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

Abstract

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 γ-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-β1) 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 γ-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-β1. As TGF-β1 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.