DNA Decatenation Research Articles

Design and synthesis of the novel DNA topoisomerase II inhibitors: Esterification and amination substituted 4′-demethylepipodophyllotoxin derivates exhibiting anti-tumor activity by activating ATM/ATR signaling pathways

According to the structure–activity relationship, drug combination principle and bioisosterism, a series of the novel esterification and amination 4′-demethylepipodophyllotoxin derivates were rationally designed in order to discover the potential antitumor prodrug. And then these compounds were tested by the drug-topoisomerase II docking models for virtual screening. Thus, twelve target compounds were screened out and synthesized. Most of compounds exhibited promising in vitro anti-tumor activity, particularly 4-N-tris(hydroxymethyl)metylaminomethane-4-deoxy-4′-demethylepipodophyllotoxin (Compound 1). The anti-tumor activity of Compound 1 against the tumor cell lines BGC-823 (i.e., the IC50 value of 5.35 ± 0.77 μM), HeLa (i.e., the IC50 value of 160.48 ± 14.50 μM), and A549 (i.e., the IC50 value of 13.95 ± 5.41 μM) was significantly improved by 706%, 31% and 900% than that of etoposide (i.e., the IC50 values of 43.74 ± 5.13, 209.90 ± 13.42, and 139.54 ± 7.05 μM), respectively. Moreover, the IC50 value of Compound 1 against the normal human cell line HK-2 (i.e., 16.3 ± 3.77 μM) was 78% lower than that of etoposide (i.e., 9.17 ± 1.58 μM). Compound 1 could diminish the relaxation reaction topoisomerase II DNA decatenation at a concentration of 10 μM and induce BGC-823 apoptosis by breaking DNA double-strand and activating ATM/ATR signaling pathways.

Structural determinants of the catalytic inhibition of human topoisomerase IIα by salicylate analogs and salicylate-based drugs

We previously identified salicylate as a novel catalytic inhibitor of human DNA topoisomerase II (topo II; EC 5.99.1.3) that preferentially targets the alpha isoform by interfering with topo II-mediated DNA cleavage. Many pharmaceuticals and compounds found in foods are salicylate-based. We have now investigated whether these are also catalytic inhibitors of topo II and the structural determinants modulating these effects. We have determined that a number of hydroxylated benzoic acids attenuate doxorubicin-induced DNA damage signaling mediated by the ATM protein kinase and inhibit topo II decatenation activity in vitro with varying potencies. Based on the chemical structures of these and other derivatives, we identified unique properties influencing topo II inhibition, including the importance of substitutions at the 2′- and 5′-positions. We extended our findings to a number of salicylate-based pharmaceuticals including sulfasalazine and diflunisal and found that both were effective at attenuating doxorubicin-induced DNA damage signaling, topo II DNA decatenation and they blocked stabilization of doxorubicin-induced topo II cleavable complexes in cells. In a manner similar to salicylate, we determined that these agents inhibit topo II-mediated DNA cleavage. This was accompanied by a concomitant decrease in topo II-mediated ATP-hydrolysis. Taken together, these findings reveal a novel function for the broader class of salicylate-related compounds and highlight the need for additional studies into whether they may impact the efficacy of chemotherapy regimens that include topo II poisons.

Prospects for Developing New Antibacterials Targeting Bacterial Type IIA Topoisomerases

The modulation of DNA topology by DNA gyrase and topoisomerase IV, both of which are type IIA topoisomerases and found in most bacteria, is a function vital to DNA replication, repair and decatenation. Despite the potential for resistance development, DNA gyrase and/or topoisomerase IV have been proven to be and remain highly attractive targets in antibacterial drug discovery due to their potential for dual targeting. The search for new GyrA and/or ParC inhibitors that can overcome the increasing spread of multidrug-resistant bacteria has been successfully focused in the last decades on the modification of the known fluoroquinolone scaffold as primarily guided by ligand-based design via classical structure-activity relationship studies and the optimisation of physicochemical properties. This focus has resulted in several novel fluoroquinolones that have been introduced into clinical practice since 2000, and several of these new compounds are currently in different phases of clinical trials. Due to increasing resistance to fluoroquinolones, a significant part of DNA gyrase research has shifted to the discovery of new GyrB and/or ParE inhibitors, which are commonly identified through fragment-based design as well as virtual screening techniques and structure-based hit optimisation programs. This research often results in lead compounds with potent inhibitory activity and promising antibacterial activity profiles. Nevertheless, it is important to understand how different physicochemical properties (e.g., logD and total polar surface area) and different structural motifs influence the compounds' permeability to ensure the efficient discovery of potent, small-molecule antibacterials particularly against Gram-negative strains.

Purification of GidA protein, a novel topoisomerase II inhibitor produced by Streptomyces flavoviridis

The presence of topoisomerase II inhibition activities in the intracellular extract of Streptomyces flavoviridis was investigated. One active compound inhibiting relaxation activity of topoisomerase II was determined to be a protein. This active principle was purified to homogeneity by gel filtration followed by ion exchange chromatography. The apparent molecular mass was 42 kDa as determined by SDS-PAGE. MALDI TOF peptide mass fingerprinting analysis confirmed this topoisomerase II inhibitor, as glucose-inhibited division protein A (GidA) by MOWSE score of 72. The effects of purified GidA protein on DNA relaxation and decatenation by topoisomerase II were investigated. It inhibited topoisomerase II activity and acted as a topoisomerase poison that significantly stabilized the covalent DNA-topoisomerase II reaction intermediate "cleavable complex", as observed with etoposide. Collectively, these findings indicate that GidA is a potent inhibitor of topoisomerase II enzyme, which can be exploited for rational drug design in human carcinomas.

A Chromatin Remodeling Complex Regulates Topoisomerase IIα Function

TOP2A-mediated DNA decatenation during mitosis requires the BAF subunits BRG1 and BAF250A.

HY‐1 induces G2 / M cell cycle arrest in human colon cancer cells through the ATR‐Chk1‐Cdc25C and Weel pathways

The novel aroylthiourea analogue of podophyllotoxin HY-1 (4β-[benzoyl-thioureido]-4-deoxypodophyllotoxin) was synthesized in our laboratory with the aim of developing multitargeted DNA topoisomerase II inhibitors. The compound showed significant antiproliferative effects on seven cancer cell lines and induced G2 /M phase arrest in HCT116 cells. Moreover, HY-1 showed a potent inhibitory effect on topoisomerase II-mediated kinetoplast DNA decatenation in a dose-dependent manner. Our results showed that cdc2 phosphorylation and decreased cdc2 kinase acitivity through the ATR-Chk1-Cdc25C and Weel pathways were the central mechanisms for G2 /M phase arrest in human colon cancer cells.

BAF complexes facilitate decatenation of DNA by topoisomerase IIα

Recent exon sequencing studies of human tumors have revealed that subunits of mSWI/SNF or BAF complexes are mutated in more than 20% of all human malignancies,1,2 yet the mechanisms involved in tumor suppression are unclear. BAF chromatin remodeling complexes are polymorphic assemblies that use energy provided by ATP hydrolysis to regulate transcription through the control of chromatin structure3 and the placement of Polycomb (PcG) across the genome4,5. Several proteins dedicated to this multi-subunit complex, including SMARCA4 (BRG1) and BAF250A (ARID1A), are mutated at frequencies similar to that of recognized tumor suppressors. In particular, the core ATPase BRG1 is mutated in 5-10% of childhood medulloblastoma6-9 and greater than 15% of Burkitt's Lymphoma.10,11 Here we find a novel function of BAF complexes in decatenating newly replicated sister chromatids, a requirement for proper chromosome segregation during mitosis. We find that deletion of Brg1, as well as the expression of Brg1 point mutants identified in human tumors leads to anaphase bridge formation (sister chromatids linked by catenated strands of DNA), and a G2/M phase block characteristic of the decatenation checkpoint. Endogenous BAF complexes directly interact with endogenous TopoIIα through BAF250a and are required for TopoIIα binding to about 12,000 sites over the genome. Our results demonstrate that TopoIIα's chromatin binding is dependent on the ATPase activity of Brg1, which is compromised in oncogenic Brg1 mutants. These studies indicate that the ability of TopoIIα to prevent DNA entanglement at mitosis requires BAF complexes and suggest that this activity contributes to the role of BAF subunits as tumor suppressors.

Abstract 3428: Effect of a non cardiotoxic Doxorubicin analog, 13-deoxy, 5-imino doxorubicin on decatenation of DNA by Topoisomerase II.

Abstract Anthracyclines such as doxorubicin are widely used to treat solid and hematological cancers. However, within the course of treatment they have the potential to cause life threatening chronic cardiotoxicity which limits their potential use. The cause of cardiotoxicity is not thoroughly understood, with previous research indicating the generation of reactive oxygen species and free iron accumulation as possible causes for cardiomyocyte death. Recent publications have shown the interaction between doxorubicin and Topoisomerase IIβ plays a key role in anthracycline induced cardiotoxicity. Topoisomerase enzymes are utilized during the coiling and uncoiling of DNA within the cell and are active targets of anthracyclines not only in tumoricidal action (Topoisomerase IIα) but also in the pathogenesis of cardiotoxicity (Topoisomerase IIβ). In this research we seek to further evaluate the interaction between doxorubicin, doxorubicin analogues and Topoisomerase IIβ. The analogues were selected due to previous chronic in vivo experiments in rabbits where classical manifestations of cardiotoxicity (including reduced left ventricular shortening fraction and pathological changes in cardiac tissue) were not observed. Furthermore, one analogue (13-deoxy, 5-imino doxorubicin) has shown promising clinical data suggesting a strong antineoplastic effect without the cardiotoxic effects. Doxorubicin and this analogue were placed in the presence of catenated DNA for 6 hours at 37°C prior to exposure to Topoisomerase IIα and β enzymes. The reaction was allowed to proceed for 30 minutes at 37°C and analyzed for decatenation of the DNA by the enzyme. This experiment was then repeated in cardiac H9c2 cells to assay possible differential inhibition at the cellular level. Citation Format: Nicole Frank, Richard Olson, Gerald M. Walsh, Todd Talley, Barry Cusack. Effect of a non cardiotoxic Doxorubicin analog, 13-deoxy, 5-imino doxorubicin on decatenation of DNA by Topoisomerase II. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2013-3428

Synthesis and evaluation of novel ellipticines as potential anti-cancer agents

Drugs that inhibit DNA topoisomerase I and DNA topoisomerase II have been widely used in cancer chemotherapy. We report herein the results of a focused medicinal chemistry effort around novel ellipticinium salts which target topoisomerase I and II enzymes with improved solubility. The salts were prepared by reaction of ellipticine with the required alkyl halide and evaluated for DNA intercalation, topoisomerase inhibition and growth inhibition against 12 cancer cell lines. Results from the topoisomerase I relaxation assay indicated that all novel ellipticine derivatives behaved as intercalating agents. At a concentration of 100 μM, specific topoisomerase I inhibition was not observed. Two of the derivatives under investigation were found to fully inhibit the DNA decatenation reaction at a concentration of 100 μM, indicative of topoisomerase II inhibition. N-Alkylation of ellipticine was found to enhance the observed growth inhibition across all cell lines and induce growth inhibition comparable to that of Irinotecan (CPT-11; GI(50) 1-18 μM) and in some cell lines better than Etoposide (VP-16; GI(50) = 0.04-5.2 μM). 6-Methylellipticine was the most potent growth inhibitory compound assessed (GI(50) = 0.47-0.9 μM). N-Alkylation of 6-methylellipticine was found to reduce this response with GI(50) values in the range of 1.3-28 μM.

Synthesis, topoisomerase-targeting activity and growth inhibition of lycobetaine analogs

The plant alkaloid lycobetaine has potent topoisomerase-targeting properties and shows anticancer activity. Based on these findings, several lycobetaine analogs were synthesized mainly differing in their substituents at 2, 8 and 9 position and their biological activities were evaluated. The topoisomerase-targeting properties and cytotoxicity of these structural analogs were assessed in the human gastric carcinoma cell line GXF251L. Performing a plasmid relaxation assay, an increased inhibition of topoisomerase I was found with N-methylphenanthridinium chlorides bearing a 8,9-methylenedioxy moiety or a methoxy group in 2-position. Furthermore, quaternized phenanthridinium derivatives bearing either a 2-methoxy or a 8,9-methylenedioxy moiety in conjunction with a 2-hydroxy or 2-methoxy group display potent topoisomerase II inhibition as shown by decatenation of kinetoplast DNA. In general, the N-methylphenanthridinium chlorides possess more potency in inhibiting topoisomerase I than topoisomerase II. All quaternized derivatives also exhibited potent inhibition of tumor cell growth in the low micromolar concentration range. Hence, N-methylphenanthridinium compounds were found to represent a promising class of compounds, potently inhibiting both, topoisomerases I and II, and may be further developed into clinically useful topoisomerase inhibitors.

Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA Complex: A Mechanism for Disentangling Chromosomes

Genetic evidence indicates that Saccharomyces cerevisiae Sgs1, Top3, and Rmi1 resolve topologically linked intermediates arising from DNA replication and recombination. Using purified proteins, we show that Sgs1, Top3, Rmi1, and replication protein A (RPA) coordinate catenation and decatenation of dsDNA through sequential passage of single strands of DNA, establishing a unique pathway for dsDNA decatenation in eukaryotic cells. Sgs1 is required for dsDNA unwinding and, unexpectedly, also has a structural role in DNA strand passage. RPA promotes DNA unwinding by Sgs1 by trapping ssDNA, and it stimulates DNA strand passage by Top3. Paradoxically, Rmi1 has a unique regulatory capacity that slows DNA relaxation by Top3 but stimulates DNA decatenation. We establish that Rmi1 stabilizes the "open" Top3-DNA covalent complex formed as a transient intermediate of strand passage. This concerted activity of the Sgs1-Top3-Rmi1-RPA represents an important mechanism for disentangling structures resulting from the topological features of duplex DNA.

The role of Ca 2+ in the activity of Mycobacterium tuberculosis DNA gyrase

DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca2+-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca2+ cannot support topoisomerase reactions in the absence of Mg2+, but partial removal of Ca2+ from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca2+. More extensive removal of Ca2+ by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca2+-binding residues also leads to loss of activity. We propose that Ca2+ has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca2+ binding.

Topoisomerase Assays

Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. The protocols described in this unit are for assays used to assess new chemical entities for their ability to inhibit both forms of DNA topoisomerase. Included are an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA, and an assay for topoisomerase II based on the decatenation of double-stranded DNA. The preparation of mammalian cell extracts for assaying topoisomerase activity is described, along with a protocol for an ICE assay to examine topoisomerase covalent complexes in vivo, and an assay for measuring DNA cleavage in vitro.

Mechanism of Generation of Therapy Related Leukemia in Response to Anti-Topoisomerase II Agents

Type II DNA topoisomerases have the ability to generate a transient DNA double-strand break through which a second duplex can be passed; an activity essential for DNA decatenation and unknotting. Topoisomerase poisons stabilize the normally transient topoisomerase-induced DSBs and are potent and widely used anticancer drugs. However, their use is associated with therapy-related secondary leukemia, often bearing 11q23 translocations involving the MLL gene. We will explain recent discoveries in the fields of topoisomerase biology and transcription that have consequences for our understanding of the etiology of leukemia, especially therapy-related secondary leukemia and describe how these findings may help minimize the occurrence of these neoplasias.

Conformational Change in the ATPase-Gate of Topoisomerase VI Induced by Nucleotide and DNA

Archaeal topoisomerase VI catalyzes the relaxation and decatenation of DNA. In an ATP-dependent manner it transports a DNA double strand through a gap in a second duplex by a sequential opening and closing of two gates. Conformational change in the ATPase-gate of M. mazei topoisomerase VI was monitored in single molecule FRET experiments, using fluorescently labeled enzyme with one dye attached to each nucleotide-binding domain.We have observed four different conformational states of the ATP-binding domains: In the absence of nucleotide or DNA the ATPase-gate exhibits conformational flexibility, indicating gate opening and closing. Binding of supercoiled plasmid to TopoVI forces the ATPase-gate to open up even wider, although considerable flexibility of the domains is retained; both these states have been proposed on the basis of two crystal structures of intact TopoVI holoenzymes, showing a closed and an open gate conformation. Addition of ADPNP - a non-hydrolysable ATP analog - induces a well-defined conformation of the ATPase domains close to each other, indicating dimerization; this is in agreement with crystal structure from isolated TopoVI-B domains which show tight association when bound to ADPNP. Addition of both nucleotide and plasmid immobilizes the domains in a conformation different from the one observed for the closed gate, indicating that the presence of a DNA segment in the central cavity induces physical strain to the TopoVI complex; this state has not been proposed so far, but should be due to an internal rotation in the ATP-binding domains mediated by a conserved lysine residue acting as a gamma-phosphate sensor.Topoisomerase VI exhibits a different conformational behavior than type II topoisomerases (e.g. gyrase) in that it does not show DNA-induced pre-closure but rather opening of the gate in presence of a supercoiled DNA.

P3-01-01: Geminin Overexpression Prevents the Completion of Topoisomerase IIa Chromosome Decatenation Leading to Aneuploidy in Human Mammary Epithelial Cells.

Abstract Topoisomerase IIα (TopoIIα) cleaves DNA in a reversible manner, making it a valuable target for agents such as etoposide that trap the enzyme in a covalent bond with the DNA end it cleaves and prevents DNA re-ligation and triggers cell death in cancer cells. However, development of resistance to these agents limits their therapeutic use. In this study, we examined therapeutic targeting of geminin for improving the therapeutic potential of TopoIIa agents. Human mammary epithelial (HME) and breast cancer cell lines were used. Geminin, TopoIIα, Cdc7 silencing was done using specific siRNAs. Transit or stable inducible overexpression of these proteins and CKI∈ were also used, as well as several pharmacological inhibitors that target TopoIIα, Cdc7, or CKI∈. We manipulated HME cells expressing H2B-GFP, in order to detect chromosome bridges. Immunoprecipitation and direct western blot were used to detect interactions between these proteins and their total expression, respectively, whereas interactions on chromosomal arms were detected using the TARDIS assay. TopoIIα phosphorylation by Cdc7 or CKI∈ was done using in vitro kinase assay. The TopoGen decatenation kit was used to measure TopoIIα decatenation activity. Finally, comet assay and metaphase chromosome spread were used to detect chromosome breakages and changes in chromosome condensation or numbers, respectively. We found that geminin and TopoIIα interact in G2/M/early G1 cells on chromosomes, that geminin recruits TopoIIα to chromosomal decatenation sites or vice versa, and that geminin silencing in HME cells triggers the formation of chromosome bridges through suppressing TopoIIα access to chromosomal arms. CKIε kinase phosphorylates and positively regulates TopoIIα chromosome localization and function. CKIε kinase overexpression or Cdc7 kinase silencing (also phosphorylates TopoIIα in vitro), restored DNA decatenation and chromosome segregation in geminin-silenced cells before triggering cell death. In vivo, at normal concentration, geminin recruits the desumoylating enzymes SENP1 and SENP2 to desumoylate chromosomal bound TopoIIα and promote its release from chromosomes following completion of DNA decatenation. In cells overexpressing geminin, premature departure of TopoIIα from chromosomes is thought to be due to the fact that geminin recruits more of these desumoylating enzymes, or recruits them earlier, to chromosomal bound TopoIIα. This triggers premature release of TopoIIα from chromosomes, which we propose induces aneuploidy in HME cells, since chromosome breakages generated through were not sensed and/or repaired and the cell cycle was not arrested. TopoIIα recruitment and its chromosome decatenation function require normal level of geminin. Geminin silencing induces a cytokinetic checkpoint in which Cdc7 phosphorylates TopoIIα and inhibits its chromosomal recruitment and decatenation function. Geminin overexpression prematurely desumoylates TopoIIα, triggering its premature departure from chromosomes and leading to chromosomal abnormalities and the formation of aneuploid, drug resistant cancer cells. We propose that therapeutic targeting of geminin is essential for improving the therapeutic potential of TopoIIα agents. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-01.

Neoamphimedine Circumvents Metnase-Enhanced DNA Topoisomerase IIα Activity Through ATP-Competitive Inhibition

Type IIα DNA topoisomerase (TopoIIα) is among the most important clinical drug targets for the treatment of cancer. Recently, the DNA repair protein Metnase was shown to enhance TopoIIα activity and increase resistance to TopoIIα poisons. Using in vitro DNA decatenation assays we show that neoamphimedine potently inhibits TopoIIα-dependent DNA decatenation in the presence of Metnase. Cell proliferation assays demonstrate that neoamphimedine can inhibit Metnase-enhanced cell growth with an IC50 of 0.5 μM. Additionally, we find that the apparent Km of TopoIIα for ATP increases linearly with higher concentrations of neoamphimedine, indicating ATP-competitive inhibition, which is substantiated by molecular modeling. These findings support the continued development of neoamphimedine as an anticancer agent, particularly in solid tumors that over-express Metnase.

RECQL5 cooperates with Topoisomerase II alpha in DNA decatenation and cell cycle progression

DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase IIα with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase IIα stimulates the decatenation activity of Topoisomerase IIα. Consistent with these observations, RECQL5 co-localizes with Topoisomerase IIα during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process.

Identification of a Small-Molecule Inhibitor of DNA Topoisomerase II by Proteomic Profiling

BNS-22, a chemically synthesized derivative of the natural plant product GUT-70, has antiproliferative activity against human cancer cells, the mechanism of which is unknown. Here, we identify a target of BNS-22 by proteomic profiling analysis, which suggests that BNS-22 belongs to the same cluster as ICRF-193, a DNA topoisomerase II (TOP2) catalytic inhibitor. BNS-22 inhibits kinetoplast DNA decatenation that is mediated by human TOP2α and TOP2β invitro at an IC(50) of 2.8 and 0.42μM, respectively. BNS-22 does not affect DNA damage and antagonizes TOP2 poison-mediated DNA damage. Like ICRF-193, BNS-22 induces mitotic abnormalities, characterized by impairments in chromosome alignment and segregation, thereby causing polyploidy in HeLa cells. These results indicate that BNS-22 targets TOP2 and acts as its catalytic inhibitor.

Geminin overexpression prevents the completion of topoisomerase IIα chromosome decatenation, leading to aneuploidy in human mammary epithelial cells

IntroductionThe nuclear enzyme topoisomerase IIα (TopoIIα) is able to cleave DNA in a reversible manner, making it a valuable target for agents such as etoposide that trap the enzyme in a covalent bond with the 5′ DNA end to which it cleaves. This prevents DNA religation and triggers cell death in cancer cells. However, development of resistance to these agents limits their therapeutic use. In this study, we examined the therapeutic targeting of geminin for improving the therapeutic potential of TopoIIα agents.MethodsHuman mammary epithelial (HME) cells and several breast cancer cell lines were used in this study. Geminin, TopoIIα and cell division cycle 7 (Cdc7) silencing were done using specific small interfering RNA. Transit or stable inducible overexpression of these proteins and casein kinase Iε (CKIε) were also used, as well as several pharmacological inhibitors that target TopoIIα, Cdc7 or CKIε. We manipulated HME cells that expressed H2B-GFP, or did not, to detect chromosome bridges. Immunoprecipitation and direct Western blot analysis were used to detect interactions between these proteins and their total expression, respectively, whereas interactions on chromosomal arms were detected using a trapped in agarose DNA immunostaining assay. TopoIIα phosphorylation by Cdc7 or CKIε was done using an in vitro kinase assay. The TopoGen decatenation kit was used to measure TopoIIα decatenation activity. Finally, a comet assay and metaphase chromosome spread were used to detect chromosome breakage and changes in chromosome condensation or numbers, respectively.ResultsWe found that geminin and TopoIIα interact primarily in G2/M/early G1 cells on chromosomes, that geminin recruits TopoIIα to chromosomal decatenation sites or vice versa and that geminin silencing in HME cells triggers the formation of chromosome bridges by suppressing TopoIIα access to chromosomal arms. CKIε kinase phosphorylates and positively regulates TopoIIα chromosome localization and function. CKIε kinase overexpression or Cdc7 kinase silencing, which we show phosphorylates TopoIIα in vitro, restored DNA decatenation and chromosome segregation in geminin-silenced cells before triggering cell death. In vivo, at normal concentration, geminin recruits the deSUMOylating sentrin-specific proteases SENP1 and SENP2 enzymes to deSUMOylate chromosome-bound TopoIIα and promote its release from chromosomes following completion of DNA decatenation. In cells overexpressing geminin, premature departure of TopoIIα from chromosomes is thought to be due to the fact that geminin recruits more of these deSUMOylating enzymes, or recruits them earlier, to bound TopoIIα. This triggers premature release of TopoIIα from chromosomes, which we propose induces aneuploidy in HME cells, since chromosome breakage generated through this mechanism were not sensed and/or repaired and the cell cycle was not arrested. Expression of mitosis-inducing proteins such as cyclin A and cell division kinase 1 was also increased in these cells because of the overexpression of geminin.ConclusionsTopoIIα recruitment and its chromosome decatenation function require a normal level of geminin. Geminin silencing induces a cytokinetic checkpoint in which Cdc7 phosphorylates TopoIIα and inhibits its chromosomal recruitment and decatenation and/or segregation function. Geminin overexpression prematurely deSUMOylates TopoIIα, triggering its premature departure from chromosomes and leading to chromosomal abnormalities and the formation of aneuploid, drug-resistant cancer cells. On the basis of our findings, we propose that therapeutic targeting of geminin is essential for improving the therapeutic potential of TopoIIα agents.