We used confocal microscopy to detect prefixed immunfluorescently labeled histones and laser scanning cytometer to detect histone aggregation. Results: Using Confocal Microscopy (CLSM), we observed marked differences between the effect of doxorubicin (Dox), applied in a concentration range between 0.6-36 µM, on the intracellular distribution of H2A vs. H2B in Jurkat cells. Aggregation was assessed by Laser Scanning Cytometry (LSC), via the retention and consequential accumulation of histones in permeabilized nuclei of cells showing no signs of apoptosis. Aggregation was observed only in the case of H2A, while the dominant effect of the anthracyclin on H2B was the massive accumulation of the histone in the cytoplasm concommitant with its disappearance from the nuclei. The latter phenomenon was not affected by inhibitors of protein and RNA synthesis, by Tanespimycin, an hsp70 inhibitor, and by leptomycin B, a nuclear export inhibitor. On the other hand, cytoplasmic accumulation was completely diminished by PYR41, an inhibitor of ubiquitylation, when H2B showed a H2A-like aggregated pattern in the nuclei. The cytoplasmic accumulation of H2B was confirmed also by mass spectrometric identification of elevated levels of H2B following Dox treatment in the dechro-matinized samples. Conclusions: Anthracyc-lines are widely used anti-cancer drugs exhibiting pleiotropic effects. At the chromatin level these include topoisomerase inhibition, DNA intercalation, aggregation of chromatin, histone eviction as well as direct binding to histones. The above large-scale effects were detected already at Dox concentrations that overlap serum peak levels reached in the typical clinical setting and therefore can be a factor both in the anticarcinogenic mechanism and in the side-effects of this anthracyclin.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)