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 aerodigestiv e tract, but the biological mechanisms of this ethanol effect are still, un clear. We recently reported that ethanol is able to induce in vitro prolife ration accompanied by an increased number of cells in the S phase of the ce ll 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 limi ted 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 v;ell as HaCaT (human immortalized keratinocytes)-used as a control-were cultured for 96 hr in the presence or absence of ethanol (10(-3)M). At several time intervals the expression of cyclin D1 and p21 a nd the phosphorylation status of the retinoblastoma protein (pRb) were dete rmined by Western or Northern Blot analysis, or both. Results: Ethanol had no influence on the protein expression of cyclin D1. I n contrast, a distinct downregulation of p21 at the protein as well as the mRNA level could be detected. Furthermore, as a downstream event, the hyper phosphorylated form of the pRb increased. Conclusions: In the acute alcohol in vitro experiments, the marked downregu lation of the important cell cycle inhibitor p21 and the corresponding incr ease of hyperphosphorylated pRb accelerate the progression of cells from th e GI to the S phase in the cell cycle. The importance of these data and the ir relevance to in vivo conditions remain speculative, but it could be a cr itical step in the multistep process of SCCPHN carcinogenesis induced by et hanol.