Effect of ionizing radiation on cell-cycle progression and cyclin B1 expression in human melanoma cells

In the present study we investigated the effect of γ-irradiation (2.5 and 10 Gy) on cell-cycle progression of a human melanoma cell line, M14, characterized by a moderate radiosensitivity (SF2 = 0.5). Flow cytometric analysis showed a dose-dependent S-phase accumulation, which was detectable 8 hr after treatment with 2 and 5 Gy and was still persistent at 12 hr after 10 Gy exposure. Such a delay in S-phase was paralleled or followed by an accumulation of cells in G₂M, which was transient at the lowest radiation doses and still persistent at 72 hr after 10 Gy. Such an accumulation was, at least in part, due to a block in G₂-M transition, as demonstrated by mitotic index analysis. Bivariate flow cytometric analysis of DNA content and cyclin B₁ expression showed that, following 2 and 5 Gy, the fraction of cyclin B₁-expressing cells was superimposable upon that of G₂M cells. Conversely, in cells treated with 10 Gy, the fraction of cyclin B₁-expressing cells was half the G₂M cell fraction. Northern-blot analysis indicated that the radiation-induced decrease in cyclin B₁ protein expression was accompanied by a reduced cyclin B mRNA level. On the whole, our results indicate a direct inhibitory effect of 10 Gy irradiation on cyclin B₁ expression as a possible cause for the persistent G₂ block in irradiated M14 cells.