1,25(OH)(2) vitamin D-3 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 D-3, 1,25-dihydroxyvitamin D-3 [1,2 5(OH)(2)D-3], inhibits proliferation and induces differentiation for Variou s 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 re ceptor. To further explore the molecular mechanisms of the antiproliferativ e activity in SCCHN we studied the influence of 1,25(OH)(2)D-3 on the expre ssion of the G1 phase-regulating proteins cyclin D1, p21 and p27. Furthermo re, as a direct target of G1 protein complexes, we investigated the phospho rylation 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 th e presence or absence (ethanol as control) of 1,25(OH)(2)D-3 (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 ce ll cycle-regulating proteins was determined at the protein and mRNA levels. In all cell lines tested 1,25(OH)(2)D-3 caused an arrest of cells in the G 0/G1 phase of the cell cycle and markedly induced the expression of the inh ibitors p21 and p27. No influence was detectable on the expression of cycli n D1. Induction of p21 and p27 mRNA revealed transcriptional regulation by the vitamin D receptor. Simultaneously, hyperphosphorylated pRb was transfo rmed to the hypophosphorylated form. Our results demonstrate that the biolo gically active form of vitamin D-3 directly regulates the expression of p21 and p27, inducing a G0/G1 phase arrest: one mechanism by which 1,25(OH)(2) D-3 controls cell proliferation in SCCHN.