Changes in the Protein Pattern of H1 Histones Associated with Apoptotic DNA Fragmentation

Characteristic steps in the course of cellular apoptosis are the induction of chromatin condensation and cleavage of the DNA, leading to the formation of oligomers of nucleosomes. Since the H1 histones represent functional elements that are essential for the generation of highly condensed chromatin structures, we analysed the total cellular H1 histones of five leukaemic and three solid human tumour cell lines, comparing the H1 pattern of exponentially growing cells with that of apoptotic cells. For the induction of apoptosis, cell lines were treated with the water-soluble camptothecin derivative, topotecan (a topoisomerase I inhibitor), or with an apoptosis-inducing monoclonal anti-CD95 (Fas/APO-1) antibody. Total histone H1 proteins were isolated by extraction with 5% perchloric acid and were analysed by means of capillary zone electrophoresis (CZE) separation. The identities of the peaks representing different histone H1 subtypes on CZE electropherograms were confirmed by analysis of preparations (recombinant proteins purified from transformed yeast used as internal standards) mixed with each of the subtypes respectively. The progress of topotecan- or anti-CD95-induced cell death was monitored by flow cytometry analysis and also by agarose electrophoresis of fragmented DNA. During early apoptosis of three of these cell lines, we observed the induction of internucleosomal DNA cleavage and, simultaneously, a typical change in the histone H1 protein pattern, leading to an increase in the relative amounts of histone subtypes H1.4 and H1.5. Upon apoptosis induction, these changes were only observed in correlation with the occurrence of DNA fragmentation, thus possibly reflecting a prerequisite for DNA accessibility and/or endonuclease activity.