1. Introduction
When a DNA double helix is unwound for strand separation, torsional strain is produced, which leads to various topological problems for DNA [1,2]. An ATP-dependent enzyme called DNA topoisomerase II (TOP2) solves the topological problems caused by DNA unwinding [1,2,3]. TOP2 plays a critical role in ensuring the execution of various nuclear processes accompanied by DNA unwinding, such as transcription, DNA replication, and chromosome segregation [1,2,3]. Human cells have two TOP2, namely TOP2A and TOP2B [4,5]. TOP2A is expressed in a cell-cycle-dependent manner and is present in proliferating cells [6,7]. TOP2A plays an essential role in cell division, particularly chromosome segregation [7,8]. TOP2B exists throughout the cell cycle and functions in both dividing and nondividing cells [7]. TOP2B is involved in the control of transcription [9,10,11] and the chromatin architecture [12,13,14]. Although TOP2A and B have different roles in cell physiology [2,3], they are structurally similar: They consist of the N-terminal ATPase and the central catalytic domains [1,15]. They form homo dimers [1] and exhibit similar enzymatic properties in vitro [16,17].
To relieve the torsional strain of DNA, TOP2 functions through multiple steps that form a so-called catalytic cycle [18,19]. TOP2 starts its catalytic cycle by attaching to DNA segments. Afterward, TOP2 binds to ATP and undergoes conformational changes. TOP2 and DNA form a TOP2-DNA cleavage complex, and TOP2 introduces a DNA double-strand break (DSB) in one segment of DNA. A DSB serves as a gate and allows another DNA segment to pass through it, relieving torsional strain. TOP2 religates the DSBs to restore the integrity of DNA. Finally, TOP2 hydrolyzes ATP and dissociates from DNA strands, returning to the initial step of the catalytic cycle [18,19].
Various compounds have been developed to impede the TOP2 catalytic cycle [19]. For example, etoposide stabilizes the TOP2-DNA cleavage complex and prevents the religation of DSBs [20]. In a cell, stabilized TOP2-DNA cleavage complexes are readily converted into DSBs, which can trigger cell death, particularly in rapidly dividing cells, such as cancer cells. Etoposide is referred to as a TOP2 poison and is regarded as an effective chemotherapy agent [20]. Another class of drugs targeting the TOP2 catalytic cycle is TOP2 catalytic inhibitors, such as ICRF-187. ICRF-187 inhibits the ATPase activity of TOP2 and traps a TOP2 homodimer in a closed conformation around DNA, which is called a closed clamp [21,22]. Thus, ICRF-187 tethers TOP2 to DNA strands in the late step of the catalytic cycle [1,2].
Aclarubicin is a member of the anthracyclines and was originally identified as an anti-tumor antibiotic [23]. Although both the TOP2 catalytic inhibitors and TOP2 poisons act on DNA-bound TOP2, aclarubicin exerts its inhibitory action differently [19]. Aclarubicin intercalates into DNA double helices and induces structural distortions in DNA. Gel shift assays using a purified TOP2 protein and a radiolabeled DNA probe have demonstrated that aclarubicin completely interferes with the association between TOP2 and DNA [24]. Thus, the primary inhibitory mechanism of aclarubicin on TOP2 was proposed to prevent the association of TOP2 with DNA in the early step of the catalytic cycle [19,25]. Consistent with this view, cell-based assays have shown that aclarubicin antagonizes the cellular toxicity of TOP2 poisons, such as etoposide [26,27] and doxorubicin [28].
In addition to its TOP2 inhibition, aclarubicin exhibits multiple biological actions. First, aclarubicin causes increased reactive oxygen species levels and mitochondrial dysfunction [29,30]. Second, aclarubicin alters the accessibility of RNA polymerase II to promoter regions, leading to enhanced transcriptional elongation [31]. Next, Western blot analysis of nuclear proteins showed aclarubicin-mediated degradation of RNA polymerase II [32] and increases in the chromatin-bound fractions of the histone chaperone FACT [33] and TOP2A and TOP2B [32]. Finally, aclarubicin at relatively high concentrations induces histone eviction [34,35].
Human TOP2B is highly mobile in the nucleus and localizes in both the nucleoplasm and nucleoli [36,37,38]. Previous studies have demonstrated that the subnuclear distribution of TOP2B alters in response to cellular ATP levels. When intracellular ATP is depleted by inhibiting the glycolytic and mitochondrial pathways for ATP production, TOP2B quickly accumulates in the nucleoli [37,38]. We were intrigued by this phenomenon and searched for drugs that could affect it. In the course of our research, we found that aclarubicin reduces the mobility of TOP2B. Because aclarubicin is known to interfere with the TOP2-DNA association [24], the observed reduction in the nuclear mobility of TOP2B suggests that aclarubicin augments the association of TOP2B with some nuclear components other than DNA. Disruptions of TOP2B catalytic sites did not alleviate the aclarubicin-mediated reduction in TOP2B mobility, supporting the idea that TOP2B associates with non-DNA components in the presence of aclarubicin. Our observations suggest that the inhibitory action of aclarubicin on TOP2 in the cellular environment is more complicated than currently recognized.
3. Discussion
TOP2B is known to be highly mobile in the nucleus, and its subnuclear distribution alters in response to cellular ATP levels [36,37,38]. A low ATP state triggers the translocation of TOP2B from the nucleoplasm to the nucleoli, which is proposed to be a novel proteostatic mechanism to prevent unfavorable actions of TOP2B via nucleolar sequestration [38]. In the course of research on the subnuclear translocation of TOP2B, we found that aclarubicin markedly affects the nuclear mobility of TOP2B. FRAP analysis demonstrated that aclarubicin reduces TOP2B mobility in a dose-dependent manner. A previous study using a purified TOP2 protein demonstrated that aclarubicin prevents the association of TOP2 with DNA in vitro [24]. We thus speculate that the reduced nuclear mobility of TOP2B by aclarubicin could be ascribed to the augmented association of TOP2B with a non-DNA component of chromatin. Accordingly, the enzymatic activities of TOP2B were indispensable for the effect of aclarubicin, which is in contrast to the case of ICRF-187: ICRF-187 halts TOP2 as a closed clamp on DNA [21,22], and its action requires the functional ATPase of TOP2 [37,40]. Furthermore, the disruption of topoisomerase activity by the Y821S substitution did not relieve the reduced mobility of TOP2B by aclarubicin. These observations imply that a previously unanticipated effect of aclarubicin on TOP2B exists in the cellular environment.
Although this study reveals a novel effect of aclarubicin on the nuclear mobility of TOP2B, it raises new questions for further investigation. The first question is the mechanism for the reduction in TOP2B mobility by aclarubicin. As mentioned above, we suppose that aclarubicin augments the association of TOP2B with a non-DNA component of chromatin. Proteomics analysis has identified multiple TOP2B-interacting proteins, including CCCTC-binding factor (CTCF) and the subunits of cohesin [12,44]. CTCF and cohesin colocalize at many sites across the human genome and work in coordination to control chromatin structure [45,46]. In addition to evidence from proteomics, genome-wide analysis of TOP2B binding sites has demonstrated that TOP2B is frequently localized at overlapping or adjacent positions to the CTCF/cohesin-binding sites on the genome [12,13,14,44,47]. Interestingly, the association of CTCF and cohesin with chromatin is much more stable than that of TOP2B. The half-time of fluorescence recovery of EGFP-TOP2B was estimated to be a few seconds (Figure 3e and Figure 4h), indicating the high mobility of TOP2B. On the other hand, the residence times of CTCF and cohesin in chromatin were reported to be 1 min and 22 min, respectively [48]. If aclarubicin stabilizes the association of TOP2B with either CTCF or cohesion, TOP2B mobility decreases, as observed in this study. Another possible mechanism for the reduced TOP2B mobility may be the interaction of TOP2B with stable chromatin components that are not recognized as TOP2B-interacting partners. The comparison of TOP2B-interacting factors in the presence and absence of aclarubicin will provide clues to understanding the mechanism for the reduced TOP2B mobility caused by aclarubicin.
Another question to be explored in the future is the impact of its reduced mobility on the physiological role of TOP2B. Many proteins, including TOP2B, are highly mobile in the nucleus and exhibit rapid association and dissociation with their target sites [49,50,51]. High protein mobility in the nucleus is generally beneficial for cellular physiology [51,52,53]. First, high mobility can facilitate targeting a protein to its specific binding sites. Second, the rapid association with and dissociation of a protein from its target site enables the efficient redistribution of a protein to different sites in response to cellular signaling and environmental changes. Thus, aclarubicin may restrain the timely distribution to target sites and regulatory plasticity of TOP2B in a cell. Aclarubicin is well known to interfere with the TOP2-DNA association in vitro, and future research will further advance our understanding of how aclarubicin affects the function of TOP2B in the cellular environment.
4. Materials and Methods
4.1. Reagents, Antibodies, and Plasmid
Aclarubicin was purchased from Focus Biomolecules (Plymouth Meeting, PA, USA). 2-deoxy-glucose (2DG) and etoposide were obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan). ICRF-187 and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Anti-TOP2B mouse monoclonal antibody was obtained from BD Biosciences (#611492, Franklin Lakes, NJ, USA). Goat anti-mouse IgG-Alexa Fluor488 antibody was from Thermo Fisher Scientific (A11029, Waltham, MA, USA). The expression plasmids for EGFP-TOP2B WT, G180I, and Y821S were described previously [54].
4.2. Cell Culture and Transfection
HeLa cells were obtained from RIKEN BioResource Research Center (Wako, Japan). Cells were grown in α-modified minimum essential medium (αMEM, FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal bovine serum (Corning, NY, USA), 100 µg/mL streptomycin, and 100 units/mL penicillin (FUJIFILM Wako Pure Chemical, Osaka, Japan). Cells were cultured under standard conditions at 37 °C in a humidified incubator containing 5% CO2.
For plasmid transfection, cells (1 × 105) were plated in a 35 mm glass-bottomed dish (Matsunami, Osaka, Japan). After 24 h of incubation at 37 °C, a plasmid (300 ng) was transfected using a FuGENE HD reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions. At 30 h after transfection, cells were used for live cell imaging and FRAP analysis.
4.3. ATP Measurement
Cells were plated in a 96-well plate (5 × 103 cells/well). After 24 h of incubation at 37 °C, cells were pretreated with or without 1 µM aclarubicin for 1 h. Cells were subsequently treated with 25 mM 2DG and 10 µM CCCP for periods indicated in Figure 1d. Cell lysis and ATP measurement were performed using a CellTiter-Glo luminescent cell viability assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Luminescence was measured using a 2030 ARVO X multilabel reader (Perkin Elmer, Shelton, CT, USA).
4.4. Fluorescence Microscopy and Live Cell Imaging
Fluorescence microscopy and live cell imaging were carried out as described previously [39]. Briefly, an FV1200-IX83 laser scanning confocal microscope with an oil-immersed 60× objective (Olympus, Tokyo, Japan) was used for fluorescence microscopy. For live cell imaging, cells plated in a glass-bottomed 35 mm dish were placed on a stage top incubator (Tokai Hit, Fujinomiya, Japan) that maintained a humidified atmosphere of 5% CO2 at 37 °C. Images were captured and analyzed using FLUOVIEW software (Version 4.1, Olympus, Osaka, Japan).
4.5. Immunofluorescence Staining
Cells were fixed with 4% paraformaldehyde dissolved in Dulbecco’s phosphate-buffered saline (D-PBS) for 15 min at 4 °C. Fixed cells were washed with D-PBS three times and subsequently permeabilized with 0.4% Triton X-100 in D-PBS at room temperature for 5 min. Fixed and permeabilized cells were blocked with 1% bovine serum albumin in D-PBS for 15 min and reacted with mouse anti-TOP2B monoclonal antibody at 4 °C for 4 h. After washing with D-PBS, cells were incubated with goat anti-mouse IgG-Alexa Fluor488 antibody at 4 °C for 1 h. Cells were mounted in a Vectashield mounting medium with DAPI (Vector Laboratories, Newark, CA, USA).
For membrane permeabilization prior to fixation, detergent treatment was performed according to previously described procedures [55] with slight modifications. Cells were incubated twice for 3 min at room temperature with cytoskeleton buffer (CSK) containing 10 mM Pipes (pH 7.0), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 0.4% Triton X-100. After washing with D-PBS, cells were fixed with 4% PFA and subsequently subjected to immunofluorescence staining as described above.
4.6. FRAP Analysis
FRAP analysis was performed as reported previously [39]. Briefly, transfection and live cell imaging were carried out as described above. A spot in the nucleoplasm of a transfected cell was photobleached with a 473 nm laser at 35% output for 1 s. Before and after photobleaching, fluorescent images were captured at 2 s intervals. The intensities of fluorescence of photobleached and unbleached areas in the same cell were quantified using FLUOVIEW software (Version 4.1, Olympus, Tokyo, Japan). The intensities of fluorescence before and immediately after photobleaching were set to 1 and 0, respectively. Fluorescence recovery was expressed as a ratio of fluorescence before and after photobleaching. FRAP curve fitting by means of non-linear regression was carried our using the Stowers ImageJ plugins (https://research.stowers.org/imagejplugins/ImageJ_tutorial2.html. Accessed in 2023 and 2024.) in Fiji (ImageJ version 1.53t) [56] according to the procedures described previously [57]. The normalized fluorescence recovery curves were fit with an exponential recovery function using the ImageJ plugin ‘batch FRAP fit jru v1’ in the Stowers ImageJ plugins (https://research.stowers.org/imagejplugins/ImageJ_tutorial2.html. Accessed in 2023 and 2024). The half-time of fluorescence recovery (t1/2) was calculated for each curve.
4.7. Statistical Analysis
Welch’s t-test was used for statistical analysis. The number of experiments and p-values are indicated in the figure legends.
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Keiko Morotomi-Yano www.mdpi.com
