G1 Cell Cycle Arrest Associated With Upregulation of p16 and p21 by Procaine Hydrochloride

Laura A. Lambert, Kelly K. Hunt, Khandan Keyomarsi
University of Texas, MD Anderson Cancer Center, Houston, TX

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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