Histone H1 subtype preferences of DFF40 and possible nuclear localization of DFF40/45 in normal and trichostatin A-treated NB4 leukemic cells.

A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.

Differential sensitivity of human leukemic cell lines to the histone deacetylase inhibitor, trichostatin A.

Histone deacetylase inhibitors (HDACIs) inhibit deacetylases and the accumulation of high levels of acetylation results in chromatin remodeling events which may lead to cell cycle arrest and apoptosis. This work investigates the sensitivity of four leukemic cell lines to the HDACI, trichostatin A (TSA) as compared to normal lymphocytes with respect to acetylation and apoptotic levels. Specifically, this study analyzes the time kinetics of histone H4 and alpha-tubulin acetylation and associates these findings to the time course of TSA-induced PARP cleavage and DFF45 proteolysis. The results of this study show (1) that a non-responsive leukemic cell line to the apoptotic effects of TSA does not have increased acetylation levels in contrast to the responsive leukemic cell lines that show a hyperacetylated profile. This indicates that acetylation levels may be of special importance in accessing the potential sensitivities of leukemic cells to HDACIs, (2) TSA induced apoptosis in lymphocytes but at lower levels and (3) the lack of PARP cleavage and DFF45 proteolysis found in lymphocytes clearly differentiates the final stages apoptosis of human peripheral blood lymphocytes from those of the TSA-sensitive leukemic cell lines. Of value is that the results of this study show that the evaluation of the acetylation levels of target proteins may possibly have the potential of being used as additional indicators of the responsiveness or sensitivity of different cancer cell types to this continuously growing class of anticancer agents.