Molecular analysis of p21 promoter activity isolated from squamous carcinoma cell lines of the head and neck under the influence of 1,25(OH)(2) vitamin D-3 and its analogs

Objective - The biologically active 1,25(OH)(2) vitamin D-3 and its analogs have been shown to have antiproliferative and differentiating effects in a variety of malignant and non-malignant cells. For squamous carcinoma cell lines of the head and neck (SCCHN) we could show that this antiproliferative activity of 1,25(OH)(2) vitamin D-3 is due to induced expression of the cell-cycle inhibitory proteins p21 and p27, causing an arrest in the G0/G1 cell-cycle phase. Material and Methods - In this work we investigated the effects of three vitamin D-3 analogs, EB1089, MC1288 and CB1093, on proliferation behavior and cell-cycle status in a laryngeal carcinoma cell line (JPPA) as well as in control human immortalized keratinocytes (HaCaT). To study the molecular mechanism the functional activity of the promoter region of p21, a potential target gene of vitamin D-3 transcriptional regulation, was investigated. For this reason a 2.7-kb fragment of the p21 promoter was isolated by polymerase chain reaction from HaCaT, JPPA and SCC9 (tongue carcinoma) cells and directionally cloned into an enhanced green fluorescence protein (EGFP) reporter gene vector system. A construct was used to stably transfect HaCaT cells and to monitor the expression of the EGFP gene by confocal microscopy. Results - Analysis of proliferation and cell-cycle status revealed decreased growth rates and G0/G1 cell-cycle arrest in cells treated with 1,25(OH)(2) vitamin D-3 and its analogs. The EGFP reporter gene-transfected cells showed distinct fluorescence under the influence of 1,25(OH)(2) vitamin D-3 and its analogs compared to control cells. Conclusion - These results demonstrate that the cell-cycle inhibitor protein p21 is a direct target gene of biologically active 1,25(OH)(2) vitamin D-3, inducing G0/G1 cell-cycle arrest. The ability of vitamin D analogs to act via the same molecular mechanism as the natural hormone but with less hypercalcemic activity may have therapeutic implications for patients with SCCHN malignancy.