X-irradiation-induced cell cycle delay and DNA double-strand breaks in the murine osteoblastic cell line OCT-1

Radiation response of bone cells, especially the bone-forming osteoblasts, is an important issue for radiotherapy in young age. A radiation-induced cell cycle arrest may enhance or accelerate osteoblastic differentiation. To analyze radiation response of osteoblastic cells, the correlation between DNA double-strand break induction (DSB), cell cycle alterations and gene expression modifications after X-irradiation was investigated in the osteoblast-like cell line OCT-1. As marker of the cellular response to DSB, the temporal appearance of gamma-H2AX foci after X-irradiation was visualized. Gene expression profiles of the key cell cycle regulatory protein p21 (CDKN1A), and the most abundant growth factor in human bone, transforming growth factor beta 1 (TGF-beta1) were recorded using quantitative real-time reverse transcription PCR (qRT-PCR). The distribution of cells in the cell cycle phases G1, S and G2 was determined by propidium iodide (PI) staining and flow cytometry. Initial studies show a strong dose dependency in the number of gamma-H2AX foci shortly after X-irradiation. Exposure to 1 Gy yields approximately 36 small foci in OCT-1 cells after 30 min that became larger after 1 h of incubation; after 24 h most of the foci had disappeared. X-rays provoked a dose-dependent arrest in G2 phase of the cell cycle, accompanied by a dose-dependent gene expression regulation for p21 and TGF-beta1. As TGF-beta1 is known to affect osteoblast differentiation, matrix formation and mineralization, modulation of its expression could influence the expression of the main osteogenic transcription factor Runx2 (Cbfa1) and other osteoblast differentiation markers.