Hypophosphorylated Retinoblastoma Protein Is Associated with G2Arrest in Esophageal Squamous Cell Carcinoma

Hypophosphorylated retinoblastoma (Rb) gene product binds critical transcription factors, leading to G1arrest in a number of conditions, including following DNA damage. We have previously shown that irradiated esophageal squamous cell carcinoma (ESSC) cells undergo predominantly G2arrest, with increases in inhibitors of Rb phosphorylation. We thus hypothesized that this G2arrest would be accompanied by increases in hypophosphorylated Rb protein (pRb). We sequenced the Rb genes of three human ESSC lines (KYSE) following reverse transcription polymerase chain reaction of exons A–E. Western gels were performed on protein extracts for pRb. Cells were irradiated at 6 Gy, and protein was extracted at 6 h. ELISA was used to measure hypophosphorylated pRb in radiated versus control cells. Student'sttest was used to compare results. All lines had wild-type Rb genes. Western gels confirmed the presence of pRb. There were significant increases in hypophosphorylated pRb in all three lines following irradiation (no line with less than a 100% increase). We have thus shown that irradiation-induced G2arrest occurs in association with wild-type Rb genes and that there is associated hypophosphorylation of pRb. This supports our data describing a further role for other G1mediators, such as p21, in G2arrest. Further investigations into therapies to expoit this cell cycle checkpoint are warranted and planned.