Lack of inverse dose‐rate effect and binding of the retinoblastoma gene product in the nucleus of human cancer T‐47D cells arrested in G2 by ionizing radiation

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Abstract
Purpose: To investigate the radiosensitivity of human breast cancer cells, T‐47D, irradiated with low dose‐rates and to study activation of the retinoblastoma gene product in the G1 and G2 phases during irradiation. Materials and methods: Cells were irradiated with 60Co γ‐rays with dose‐rates of 0.37 and 0.94 Gy h−1. Cell survival was measured as the ability of cells to form colonies. Cells were extracted, fixed and stained for simultaneous measurements of nuclear‐bound pRB content and DNA content. Cell nuclei were stained with monoclonal antibody PMG3‐245 and Hoechst 33258 was used for additional staining of DNA. Two‐parametric flow cytometry measurements of pRB and DNA content were performed using a FACSTARPLUS flow cytometer. Results: It was observed that irradiated cells were arrested in G2. No increase in radiation sensitivity was observed when the cells accumulated in G2. Irradiation of cells at both 0.37 and 0.94 Gy h−1 resulted in exponential dose–survival curves with nearly equal α values, i.e. the same radiosensitivity. However, the retinoblastoma gene product was bound in the nucleus, i.e. hypophosphorylated, in about 15% of the cells arrested in G2. Conclusions: T47‐D cells accumulate in G2 during low dose irradiation, but no inverse dose‐rate effect, i.e. a more efficient inactivation of cells at lower than at higher dose‐rates, was observed. A population of arrested G2 cells has pRB protein bound in the nucleus, and pRB therefore could play a role in protecting cells against radiation‐induced cell death in G2.