Antisense blockade of p21/WAF1 decreases radiation-induced G2 arrest in esophageal squamous cell carcinoma.

Introduction. The p21 cyclin-dependent kinase inhibitor arrests the cell cycle following DNA damage at the G1-S checkpoint. Recent literature has also demonstrated a role for p21 in G2 arrest. Studies with esophageal squamous cell carcinoma (ESSC) lines have shown that radiation-induced p21 protein induction is associated with G2 arrest. The aim of this study was to determine if p21 blockade would affect this G2 arrest pattern. Method. We transfected the ESSC line KYSE 170 with antisense p21 mRNA oligonucleotides or scrambled 20-mer p21 control oligonucleotides using a lipofectant reagent. Cells were exposed to 6 Gy or used as controls. p21 levels were determined by ELISA. Cell cycle arrest pattern was determined via flow cytometry. Student's t test and ANOVA were used to compare p21 levels and percentages of G2 arrest. Results. Irradiated/scrambled cells expressed 10.1 ng/ml of p21 protein compared to irradiated/antisense cells at 2.1 ng/ml (P < 0.05), demonstrating successful blockade of p21. Irradiated cells displayed prominent G2 arrest following 6 Gy doses, but there was a decrease from 65 to 44% G2 phase when p21 was blocked (P < 0.05). Conclusions. We have demonstrated that G2 arrest accompanying irradiation of ESSC cells decreases when p21 protein production is blocked via antisense oligonucleotides. These data support a role for p21 in mediating G2 arrest in these cells. Copyright 1999 Academic Press.