Hager G, Formanek M, Gedlicka C, Thurnher D, Knerer B, Kornfehl J
Department of Oto-Rhino-Laryngology, Head and Neck Surger, University of Vienna, Austria.
Acta Otolaryngol. 2001 Jan;121(1):103-9. doi: 10.1080/000164801300006353.
The biologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], inhibits proliferation and induces differentiation for various malignant cells, including squamous cell carcinoma cell lines of the head and neck (SCCHN). These effects are due to an arrest of cells in the G0/G1 phase of the cell cycle and are predominantly mediated by the vitamin D receptor. To further explore the molecular mechanisms of the antiproliferative activity in SCCHN we studied the influence of 1,25(OH)2D3 on the expression of the G1 phase-regulating proteins cyclin D1, p21 and p27. Furthermore, as a direct target of G1 protein complexes, we investigated the phosphorylation status of the retinoblastoma protein (pRb). Synchronized cells of 2 SCCHN cell lines [JPPA (laryngeal carcinoma) and SCC 9 (tongue carcinoma)] and human immortalized keratinocytes (HaCaT) were cultured for 96 h in the presence or absence (ethanol as control) of 1,25(OH)2D3 (10(-7) M). At various time intervals the cell cycle status was detected by fluorescence-activated cell sorting (FACS) analysis and in parallel the expression of cell cycle-regulating proteins was determined at the protein and mRNA levels. In all cell lines tested 1,25(OH)2D3 caused an arrest of cells in the G0/G1 phase of the cell cycle and markedly induced the expression of the inhibitors p21 and p27. No influence was detectable on the expression of cyclin D1. Induction of p21 and p27 mRNA revealed transcriptional regulation by the vitamin D receptor. Simultaneously, hyperphosphorylated pRb was transformed to the hypophosphorylated form. Our results demonstrate that the biologically active form of vitamin D3 directly regulates the expression of p21 and p27, inducing a G0/G1 phase arrest: one mechanism by which 1,25(OH)2D3 controls cell proliferation inSCCHN.
维生素D3的生物活性形式，即1,25 - 二羟基维生素D3 [1,25(OH)2D3]，可抑制多种恶性细胞的增殖并诱导其分化，包括头颈部鳞状细胞癌细胞系（SCCHN）。这些作用归因于细胞在细胞周期的G0/G1期停滞，且主要由维生素D受体介导。为进一步探究SCCHN中抗增殖活性的分子机制，我们研究了1,25(OH)2D3对G1期调节蛋白细胞周期蛋白D1、p21和p27表达的影响。此外，作为G1蛋白复合物的直接靶点，我们研究了视网膜母细胞瘤蛋白（pRb）的磷酸化状态。将2种SCCHN细胞系[JPPA（喉癌）和SCC 9（舌癌）]以及人永生化角质形成细胞（HaCaT）同步化处理后，在存在或不存在（以乙醇作为对照）1,25(OH)2D3（10(-7) M）的情况下培养96小时。在不同时间间隔，通过荧光激活细胞分选（FACS）分析检测细胞周期状态，并同时在蛋白质和mRNA水平测定细胞周期调节蛋白的表达。在所有测试的细胞系中，1,25(OH)2D3导致细胞在细胞周期的G0/G1期停滞，并显著诱导抑制剂p21和p27的表达。未检测到对细胞周期蛋白D1表达的影响。p21和p27 mRNA的诱导显示出由维生素D受体介导的转录调控。同时，高磷酸化的pRb转变为低磷酸化形式。我们的结果表明，维生素D3的生物活性形式直接调节p21和p27的表达，诱导G0/G1期停滞：这是1,25(OH)2D3控制SCCHN细胞增殖的一种机制。
