G1 Cell Cycle Arrest Associated With Upregulation of p16 and p21 by Procaine Hydrochloride
Objective: Procaine hydrochloride (PH) has been shown to act as a demethylating agent capable of causing G1-arrest in breast cancer cell lines. Down-regulation of p16 in gastrointestinal stromal tumors (GIST) frequently occurs through promoter hypermethylation and is associated with a poor outcome. We hypothesized that treatment of GIST cells negative for p16 with PH would alter the cell cycle through demethylation and up-regulation of p16.
Design: p16-negative GIST cells (AR04), p16-negative synovial sarcoma cells (SW982) and p16+ MDA-MB-436 breast cancer cells were treated with PH. Expression of p16, p21, and other cell cycle modulators were evaluated by western blot analysis. Cell cycle distribution was determined by flourescence-activated cell sorting analysis (FACS).
Results: Treatment with 0.1-4.0 mM PH for 24 hours caused a dose-dependent up-regulation of p16 and p21 in AR04. A dose-independent up-regulation of p21, but not p16, occurred in SW982. MDA-MB-436 did not express p21. There was no significant change in other cell cycle regulators following treatment with PH. FACS showed an increased percentage of PH-treated AR04 cells in G1 phase (53% untreated versus 71% treated, p < 0.0001), but not in the PH-treated SW982 or MDA-MB-436.
Conclusions: This is the first report of G1-arrest associated with p16 and p21 up-regulation following treatment of a p16-negative GIST cell line with PH. G1-arrest did not occur in SW982 (p16-/p21+) or MDA-MB-436 (p16+/p21-), indicating that up-regulation of both p16 and p21 may be required. Because loss of p16 in GIST cells is associated with a poorer prognosis and decreased survival, p16 is a potential therapeutic target. The clinical usefulness of PH as a demethylating agent is currently being evaluated.
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