1,25(OH)2 Vitamin D3 Induces Elevated Expression of the Cell Cycle-regulating Genes P21 and P27 in Squamous Carcinoma Cell Lines of the Head and Neck

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 in SCCHN.