将25(OH)D3作为底物生成1,25(OH)D3。在MCF10A细胞中，鉴定出更多的剪接变体，可能是恶性细胞含有无活性的变体。由于未进行活性测量，它们的活性降低程度尚不清楚。
