Loss of drug-stimulated topoisomerase II DNA breaks in living cells is different at two unrelated loci

Abstract

Topoisomerase II (top2) has been implicated in the initial steps of chromosomal translocations leading to leukemias and lymphomas, since it can generate DNA cleavage. To evaluate the effects of chromatin structure on enzyme-mediated cleavage, we determined the kinetics of loss of double-stranded DNA breaks stimulated by top2 poisons in Drosophila melanogaster Kc cells at two genomic regions that differ in chromatin structure. Moreover, cleavage loss was determined at 25°C as well as after heat shock. Kinetics were dependent on the poison, nevertheless, loss rate overall was slow at the histone gene cluster, an active chromatin domain. At the repressed satellite III DNA, loss of cleavage was much faster and complete after 5 min in drug-free medium. In addition, differences were noted among sites that were closely spaced and equally intense. Following heat shock at 37°C, we observed reduced cleavage levels and faster loss of breaks at the histone gene cluster. In vitro reversal could only partially explain the in vivo kinetics. Thus, the chromatin context of DNA breaks might play a role in the loss of top2 DNA breaks. The present findings suggest that irreversible cuts may more likely occur in active than silent loci.

INTRODUCTION

Eukaryotic cells have developed a network of highly conserved surveillance mechanisms ensuring that damaged chromosomes are repaired before being replicated and segregated. Nevertheless, non-homologous recombination or other types of incorrect repair may lead to chromosomal translocations thus generating fusion proteins involved in the development of cancer (1–4). In particular, specific translocations of chromosome 11q23 have been consistently found in therapy-related myeloid leukemias and lymphomas (5–7). Chromosomal translocations have been proposed to originate from the action of topoisomerase II (top2) poisons included in the chemotherapeutic regimen for a previous tumor (8–10).

top2 is the target of effective antitumor agents, such as anthracyclines and demethylepipodophyllotoxins (11–14). The drugs stabilize a covalent enzyme–DNA intermediate by forming a ternary enzyme–poison–DNA complex in which DNA strands are cut and covalently linked to enzyme subunits. top2 DNA cleavage is reversible, showing that drug-blocked top2 is able to reseal DNA ends and complete the catalytic cycle upon drug removal. Therefore, cellular repair processes are likely not triggered by top2-mediated cleavage, unless the lesion has not been repaired by the enzyme and has then become irreversible. The mechanisms by which transient top2 DNA breaks eventually result in frank and irreversible cuts have not been fully established, however, one would expect that DNA breaks are less likely to become irreversible if top2 is efficient in rejoining the strand breaks. In vivo DNA break reversibility has not been fully studied and it remains to be established whether chromatin has any role in the efficiency of break reversion. Therefore, we have here measured the kinetics of the loss of breaks upon drug removal from the medium at two genomic loci of Drosophila melanogaster Kc cells. We chose to investigate two repeated sequences, the histone gene and satellite III clusters, since their chromatin structures have been extensively studied (15,16) and top2-promoted DNA cleavage in living cells can be more easily detected in multi-copy DNA segments (16–19). Moreover, cleavage loss was investigated after heat shock of cells, since this treatment is known to modulate chromatin structure and top2-dependent DNA breakage (16,20–22).

MATERIALS AND METHODS

Drugs, enzymes and other materials

The anthracycline analog 4′-demethoxy-3′-deamino-3′-hydroxy-4′-epi-doxorubicin (dh-EPI) was kindly donated by Dr A. Suarato (Pharmacia-Upjohn, Milan, Italy). VM-26 was purchased from Bristol Italiana (Latina, Italy). Drosophila topoisomerase II was purchased from US Biochemical (Cleveland, OH). T4 polynucleotide kinase and polyacrylamide were purchased from Life Technologies (Basel, Switzerland). Restriction enzymes were from New England Biolabs (Taunus, Germany) and [γ-³²P]ATP was obtained from Amersham (Milan, Italy). Human recombinant DNA topoisomerase IIα was purified as reported earlier (18,19).

DNA cleavage assay in living cells

Drosophila Kc cells (3–4 × 10⁶ cells/ml) were treated with 10 µM dh-EPI or 50 µM VM-26 for 30 min and then incubated in drug-free medium for the indicated time at 25°C. DNA was then purified and analyzed by the indirect end-labeling method. In heat shock experiments, cells were pretreated at 37°C for 1 h, then drug was added for 30 min at 37°C. To measure cleavage loss, treated cells were washed and resuspended in fresh drug-free medium and further incubated at 37°C for the indicated time. After treatment, DNA was purified by standard procedures and DNA cleavage was analyzed by the indirect end-labeling method using HB or HP probes (18). Cleavage sites were numbered according to previous reports (16,18). To measure cleavage levels, blots were exposed to phosphor screens and analyzed with a PhosphorImager model 425 (Molecular Dynamics).

In vitro cleavage assay

For dh-EPI, DNA fragments containing the major histone gene repeat were incubated with human top2α or Drosophila top2 without or with dh-EPI for 20 min at 37°C in 20 µl of 10 mM Tris–HCl (pH 7.9), 50 mM NaCl, 50 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 1 mM ATP, 15 µg/ml BSA. DNA cleavage was then analyzed by Southern blotting and the indirect end-labeling technique, as described in the legend to Figure 1. For VM-26, DNA fragments were uniquely 5′-end-labeled with T4 kinase and then incubated with Drosophila top2 with or without VM-26 (25 µM) for 20 min at 25°C. Cleavage was examined by agarose gel electrophoresis and dried gels analyzed as described earlier (18). Cleavage reversibility was assessed by adding 0.6 M NaCl before stopping the reaction with 1% SDS and 0.1 mg/ml proteinase K at 42°C for 45 min.

Figure 1.

Top2 DNA cleavage stimulated by dh-EPI at the histone gene cluster. (A) Top2 DNA cleavage intensity patterns and cleavage loss at 25 and 37°C (as indicated). Lanes C, untreated cells; lanes D, cells lysed immediately after drug treatment. Numbers are minutes after drug removal. On the right of the panels, numbers and bars indicate cleavage sites; site numbers are assigned according to Käs and Laemmli and Borgnetto et al. (16,18). Asterisks indicate minor repeat variants. (B) Map of the 5 kb histone repeat of D.melanogaster, shown as a thin line with arrows indicating coding regions and transcription orientation. Circles between H1 and H3 are fixed nucleosomes at the SAR. The H4 to H1 region has been enlarged in the middle. dh-EPI-dependent cleavage sites are indicated by black arrows. Relative cleavage intensity is indicated by arrow size.

RESULTS AND DISCUSSION

top2 cleavage sites are poison-specific in living cells and, in particular, site patterns are markedly distinct at the histone gene locus between VM-26 and dh-EPI (18). dh-EPI stimulates the most intense cleavage at site 10 (Fig. 1), which maps in the TATA box of the H2A gene (18). In the present report, we have mapped other sites in this particular segment of the histone repeat. dh-EPI was very active, stimulating cleavage at many sites (Fig. 1, sites 9–12) within a segment of ∼2000 bp, whereas VM-26 was only weakly effective in the same repeat segment (18). Taken together, the results indicate that top2 activity was intense in that region under conditions of exponential cell growth.

To evaluate molecular aspects of in vivo cleavage reversibility, we have then measured the kinetics of loss of double-stranded DNA breaks over a wide time range (from 5 min to 5 h) at 25°C (Fig. 1A). First, dh-EPI-stimulated DNA cleavage persisted for very long periods. Moreover, reversal kinetics were markedly different among sites mapped in the H2A–H2B region (Fig. 1) and the histone cluster SAR (Fig. 2). Most persistent cleavage was observed at sites 10, 9 and 6a, where double-stranded DNA breaks were 43–60% of initial levels 4 h after drug treatment (Fig. 2). In contrast, cleavage was completely gone after 2 h at sites 5, 6b, 7b, 11 and 12 (Fig. 2A). Thus, the results demonstrate that dh-EPI-stimulated DNA cleavage remained even after 4–5 h and that this slow cleavage loss was site specific.

Figure 2.

Loss rates of dh-EPI-stimulated DNA cleavage. (A) Loss of DNA cleavage at the histone cluster SAR. Cells were treated with 10 µM dh-EPI for 30 min and then cultured in drug-free medium. Lane C, untreated cells; numbers indicate the time in drug-free medium. Asterisks indicate minor repeat variants. Sites 7b, 6a and 6b are indicated by the three closely spaced bars on the left. Sites 6a and 10 (also indicated on the right) are unique, since DNA cleavage is much more persistent than cleavage at the other sites. (B) Kinetics of loss of DNA cleavage stimulated by dh-EPI. Percentage of cleaved DNA is reported relative to that immediately after drug treatment. Symbols indicate sites (see also Fig. 1) as follows: closed square, 10; closed diamond, 9; open triangle, 11a; closed triangle, 11b and 11c; open circle, 12 (a–c).

Next, we determined cleavage levels and kinetics of break loss under heat shock conditions. It is well known that heat shock influences top2 DNA cleavage in Drosophila cells and induces a reorganization of chromatin structures (16,20–22). A comparison between 25 and 37°C showed that cleavage levels were reduced at the latter temperature (Fig. 1), even though the main sites were the same (sites 9 and 10). Recovery from cleavage was almost complete at 60–120 min (Fig. 2), showing a marked reduction in cleavage persistence as compared with the lower temperature. The results thus indicated that heat shock strongly affected the loss of top2 DNA cleavage at the histone gene cluster.

In contrast to dh-EPI, VM-26 can stimulate DNA cleavage at both histone gene and satellite III repeats (16,18), thus allowing us to directly compare the loss of drug-stimulated DNA breaks in distinct chromatin contexts. For VM-26, we compared cleavage sites in the histone cluster SAR to the satellite III sites, since this poison stimulated strongest cleavage in SAR and not in the H2A–H2B region (16,18). The reversal rate of VM-26-dependent cleavage was markedly slower in the histone repeat region than in satellite DNA (Fig. 3). All DNA cuts in the satellite III DNA were essentially gone only 5 min after drug removal, whereas cleavage persisted up to 120 min in histone cluster SAR. We also observed differences among VM-26-stimulated sites: cleavage was still readily detectable at sites 4 and 5 after 60–120 min, whereas cleavage reversal was faster at sites 3, 6 and 7, and more similar to that of the satellite repeats (Fig. 3). The initial level of cleavage was not correlated with cleavage reversibility, since cleavage levels were similar at sites 3, 4 and 6, whereas their reversal rates were clearly different (Fig. 3). Moreover, the two genomic loci studied were highly and similarly affected by VM-26, as indicated by pulsed field gel electrophoresis results (23), whereas reversal was markedly different (Fig. 3). Reversal of VM-26-stimulated DNA cleavage was also determined under heat shock conditions and, in agreement with dh-EPI, an enhancement of reversal rate was detected at the histone cluster (not shown).

Figure 3.

In vivo loss of VM-26-stimulated DNA cleavage at the histone gene cluster (left) and the centromeric satellite III DNA (right). Cells were treated with 50 µM VM-26 for 30 min and then cultured in drug-free medium. Lanes C, untreated cells; lanes V, cells treated with VM-26 and immediately lysed; lanes 5, 10, 15, 20, 30, 60 and 120, VM-26-treated cells after the indicated time (min) in drug-free medium. (Left) Numbers and bars indicate main cleavage sites in the SAR. (Right) top2 cleaves DNA once per repeat in the satellite cluster (16,18), therefore it generates a ladder of ∼300 bp, as seen in lane V.

The results suggest that diverse molecular aspects may affect loss of top2-mediated DNA cuts in living cells. To establish whether the observed in vivo heterogeneity was due to variability in the intrinsic stability of poisoned top2 at different DNA sites, we compared the kinetics of cleavage reversal upon salt addition with purified top2 and the same DNA fragments (Fig. 4). VM-26-stimulated DNA cleavage reverted in vitro with much slower kinetics than top2 DNA cleavage without poison, however, no kinetic difference was observed between the two DNA templates (Fig. 4A). In the histone gene repeat, all dh-EPI-stimulated sites were still present 1 h after salt addition at 10 µM drug, whereas cleavage reverted when using 5 µM dh-EPI (Fig. 4B). Overall, cleavage reverted with similar rates, even though some differences could be detected among sites that may correspond to the in vivo sites 6a and 5 (see bands marked with arrows in Fig. 4). Thus, the present results indicate that poison/enzyme interactions with different DNA sites may in part explain the kinetic differences observed for dh-EPI in nuclear chromatin, but not those detected for VM-26.

Figure 4.

In vitro DNA cleavage reversion. (A) (Left) VM-26-stimulated DNA in the satellite III DNA (SAT) and histone gene cluster (HIS). (Right) Cleavage reversion without drugs. Numbers above the lanes indicate the time (min) after salt addition. Lanes C, untreated DNA. (B) Reversion of DNA cleavage stimulated by dh-EPI with purified top2 at a cloned histone gene fragment. Lanes C, untreated DNA; lanes T, enzyme alone; lanes 0, 5, 15, 30, 45 and 60, minutes after salt addition and before SDS stopping. (Left) Arrows on the right indicate sites with more persistent cleavage. The lower and middle bands may correspond to sites 5 and 6a observed in vivo (see Fig. 2). Cleavage bands in the upper part of the gel increased upon salt addition due to initial DNA overdigestion (lane 0) that depleted higher fragments relative to lower ones.

The satellite III DNA is a silent heterochromatic region whereas the histone gene cluster is a transcriptionally active chromatin domain (15,24). We have shown that top2 DNA cleavage can be extensive at the loci studied (18,23). Our present findings indicate that the most dramatic difference between the two loci is the rate of break loss. The breaks can be lost in vivo in one of two ways; either the poisoned top2 can reseal the cuts on drug removal or they can be resealed by a DNA repair pathway. Our data do not allow us to distinguish between the two possibilities. Nevertheless, the loss observed in the satellite III DNA is almost immediate upon drug removal, thus suggesting that top2 itself likely reseals the break. This would indicate that the trapped top2 remains fully active in this chromatin context. In contrast, the much slower loss observed in the histone gene cluster may suggest that top2 cannot reseal the breaks and in that case a repair pathway is more likely to be necessary to restore DNA integrity. We may therefore suggest that early events downstream from top2 DNA breaks are distinct at the two loci studied and that irreversible DNA breaks are much more likely to occur in active chromatin rather than heterochromatin.

The present findings may have implications for the pathway leading to chromosomal repair and/or translocation. top2 has been proposed to generate chromosome breaks that constitute the initial step of neoplastic transformation of hematopoietic precursor cells involving the human MLL gene (8). Recent reports have suggested that chromosomal translocations may be generated by ‘error-prone repair’ mechanisms and not by the specific recombinases of the immune system (2). Moreover, the authors conclude that both chromosomes most likely undergo multiple breaks before the occurrence of translocations (2). Our findings clearly show that top2-promoted cleavage in nuclear chromatin is intense and frequent in a relatively short DNA segment, such as the 5 kb major histone gene repeat. If long-lived top2-associated breaks are more likely to be converted into irreversible lesions in active chromatin, drug action would thus result in multiple breaks at the given locus. Interestingly, DNase I-hypersensitive sites were reported near the bcr of the MLL gene, suggesting an open chromatin structure (25). Our observations are therefore consistent with the above reports (2) and with the hypothesis that top2 is involved in the development of therapy-related leukemias.