Capillary zone electrophoresis

Supplementary to our HPLC analyses we approached the topic of apoptosis-related histone modifications by histone separation via capillary zone electrophoresis (CZE). This is a highly sensitive alternative method, which reaches by far better resolutions, when it comes down to histone modification analysis. Moreover, this method offers a time saving alternative to the time consuming 2D-gel electrophoresis.

HL-60 cells were treated the same way as described and histones were isolated, purified and also separated via HPLC.

Compared to just slight changes in histone modifications and subtype pattern that were observed after 2D-gel electrophoresis, changes observed by CZE were more significant than expected. For comparative analysis, core histones from butyrate treated cells were analysed in parallel, which gave us the chance to investigate a possible connection of apoptosis-related changes in the histone acetylation pattern. Although this is an often given statement in publications, it has to be qualified, as butyrate treatment of cells does not only cause hyperacetylation of some histone types but also leads to a G1-arrest in cells, which is subsequently connected with large scale dephosphorylation. So, as histones were analysed mainly on the basis of an apoptosis-related change in their state of acetylation, a possible connection to a change in their phosphorylation pattern was still kept in mind.

Analysis of the histone H3 peak separation pattern revealed a reduction from four distinct peaks in the control sample to two large peaks and a smaller one in the topotecan^® treated sample. Compared to that, the peak pattern in the butyrate treated sample appeared to be completely different. The first two peaks which were high in the topotecan^® treated sample, turned out to be low in the sample treated with butyrate and vice versa (Fig. 3.6.3 1; red arrows). So it might be assumed, that after induction of apoptosis, H3 histones are not subject to an increase in acetylation. However, those changes observed might readily be referred to a strong tendency of dephosphorylation, an observation that has already be described for both H3 at serine 10 and 28 and histone H1 (Happel et al., 2005). As this is still an assumption, it remains to be elucidated which other modifications might be responsible for the changes in peak patterns.

In the first of altogether two histone H2A HPLC fractions, the shape of the peak pattern did not show any remarkable changes, except for a slight peak shoulder in the control sample, which obviously almost disappeared from the peak pattern of topotecan^® treated samples (Fig. 3.6.3; red and black arrows). A possible apoptosis-related change in the acetylation pattern of this histone type does not seem to be the case, as its peak shape compared with that of the butyrate treated sample was completely different. As it seems, the first peak in the butyrate treated sample is much higher than the first peak in the topotecan^® treated sample.

Peak shapes of histones from butyrate treated cells obviously undergo a shift to the left (Fig.

3.6.3 3; black arrow in lower panel), a fact that was not observed with peaks from topotecan^® treated cells. However, a possible dephosphorylation of histone H2A(1) might be assumed, all sorts of different modifications will most probably be involved in changes of these peak patterns.

Analysis of the second H2A (H2A(2) HPLC fraction revealed no changes at all, concerning the peak pattern. Peak shapes of all three samples, control, topotecan^® and butyrate, were congruent (Fig. 3.6.3 5).

More severe changes in histone modifications could be evidenced in histones H2B. The peak pattern of the control sample turned out to be a mirror image of the pattern in topotecan^® treated samples. Peak shapes and heights almost remained unchanged but appeared in reverse order (Fig. 3.6.3 2). Apoptosis-related changes of peak patterns in histone H2B were the most severe observed. The first peak of the control sample seems to resemble the last peak in the topotecan^® treated sample, with an additional small peak shoulder towards the end (Fig. 3.6.3 2; bend red and black arrows). Whereas control and butyrate samples both showed certain similarities in their peak pattern and peak shape, the topotecan^® sample shows remarkable deviations, a fact which supports the assumption that here, too, treatment with topotecan^® does not seem to be connected with an increase in acetylation of histone H2B.

Fig. 3.6.3 Capillary zone electrophoresis analyses of core histones. HL-60 cells were either treated with topotecan or with the HDAC inhibitor butyrate to achieve a hyperacetylation of histones. After purification and separation via HPLC single histone extracts were loaded onto the capillary zone electrophoresis unit and run for 2 h. (1) Separation of histone H3 control samples and with topotecan^® or butyrate treated samples. A strong shift of H3 histones was observed which were pre-treated with butyrate for 24 h. Apoptosis-related changes in the peak pattern were also monitored. Peaks marked with *Ac0-Ac3 are putative acetylated forms of H3 following published data by (Lindner et al., 2003;

Lindner et al., 1996). (2) Separation of the first H2A(1) of altogether two HPLC histone H2A fractions.

The peak pattern of control histones and apoptosis induced H2A(1) histones was almost identical, except for a very small peak shoulder in control cells (black arrow compared to red arrow). (3) The peak pattern of H2B histones of control cells turned out to be a mirror image of topotecan^® treated sample (black and red arrow). All peak heights were identical but appeared in a reverse order. All peaks of the butyrate treated sample bear quite a few similarities in shape and pattern compared to histone H2B from control cells. (4) Separation of the second H2A(2) HPLC fractions. No changes in the peak pattern was observed

H3 H2A(1) 1

0h control 8h topotecan

24h butyrate 24h butyrate

24h butyrate 24h butyrate

H2B 4 H2A(2)

0h control 8h topotecan

0h control 8h topotecan

0h control 8h topotecan

→

→

→

→

3

2

Ac0 Ac1 Ac2

Ac3

*

H4 5

0h control 8h topotecan

24h butyrate

→ →

Ac0

Ac1 Ac2

Ac3

Ac4 Ac1

Ac0

Ac3

Ac2

*

*

Fig. 3.6.3 (cont. 1) Capillary zone electrophoresis analyses of core histones. HL-60 cells were either treated with topotecan or with the HDAC inhibitor butyrate to achieve a hyperacetylation of histones.

After purification and separation via HPLC single histone extracts were loaded onto the capillary zone electrophoresis unit and run for 2 h. (5) Separation pattern of H4 histones. Two out of altogether four H4 control histone peaks vanished completely after topotecan^® treatment (red arrows). Peaks marked with *Ac0-Ac3 are putative acetylated forms of H4 following published data by Lindner et al. (Lindner et al., 2003; Lindner et al., 1996).

In analyses of histone H4 via CZE we evidenced that two out of altogether four H4 control histone peaks seemingly vanished completely after topotecan^® treatment, which went ahead with a slight shift of the two remaining H4 peaks to the right (Fig. 3.6.3 5, red arrows). The peak pattern of H4 from the topotecan^® treated sample showed great differences compared with those patterns of control cells and butyrate treated cells. The very first and last peak in the butyrate sample disappeared completely from the H4 peak separation pattern in the topotecan^® sample. As a result and on the basis of the great differences between control and butyrate samples on the one hand and the topotecan^® sample on the other hand, a tendency towards hyperacetylation of H4 during apoptosis can almost be excluded.

These studies made it quite clear that during the ongoing process of apoptosis there are apparently some major changes in histone modifications of the histones H2B, H3 and H4, which can be well described by means of CZE analysis. However, it remains to be elucidated, which types of modification we are dealing with in particular, as in terms of

analysis, these results reflect the changes of all different histone modifications, as there are acetylation, methylation, phosphorylation and ubiquitylation. In a following step it would be necessary to consider one single type of modification, e.g. histone acetylation or methylation, and to analyse it by means of mass spectrometry.