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

Abstract Recently, it has been demonstrated that nuclear histone H1 could be released into cytoplasm when apoptosis is induced by DNA double-strand breaks, this process being dependent of p53 functional status. In addition, cytosolic histone H1.2 was shown to induce cytochrome C release in a Bak-dependent manner. The aims of this study were: 1) to analyze the presence of histone H1.2 in the cytosol of peripheral blood purified CLL cells during apoptosis induced by different chemotherapeutic drugs or ionizing radiation in CLL cells in culture; 2) to correlate the presence of cytosolic histone H1 with the p53 functional status. FISH analysis was used to select samples with (n=2) or without (n=4) p53 deletion. Peripheral blood purified CLL cells were cultured for 24 hours with no treatment, after irradiation (25 Gy), in the presence of fludarabine and mitoxantrone (FM) (1mg/ml and 0.5 mg/ml respectively), or with etoposide (5.8 mg/ml). Cell viability and analysis of apoptosis were performed by annexin V/PI staining and FACscan analysis. Cytosolic and nuclear fractions were separated with the Nuclear/Cytosol Fractionation kit (Biovision). The presence of histone H1 in the cytosolic fraction was assessed by Western Blott using the anti-histone H1 antibody (Upstate). Nuclear contamination of this fraction was detected by using anti-ribonucleoprotein (hnRNP) antibody (Abcam). In p53 wild-type CLL cases, increased apoptosis was observed under all stimuli, being the FM combination the most effective. In contrast, CLL cases with p53 deletion displayed a lower or no response to the different treatments. In p53 wild-type CLL cases, histone H1 was only observed in cytosolic fractions at 24 hours when irradiation or etoposide treatment was applied, but not with FM treatment. Interestingly, cytosolic histone H1 was also present in the control cells at 24h of culture, indicating that H1 was also involved in the spontaneous apoptosis observed due to the microambient deprivation of CLL cells in culture. This histone H1 traffic was also confirmed using immunofluorescence analysis. Conversely, in p53 deleted CLL cases histone H1 was not detected into cytosolic fractions with any treatment. In conclusion, cytosolic histone H1 appeared in CLL cases in a p53 dependent manner when DNA double-strand breaks were induced. These results suggest that histone H1 plays an important role in the p53-dependent cytosol apoptotic signaling.