Comparative analysis of nuclear histone release

To compare and evaluate our results in connection with one of the main present date publications by Radic et al. (Radic et al., 2004), we used the nucleus-cytosol preparation protocol of this group and tested it with our cell line HL-60. This group analysed the apoptosis-related release of core histones and nucleosomes into the cytosol in Jurkat cells.

As a result Radic et al. (Radic et al., 2004) stated that nucleosomes were exposed at the cell surface after the onset of apoptosis and this would indicate a key role for nucleosomes in the execution of apoptosis as well as a clearance of apoptotic cells.

In our own experiments we observed a time-dependent release of specifically modified core histones into the nucleoplasm of both HeLa and HL-60 cells. However, a further massive release of these nuclear components could not be confirmed. Therefore we compared our own results and the nucleus-cytosol preparation that was used, with the protocol by Radic et al. (Radic et al., 2004). We tested the HL-60 cells using the same nucleus-cytosol preparation that Radic and co-workers used. The quality of the preparation was surveyed using markers of apoptosis, such as cleavage of nucleosomal DNA and PARP. About 2 h after induction of apoptosis, cleavage of nucleosomal DNA was monitored (Fig. 3.8.3 1; lane 3). PARP cleavage was observed 4h after treatment with topotecan^® with a slight delay in time (Fig. 3.8.3 2; lane 4). To check if the nuclei prepared were intact before the actual analysis was carried out, protein lysates from nuclear and cytosolic fractions were incubated with anti-lactate dehydrogenase as a cytosolic marker and anti-PARP antibody as a nuclear marker. No cytosolic protein was found in any of the nuclear fractions (Fig. 3.8.3 3), however, various small and full length fragments of PARP were traced in different concentrations in all cytosolic fractions (Fig. 3.8.3 4; lanes 2-6). Due to this fact it cannot be denied that nuclei from this preparation seemed to have suffered some greater leaks resulting from preparation procedures. In this study this particular problem was avoided in a way that several protocols had been tested in advance for their yield of morphologically and biochemically intact nuclei.

0h

cytosolic marker pretein nuclear marker protein

Fig. 3.8.3 Monitoring the ongoing process of apoptosis with reference to the nucleus-cytosol preparation protocol according to Radic et al. (2004). HL-60 cells were induced with topotecan^® and harvested according to the protocol by Radic et al. (Radic et al., 2004). Cells harvested shortly after seeding served as negative controls, whereas cells pre-treated with the caspase 3 inhibitor z-VAD-fmk before induction, were used as positive control, to monitor the induction efficiency. Nucleosomal DNA and cell lysates for PARP cleavage were prepared. (1) Nucleosomal DNA after induction of apoptosis.

Nucleosomes were detected as early as 2 h after drug treatment and their concentration increased during apoptosis. (2) Western blot incubated with anti-PARP antibody. PARP cleavage was first monitored 4 h after induction of apoptosis. (3) Western blot incubated with anti-lactate dehydrogenase antibody. With reference to the nucleus-cytosol preparation protocol by Radic et al. (2004) the cytosolic marker lactate dehydrogenase was not detected in any of the nuclear fractions prepared. (4) Western blot incubated with anti-PARP antibody. PARP was used as a nuclear marker. Various small and full length fragments of PARP were traced in all cytosolic fractions.

5

Caspase 3 assay

0 1 2 3 4 5 6

0h control 2h topotecan 4h topotecan 6h topotecan 2h z-VAD-fmk/6h topotecan apoptosis induction with topotecan (150 ng/ml)

Caspase 3 activity [rel. units]

Fig. 3.8.3 (cont. 1) Monitoring the ongoing process of apoptosis with reference to the nucleus-cytosol preparation protocol according to Radic et al. (2004). HL-60 cells were induced with topotecan^® and harvested according to the protocol by Radic et al. (Radic et al., 2004). Cells harvested shortly after seeding served as negative controls, whereas cells pre-treated with the caspase 3 inhibitor z-VAD-fmk before induction, were used as positive control, to monitor the induction efficiency. (5) Caspase 3 assay. A strong increase in caspase 3 activity was observed 4 h after induction of apoptosis.

To investigate a putative apoptosis-related nuclear histone release on the basis of the preparation by Radic et al. (Radic et al., 2004) compared to our own experiments reported earlier (chap. 3.8.1), protein lysates from nuclear and cytosolic fractions were separated via SDS-PAGE. As a result, gradually increasing concentrations of all four histone types were detected to a certain extent in all cytosolic fractions, including the cytosolic control fraction (Fig. 3.8.3 6; lanes 2-5). Western blot analyses just confirmed these findings. Blots incubated with anti-H3, anti-H2B, anti-H2A and anti-H4 antibodies showed a gradual accumulation of all four core histones into the cytosol (Fig. 3.8.3 7-10). Depending on the quality of the antibody’s binding specificity, signal intensities varied quite a bit, however, the overall result was not influenced by it.

As a general rule, it can be assumed that the outcome of most experiments in the area of apoptosis-related nucleosomal cleavage and subsequent release of core histones from bulk chromatin into the nucleus and later on into the cytosol, always remains a question of methodical approach. Only if these differences are taken into account, it will be possible to pronounce general statements that can be properly evaluated.

cytosolic

Fig. 3.8.3 (cont. 2) Western blot analyses of apoptosis-related nucleosomal cleavage and release of core histones from bulk chromatin into the nucleus and cytosol with reference to a nucleus-cytosol preparation protocol by Radic et al. (2004). HL-60 cells were induced and harvested as described by Radic et al. (Radic et al., 2004). (6) Coomassie stained SDS-gel showing cytosolic fractions (left) and nuclear fractions (right) after nucleus-cytosol preparation. With the onset of apoptosis increasing concentrations of all four core histone types were visible in all cytosolic fractions. These could even be traced in control samples. (7, 8 and 10) Western blots incubated with anti-H3, anti-H2A and anti-H4 antibodies. The signal intensities in all three blots more or less increased gradually after induction of apoptosis, however, traces of H3 were already found in the cytosolic fraction of the control sample. (9) Western blot incubated with anti-H2B antibody. Although the antibody almost failed to detect H2B protein in the cytosolic fractions, traces could still be found.

With relation to the protocol by Radic et al. (Radic et al., 2004), all Western blot analyses of apoptosis-related nucleosomal cleavage and release of core histones from bulk chromatin into the nucleus and cytosol, of course could not have produced any different results, as the ones presented here. The fact that Radic and co-workers used cytosolic and nuclear lysates rather than cautiously prepared morphological as well as biochemically intact nuclei, could only result in a gradual increase in released histone types from the nucleus into the cytosol.

3.9 Microscopic analyses of an apoptosis-related nuclear release of core histones in