Ethanol decreases expression of p21 and increases hyperphosphorylated pRb in cell lines of squamous cell carcinomas of the head and neck

BACKGROUND: Alcohol increases the risk of cancers of the upper aerodigestive tract, but the biological mechanisms of this ethanol effect are still unclear. We recently reported that ethanol is able to induce in vitro proliferation accompanied by an increased number of cells in the S phase of the cell cycle in squamous cell carcinoma cell lines of the head and neck (SCCHN). In the current study we investigated the influence of ethanol over a limited period of time (96 hr) on cell cycle-regulating proteins involved in G1/S phase transition. METHODS: Synchronized cells of SCCHN cell lines JPPA (larynx) and SCC 9 and SCC 25 (tongue), as well as HaCaT (human immortalized keratinocytes)-used as a control-were cultured for 96 hr in the presence or absence of ethanol (10-3M). At several time intervals the expression of cyclin D1 and p21 and the phosphorylation status of the retinoblastoma protein (pRb) were determined by Western or Northern Blot analysis, or both. RESULTS: Ethanol had no influence on the protein expression of cyclin D1. In contrast, a distinct downregulation of p21 at the protein as well as the mRNA level could be detected. Furthermore, as a downstream event, the hyperphosphorylated form of the pRb increased. CONCLUSIONS: In the acute alcohol in vitro experiments, the marked downregulation of the important cell cycle inhibitor p21 and the corresponding increase of hyperphosphorylated pRb accelerate the progression of cells from the G1 to the S phase in the cell cycle. The importance of these data and their relevance to in vivo conditions remain speculative, but it could be a critical step in the multistep process of SCCHN carcinogenesis induced by ethanol.