Influence of irradiation and pentoxifylline on histone H3 phosphorylation in human tumour cell lines.

Abstract

Phosphorylation of histone H3 at Ser-10 correlates with chromatin condensation and this amino terminal modification is now recognized as a specific marker of mitosis. We have monitored the appearance of cells showing histone H3 phosphorylation in four human tumour cell lines to identify cell cycle progression after irradiation. In the human melanoma cell lines Be11 and MeWo and in the squamous cell carcinoma lines 4197 and 4451 a dose of 7 Gy of Co-gamma irradiation increases the number of cells binding anti-histone H3-P antibody 1-8-fold in a p53-independent manner. In the p53 mutant cell lines MeWo and 4451 H3-P phosphorylated cells can be detected as early as 30 min and show a maximum 1 h post-irradiation. In the cell lines Be11, 4197 and 4451 the early wave of H3 phosphorylated cells is followed by a second wave, which reaches a maximum 4.5-7 h post-irradiation and then declines. These events are attributed to damage-induced cell cycle blocks in the G1 and G2 phase of the cell cycle. Addition of the dose modifying drug pentoxifylline before irradiation increases the appearance of cells showing early and the late H3 phosphorylation. When pentoxifylline is added 12-24 h post-irradiation when the cell cycle blocks have reached their maximum the appearance of cells with phosphorylated H3 increases 3-5-fold in the p53 mutant cell lines MeWo and 4451. These observations are consistent with the function of the drug as a G2 block abrogator. The large H3 phosphorylation signal in p53 mutant cells is consistent with early entry of a cohort of G2 cells into mitosis. The smaller H3-P signal in p53 wild type cells correlates with the lower proportion of stable G2 populations in G1 blocked cells. These results indicate that pentoxifylline influences the appearance of histone H3 phosphorylated cells in a manner strongly dependent on the number of cells in G2 phase. This suggests that addition of pentoxifylline indeed abrogates the G2 block and thereby facilitates early entry into mitosis.