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Topoisomerase II alpha (TOP2A) has a crucial role in proper chromosome condensation and segregation. Here we report the interaction of TOP2A with ataxia telangiectasia mutated (ATM) and its phosphorylation in an ATM-dependent manner after DNA damage. In vitro kinase assay and site-directed mutagenesis studies revealed that serine 1512 is the target of phosphorylation through ATM. Serine 1512 to Alanine mutation of TOP2A showed increased stability of the protein, retaining TOP2A activity at least with regard to cell survival activity. Ataxia telangiectasia-derived cell lines showed high levels of TOP2A that were associated with hypersensitivity to the TOP2 inhibitor etoposide. These findings suggest that ATM-dependent TOP2A modification is required for proper regulation of TOP2 stability and subsequently of the sensitivity to TOP2 inhibitor. In a lymphoblastoid cell line derived from a patient who developed MLL rearrangement, positive infant leukemia, defective ATM expression, and increased TOP2A expression were shown. It was intriguing that hypersensitivity to TOP2 inhibitor and susceptibility to MLL gene rearrangement were shown by low-dose etoposide exposure in this cell line. Thus, our findings have clinically important implications for the pathogenesis of infantile acute leukemia as well as treatment-associated secondary leukemia following exposure to TOP2 inhibitors.
Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by a pleiotropic phenotype that includes progressive cerebellar degeneration, immunodeficiency, premature aging, genetic instability, and a high incidence of cancer. Heterozygous carriers also appear to be at increased risk of cancer.[1, 2] Cells from AT homozygotes lack multiple cell cycle check points, and this leads to hypersensitivity to double-strand breaks in the DNA.[3, 4]
DNA topology is controlled and altered by DNA topoisomerases. Topoisomerases are ubiquitous enzymes that resolve topological problems, which arise during the various processes of DNA metabolism including transcription, recombination, replication, and chromosome partitioning during cell division. Topoisomerase I introduces a transient single-strand break into the DNA, passes an intact single strand of DNA through the broken strand, and re-ligates the break. Topoisomerase II (TOP2) makes transient double-strand breaks in one segment of DNA and passes an intact duplex through the broken DNA before resealing the breaks. Human cells express two isoforms of TOP2, topoisomerase II alpha (TOP2A) and topoisomerase II beta (TOP2B). TOP2A is expressed mainly during the S to G2/M phase of the cell cycle and is likely to play a major role in DNA catenation during mitosis. In contrast, TOP2B is expressed constantly throughout the cell cycle. The function of TOP2B is unknown, but the enzyme is speculated to be involved in the metabolism of DNA and/or RNA. Topoisomerase II alpha is highly phosphorylated, and its enzymatic activity has been postulated to be regulated by phosphorylation, by its interaction with other factors, or by its subcellular localization. However, no clearly defined role of phosphorylation has been identified.
Exposure in utero to TOP2 inhibitor or a similar substance is thought to lead to infantile leukemia, which is characterized by frequent chromosomal translocations involving the mixed lineage leukemia (MLL) gene.[7-9] Rearrangement of the MLL gene also occurs in treatment-related leukemia that arises after treatment with TOP2 inhibitor. We reported that dysfunction of ataxia telangiectasia mutated (ATM), responsible for AT, plays an important role in the development of some infantile acute leukemia. Our findings led us to speculate that the pathogenesis of infantile acute leukemia involves ATM dysfunction and hypersensitivity to TOP2A inhibitor, and prompted us to investigate the physiologic relation between ATM and TOP2.
Here we report an association between TOP2A and ATM and the phosphorylation of TOP2A in an ATM-dependent manner. Cell biological data also indicate a functional relation between ATM and TOP2A. Our study provides new evidence for ATM-dependent regulation of TOP2, a factor that may be involved in the pathogenesis of infantile acute leukemia.
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We found that ATM and TOP2A bind to each other, and this finding suggested the possibility of ATM dependent TOP2A phosphorylation, nevertheless its kinase activity. We also found increased TOP2A expression in AT cells that are defective in ATM expression. This increased expression of TOP2 was associated with hypersensitivity to TOP2 inhibitor.
Ataxia telangiectasia cells are hypersensitive not only to irradiation, but also to TOP2 inhibitor,[20-22] and defective activation of the replication checkpoint machinery occurs after exposure to TOP2 inhibitor. Inhibitors of TOP2 induce DNA double-strand breaks, but TOP2 catalytic inhibitor also induces chromatin structural changes without DNA breaks. For example, ATM is activated by chromatin structural changes, which is induced by the TOP2 catalytic inhibitor chloroquine. The function of TOP2 in AT cells has been much investigated, but contradictory findings, such as decreased[25-27] or increased TOP2 activity,[22, 28] have been reported. Nevertheless, these findings strongly suggest that ATM is involved in the checkpoint of DNA damage induced by TOP2 inhibition; they also suggest a functional relationship between ATM and TOP2.
Recent research suggested that TOP2A and BRCA1 interact with each other, and TOP2A decatenation activity is regulated by BRCA1 interaction and ubiquitination. BRCA1 is one of the best characterized ATM substrates, and forms BRCA1-associated genome surveillance complex that contains ATM protein. These two reports support the potential connection of molecular interaction between ATM and TOP2A shown by our experiment. Several proteins have also been reported to interact with topoisomerase. Topoisomerase II-binding protein 1 (TOPBP1), for example, contains a breast cancer suppressor protein, C-terminal domain and is phosphorylated in response to DNA damage in an ATM-dependent manner. It has been shown that TOPBP1, TOP2A, and TOP2B interact with the C-terminus of p53, and this finding allows us to speculate that ATM, TOP2, TOPBP1, and p53 function as a protein complex.
Topoisomerase II alpha is phosphorylated on multiple Ser and Thr residues, most of which are located in the C-terminal domain.[34-39] Several potential ATM-dependent or -regulated phosphorylation sites (Ser1424, 1466, 1522, and 1524) were reported. Our findings showed phosphorylation of a new target site, Ser1512, induced by DNA damage in an ATM-dependent manner. The ATM family proteins such as ATM, ATR, and DNA-PK prefer to phosphorylate the Ser/Thr-Gln motif. Topoisomerase II alpha contains three Ser/Thr-Gln motifs, but ATM immunoprecipitants did not phosphorylate these sites. The site phosphorylated by the ATM immunoprecipitants was a Ser residue followed by Val at the C-terminal of TOP2A. DNA-PK phosphorylates proline–serine in vitro. Ataxia telangiectasia mutated also phosphorylates the Ser residue with the adjacent glycine residue of BRCA1 in vitro and in cellulo. A recent study identified numerous ATM-dependent and -regulated phosphorylation sites. Target proteins are complexly regulated in an ATM-dependent manner, involving either direct or indirect phosphorylation by ATM. Our finding that TOP2A is phosphorylated by ATM kinase-dead construct immunoprecipitants to some extent suggests the possibility that the ATM immunoprecipitants include some other ATM-like unknown protein kinases, which phosphorylate TOP2A. The analysis using phospho-specific Ser1512 TOP2A antibody supported the finding that phosphorylation of Ser1512 depends on the presence of ATM molecules in cellulo. A previous report suggested that mutations at phosphorylation sites in the C-terminal of TOP2A (Ser1106, 1247, 1354, and 1393) do not impair its function. The TOP2A Ser1512Ala mutant could also complement the loss of TOP2A function for cell survival. It remains unknown whether phosphorylation at this site contributes to TOP2 function. Several TOP2A mutations associated with TOP2 inhibitor resistance have been reported. For example, mutations in the C-terminal region of TOP2A confer defects in its nuclear localization and lead to a decreased sensitivity to TOP2 inhibitor.[44-47] Several missense mutations of TOP2A have also been shown to render TOP2A resistant to inhibitors.[48-51] Interestingly, substitution of Ser1512 residue to Ala strikingly stabilized TOP2A expression. Increased expression of TOP2A observed in AT cells may result from defective phosphorylation of Ser1512. It is still unclear whether ATM activation directly conducts TOP2A to degradation. However, it is clear that AT cells show elevated TOP2A expression by increased protein stability, and disruption of the ATM-dependent phosphorylation site of TOP2A stabilizes the protein. We can hypothesize that cell cycle progression from G2 to M, mediated by TOP2A, needs to be halted to prevent carry-over DNA breaks after DNA damage. For that purpose, TOP2A expression may be suppressed after DNA damage. Further investigation is required into the consequences of phosphorylation at the C-terminal region of TOP2A, including Ser1512.
Tumor cell lines acquire resistance to topoisomerase inhibitors through various mechanisms, including expression of multidrug-resistance genes, mutation of the TOP2 gene,[44, 48, 51] distinct extranuclear localization,[46, 47, 53] and reduction of TOP2 expression. Decreased TOP2A expression by heterozygous gene targeting confers increased resistance to TOP2A inhibitor, and increased expression of TOP2A correlates with increased sensitivity to TOP2 inhibitor.[17, 18] Ataxia telangiectasia cells expressed more TOP2A than WT cells and showed hypersensitivity to TOP2A inhibitor. We also observed that the sensitivity of AT cells to etoposide was normalized by introduction of ATM. Therefore, these results indicate that ATM-associated phosphorylation and reduction of TOP2A expression play a crucial role in the regulation of sensitivity of AT cells to TOP2A inhibitor.
Epidemiological data have suggested that the development of infantile leukemia is tightly associated with in utero exposure to TOP2 inhibitor, which causes rearrangement of the MLL gene.[6-9] We can hypothesize that increased sensitivity to TOP2 inhibitor due to genetic reasons also induces the development of infantile leukemia. Indeed, we identified one infant leukemia patient with defective ATM expression by germline ATM single nucleotide polymorphism. As expected, the EBV-LCL of this patient showed increased TOP2A sensitivity. These observations are in accord with our hypothesis that defective expression of ATM correlates with increased levels of TOP2A, which sensitizes to TOP inhibitor and leads to the susceptibility to MLL gene breaks and rearrangement. Previously, we reported our observation that MLL rearrangement is induced by treatment with etoposide in AT cells but not WT cells.
On the basis of the data presented here and our previous report, ATM-dependent TOP2A regulation appears to have important clinical implications for the pathogenesis of infantile leukemia as well as treatment-associated secondary leukemia, both of which are associated with TOP 2 inhibitor related MLL rearrangement.