Cytosolic Histone H1.2 Releasing under Different Apoptotic Stimuli in Chronic Lymphocytic Leukemia (CLL).

Abstract Recently, it has been shown that nuclear histone H1.2 is released into cytoplasm when apoptosis is induced by DNA double-strand breaks (DSB’s), this process being dependent on p53 functional status. In addition, cytosolic histone H1.2 induces cytochrome C release in a Bak-dependent manner. Thus, cytosolic histone H1.2 release represents a new mechanism that links DSB’s with activation of the apoptotic mitochondrial pathway. Against this background, we analyzed the release of histone H1.2 in the cytosol of purified CLL cells during apoptosis induced by fludarabine (F), mitoxantrone (M), etoposide, or ionizing radiation. In addition, the presence of histone H1 was correlated with p53 functional status. Cell viability and analysis of apoptosis were investigated by annexin V/PI staining and FACscan analysis. The presence of histone H1 and H1.2 in the cytosolic fraction was assessed by Western Blott using the anti-histone H1 (Upstate) and anti-histone H1.2 (Abcam) antibodies. Histone H1 traffic was also evaluated by using immunofluorescence analysis in CLL cells suspensions. FISH analysis was used to select samples with (n=3) or without (n=6) p53 deletion, and activation of p53 after treatment was assessed by Western Blot. In cases without p53 deletion, increased apoptosis was observed under all stimuli, the FM combination being the most effective. In such cases, histone H1.2 release was apparent 6 hours after the onset of irradiation or pharmacologic treatments, progressively increasing up to 24 hours. In contrast, cases with p53 deletion displayed a low cytotoxic effect upon different treatments. Interestingly, no p53 activation or histone H1.2 release into cytosol was observed. These results were also confirmed by immunofluorescence analysis, in which histone H1.2 was only visible in the cytosol of non-deleted p53 cases. These results demonstrate that, upon drug or irradiation exposure nuclear histone H1.2 is released into the cytoplasm of CLL cells in a p53-dependent manner. This suggests that, in CLL, histone H1.2 traffic contributes to the apoptosis induced by DSB’s and to drug resistance in cases with p53 deletion.