Genistein induces topoisomerase IIbeta- and proteasome-mediated DNA sequence rearrangements: Implications in infant leukemia

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Abstract

Genistein is a bioflavonoid enriched in soy products. However, high levels of maternal soy consumption have been linked to the development of infant leukemia ALL and AML. The majority of infant leukemia is linked to mixed lineage leukemia gene (MLL) translocations. Previous studies have implicated topoisomerase II (Top2) in genistein-induced infant leukemia. In order to understand the roles of the two Top2 isozymes in and the molecular mechanism for genistein-induced infant leukemia, we carried out studies in vitro using purified recombinant human Top2 isozymes, as well as studies in cultured mouse myeloid progenitor cells (32Dc13) and Top2β knockout mouse embryonic fibroblasts (MEFs). First, we showed that genistein efficiently induced both Top2α and Top2β cleavage complexes in the purified system as well as in cultured mouse cells. Second, genistein induced proteasomal degradation of Top2β in 32Dc13 cells. Third, the genistein-induced DNA double-strand break (DSB) signal, γ-H2AX, was dependent on the Top2β isozyme and proteasome activity. Fourth, the requirement for Top2β and proteasome activity was mirrored in genistein-induced DNA sequence rearrangements, as monitored by a DNA integration assay. Together, our results suggest a model in which genistein-induced Top2β cleavage complexes are processed by proteasome, leading to the exposure of otherwise Top2β-concealed DSBs and subsequent chromosome rearrangements, and implicate a major role of Top2β and proteasome in genistein-induced infant leukemia.

Research highlights

► Genistein efficiently induces both Top2α and Top2β cleavage complexes. ► Genistein induces preferential proteasome-mediated degradation of Top2β. ► Genistein-induced DNA double-strand break signals are dependent on the Top2β isozyme and proteasome activity. ► Genistein-induced DNA sequence rearrangements are Top2β- and proteasome-mediated.

Introduction

Genistein is a natural bioflavonoid (isoflavone) mostly found in soy-based foods. Genistein exhibits cancer-chemopreventive and antitumor activities, as well as anti-oxidant, anti-inflammatory and anti-in vitro angiogenesis effects [1], [2], [3]. However, clinical studies have suggested a strong link between a prior exposure to dietary bioflavonoids including genistein and infant leukemia. It was demonstrated that maternal consumption of bioflavonoid-rich foods led to an approximately 10-fold higher risk of infant acute myelogenous leukemia (AML) [4], [5].

Infant leukemia is frequently associated with chromosome translocations involving the mixed lineage leukemia 1 (MLL) gene [6]. It has been estimated that up to 80% of infant acute lymphoblastic leukemia (ALL) and 65% of infant AML are linked to MLL translocations [7]. These translocations can take place in utero and are associated with poor prognosis, especially in infant ALL [5]. Interestingly, MLL translocations are also hallmarks of more than 70% of t-AML (therapy-related acute myelogenous leukemia) associated with topoisomerase II (Top2)-based chemotherapy in cancer patients [8]. Mapping of chromosomal breakpoints of MLL translocations has revealed the clustering of breakpoints within an 8.3 kb region of the human MLL gene, known as the breakpoint cluster region (BCR) [8]. The genomic breakpoints in infant leukemia and t-AML tend to co-localize with Top2 cleavage sites, suggesting a possible link between infant leukemia and Top2 [8], [9].

Genistein is known to induce Top2-linked DNA breaks (Top2 cleavage complexes) [3]. There are two human Top2 isozymes, Top2α and Top2β, that share 70% sequence identity [10]. Top2α has been suggested to function in cell cycle events such as DNA replication and chromosome condensation/segregation [11], whereas Top2β has been shown to be involved in transcription [12], [13], [14], [15]. Recent studies have shown that cancer chemotherapeutic drugs that target Top2 poison both Top2α and Top2β[10]. It has been suggested that Top2α targeting (poisoning) is primarily responsible for the antitumor activity of these drugs while Top2β targeting could lead to secondary malignancies such as (Top2 drug) therapy-related acute myelogenous leukemia (t-AML) [16]. Since genistein is a Top2-targeting compound, we envision that genistein may induce infant leukemia through poisoning of the Top2β isozyme.

Previous studies have demonstrated that the induction of DNA double-strand breaks (DSBs) by Top2-targeting drugs characteristically requires the proteasome activity [17]. It has been shown that Top2-targeting drugs induce preferential degradation of the Top2β isozyme (Top2β down-regulation) in various cells, leading to the exposure of the otherwise Top2-concealed DSBs [16], [17]. It has been proposed that Top2β down-regulation is the underlying mechanism for Top2 drug-induced DNA sequence rearrangements and carcinogenesis [16].

In the present study, we have tested the role of Top2β and proteasome in genistein-induced DSBs and chromosome rearrangements. Our results suggest that proteasomal processing of genistein-induced Top2β cleavage complexes results in DSB formation and DNA sequence rearrangements, thus implicating an important role of Top2β and proteasome in genistein-induced MLL translocations and infant leukemia.

Section snippets

shRNA-mediated knockdown of Top2β in 32Dc13 mouse myeloid progenitor cells

The Control (Ctrl) or Top2β shRNA sequences were selected using the Whitehead Institute siRNA selection program (http://jura.wi.mit.edu/bioc/siRNAext/) and the corresponding oligo duplex DNAs were cloned into a LentiLox 3.7 vector with an inserted neomycin-resistant gene. Standard procedures were then followed to generate stable Top2β- or control-knockdown 32Dc13 cells lines.

Top2-mediated DNA cleavage assay

The Top2 cleavage assay was performed as described [18].

Measurement of plasmid integration frequency

For measuring the plasmid integration frequency in MEFs,

Genistein induces Top2 cleavage complexes and proteasomal degradation of Top2β (Top2β down-regulation)

As shown in Fig. 1A, genistein, like VP-16 (a prototypic Top2 poison), induced both Top2α and Top2β-mediated DNA cleavage in a concentration-dependent manner, most certainly reflecting the formation of cleavage complexes with the two Top2 isozymes (Fig. 1A, note the disappearance of the full length DNA (labeled ∗∗) and the appearance of the cleaved DNA fragments (labeled ∗)). Relative to VP-16, genistein appeared to be slightly more effective in inducing Top2β- than Top2α-mediated DNA

Discussion

Previous studies have demonstrated that genistein-induced chromosomal DNA cleavages as well as chromosome translocations involve the MLL BCR [9], [22]. These genistein-induced DNA cleavages and chromosome translocations have been suggested to result from genistein-trapped Top2 cleavage complexes within the MLL BCR [8], [23], [24]. However, the relative contribution of the two Top2 isozymes (i.e. Top2α and Top2β) as well as the mechanism for processing Top2 cleavage complexes into DSBs, in

Conclusions

In the present study, we have tested the role of Top2β and proteasome in genistein-induced DSBs and chromosome rearrangements. We show that genistein, like other Top2 drugs, induces proteasomal degradation of Top2β. In addition, we show that genistein-induced DNA damage and sequence rearrangements are Top2β-mediated and can be prevented by co-treatment with the proteasome inhibitor MG132. These results suggest that proteasomal processing of genistein-induced Top2β cleavage complexes results in

Acknowledgments

We thank Dr. Makoto Kimura (Laboratory for Remediation Research, Plant Science Center, Saitama, Japan) for providing the pUSCV-BSD plasmid. This work was supported in part by a NIH Grant CA102463, a New Jersey Commission on Cancer Research Grant 06-2419-CCR-EO, a Department of Defense Idea Award W81XWH-07-1-0407 and a Department of Defense Concept Award W81XWH06-1-0514.

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