Human Topoisomerase IIbeta Research Articles

Synthesis, characterization and in silico designing of diethyl-3-methyl-5-(6-methyl-2-thioxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxamido) thiophene-2,4-dicarboxylate derivative as anti-proliferative and anti-microbial agents

A series of eight compounds diethyl-3-methyl-5-(6-methyl-2-thioxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxamido) thiophene-2,4-dicarboxilate (KM10–17) analogues have been prepared by conventional methods and characterized by IR, Mass, NMR and elemental analysis. In silico docking studies on Human topoisomerase IIbeta (PDB Id: 3QX3) have been performed for all molecules (KM10–17) synthesized. The compounds were tested for in vitro anti-proliferative activity on VERO and 786-O cell lines. Out of all the synthesized compounds, KM11 &KM16 showed moderate activity on both cell lines. In vitro anti-microbial activity was also checked against Bacillus subtilis (BS), Staphylococcus aurous (SA), Pseudomonas aeruginosa (PA), Escherichia coli (EC) and Candida albicans (CA) by well diffusion method. The compound KM11 was found to have highest zone of inhibition against BS, SA, PA and EC. The molecules KM13 and KM16 exhibited good activity against CA. The compounds KM14 and KM16 indicated good zone of inhibition against BS.

Use of Divalent Metal Ions in the DNA Cleavage Reaction of Human Type II Topoisomerases

All type II topoisomerases require divalent metal ions to cleave and ligate DNA. To further elucidate the mechanistic basis for these critical enzyme-mediated events, the role of the metal ion in the DNA cleavage reaction of human topoisomerase IIbeta was characterized and compared to that of topoisomerase IIalpha. This study utilized divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that substituted a sulfur atom for the 3'-bridging oxygen or the nonbridging oxygens of the scissile phosphate. On the basis of time courses of DNA cleavage, cation titrations, and metal ion mixing experiments, we propose the following model for the use of divalent metal ions by human type II topoisomerases. First, both enzymes employ a two-metal ion mechanism to support DNA cleavage. Second, an interaction between one divalent metal ion and the 3'-bridging atom of the scissile phosphate greatly enhances enzyme-mediated DNA cleavage, most likely by stabilizing the leaving 3'-oxygen. Third, there is an important interaction between a divalent second metal ion and a nonbridging atom of the scissile phosphate that stimulates DNA cleavage mediated by topoisomerase IIbeta. If this interaction exists in topoisomerase IIalpha, its effects on DNA cleavage are equivocal. This last aspect of the model highlights a difference in metal ion utilization during DNA cleavage mediated by human topoisomerase IIalpha and IIbeta.

Ability of viral topoisomerase II to discern the handedness of supercoiled DNA: bimodal recognition of DNA geometry by type II enzymes.

Previous studies with human and bacterial topoisomerases suggest that the type II enzyme utilizes two distinct mechanisms to recognize the handedness of DNA supercoils. It has been proposed that the ability of some type II enzymes, such as human topoisomerase IIalpha and Escherichia coli topoisomerase IV, to distinguish supercoil geometry during DNA relaxation is mediated by elements in the variable C-terminal domain of the protein. In contrast, the ability of human topoisomerase IIalpha and topoisomerase IIbeta to discern the handedness of supercoils during DNA cleavage suggests that residues in the conserved N-terminal or central domain of the protein are involved in this process. To test this hypothesis, the ability of Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) topoisomerase II to relax and cleave negatively and positively supercoiled plasmids was assessed. These enzymes display a high degree of sequence identity with the N-terminal and central domains of eukaryotic topoisomerase II but naturally lack the C-terminal domain. While PBCV-1 and CVM-1 topoisomerase II relaxed under- and overwound substrates at similar rates, they were able to discern the handedness of supercoils during the cleavage reaction and preferentially cut negatively supercoiled DNA. Preferential cleavage was not due to a change in site specificity, DNA binding, or religation. These findings are consistent with a bimodal recognition of DNA geometry in which topoisomerase II uses elements in the C-terminal domain to sense the handedness of supercoils during DNA relaxation and elements in the conserved N-terminal or central domain during DNA cleavage.

Mutation P732L in Human DNA Topoisomerase IIβ Abolishes DNA Cleavage in the Presence of Calcium and Confers Drug Resistance

The anti cancer drug methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) targets human DNA topoisomerase IIbeta. We report here the first selection with mAMSA of resistant human topoisomerase IIbeta. Random mutagenesis of human DNA topoisomerase IIbeta cDNA, followed by selection in yeast for resistance to mAMSA, identified betaP732L. This mutant was 10-fold less sensitive to mAMSA and cross-resistant to other chemotherapeutic agents such as etoposide, ellipticine, methyl N-(4'-(9-acridinylamino)-2-methoxy-phenyl) carbamate hydrochloride (mAMCA), methyl N-(4'-(9-acridinylamino)-phenyl) carbamate hydrochloride (AMCA), and doxorubicin. betaP732L is functional but has reduced strand passage activities and altered DNA binding compared with the wild-type protein. It has drastically altered cleavage properties compared with the wild-type enzyme. It cleaved a 40-base pair (bp) DNA substrate in the presence of magnesium but at positions different from that of the wild-type protein. More striking is that betaP732L was unable to cleave the 40-bp DNA substrate, a 500-bp linear substrate, or a 4.3-kilobase supercoiled substrate in the presence of calcium ions. This is the first report of a topoisomerase II mutation abolishing the ability of calcium to support DNA cleavage. This provides evidence for metal ion requirement for the phosphoryltransfer reaction of topoisomerase II and a possible mechanism for drug resistance.

DNA sequence specificity for topoisomerase II poisoning by the quinoxaline anticancer drugs XK469 and CQS.

The two known antineoplastic quinoxaline topoisomerase II poisons, XK469 (NSC 697887) and CQS (chloroquinoxaline sulfonamide, NSC 339004), were compared for DNA cleavage site specificity, using purified human topoisomerase IIalpha and human topoisomerase IIbeta. The DNA cleavage intensity pattern for topoisomerase IIalpha poisoning by CQS closely resembled that of VM-26, despite the lack of any apparent common pharmacophore. In contrast, the topoisomerase IIalpha DNA cleavage intensity patterns of XK469 and CQS were very different from one another despite the similar overall structures of the two drugs. This suggests that the differences in DNA site specificity of topoisomerase II poisoning by XK469 and CQS may be caused by differences in their geometry, side chains, or electronic structure. The topoisomerase IIbeta-mediated DNA cleavage sites of CQS and XK469 were also very different from one another, adding further support to this idea. Earlier work has demonstrated that a number of specific topoisomerase II poisons show very similar patterns of DNA cleavage with either topoisomerase IIalpha or topoisomerase IIbeta, suggesting that the topoisomerase II isozymes play only a minor role in choices of DNA cleavage sites. However, both of the quinoxaline topoisomerase II poisons in this study showed distinctly different and unique DNA cleavage intensity patterns with each topoisomerase II isozyme. This indicates that topoisomerase II isozymes can play a major role in DNA cleavage site selection for some classes of topoisomerase II poisons.

Characterisation of the DNA-dependent ATPase activity of human DNA topoisomerase IIbeta: mutation of Ser165 in the ATPase domain reduces the ATPase activity and abolishes the in vivo complementation ability.

We report for the first time an analysis of the ATPase activity of human DNA topoisomerase (topo) IIbeta. We show that topo IIbeta is a DNA-dependent ATPase that appears to fit Michaelis-Menten kinetics. The ATPase activity is stimulated 44-fold by DNA. The k(cat) for ATP hydrolysis by human DNA topo IIbeta in the presence of DNA is 2.25 s(-1). We have characterised a topo IIbeta derivative which carries a mutation in the ATPase domain (S165R). S165R reduced the kcat for ATP hydrolysis by 7-fold, to 0.32 s(-1), while not significantly altering the apparent K(m). The specificity constant for the interaction between ATP and topo IIbeta (kcat/K(mapp)) showed a 90% reduction for betaS165R. The DNA binding affinity and ATP-independent DNA cleavage activity of the enzyme are unaffected by this mutation. However, the strand passage activity is reduced by 80%, presumably due to reduced ATP hydrolysis. The mutant enzyme is unable to complement ts yeast topo II in vivo. We have used computer modelling to predict the arrangement of key residues at the ATPase active site of topo IIbeta. Ser165 is predicted to lie very close to the bound nucleotide, and the S165R mutation could thus influence both ATP binding and ADP dissociation.

Topoisomerase II Poisoning by ICRF-193

Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerase-targeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very significant topoisomerase II poison. Detection of topoisomerase II poisoning by ICRF-193 required the use of a chaotropic protein denaturant in the topoisomerase poisoning assays. ICRF-193 caused dose-dependent cross-linking of human topoisomerase IIbeta to DNA and stimulated topoisomerase IIbeta-mediated DNA cleavage at specific sites on (32)P-end-labeled DNA. Human topoisomerase IIalpha-mediated DNA cleavage was stimulated to a lesser extent by ICRF-193. In vivo experiments with MCF-7 cells also showed the requirement of a chaotropic protein denaturant in the assays and selectivity for the beta-isozyme of human topoisomerase II. Studies with two topoisomerase IIbeta-negative cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with beta-isozyme selectivity. Common use of only the detergent, SDS, in assays may have led to failure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.

Keratinocytes Exposed to Ultraviolet Radiation Reveal Three Down-regulated Genes with Potential Function in Differentiation and Cell Cycle Control

The incidence of skin cancer is increasing in epidemic proportion. Although solar UV radiation is known to be the major risk factor, much information is lacking about the molecular mechanisms leading to skin cancer. To gain a deeper insight into these mechanisms, we have examined cells of a human keratinocyte cell line (HaCat) after exposure to 0.16 minimal erythema doses of UVB radiation. This dose led to an S-phase delay that was reversible 22 h postirradiation. To examine gene expression 10 h after UV irradiation, a nonradioactive differential display was employed. Three genes were identified as being down-regulated significantly. The first encodes for topoisomerase-IIbeta-binding protein 1 (expression level 5% 6 h after irradiation). This protein is associated with human topoisomerase IIbeta and appears to be necessary for DNA replication during the onset of S phase. The second gene product has previously been reported to be involved in differentiation and is therefore known as differentiation-dependent A4 protein (28% 8 h after irradiation). The third gene is XPO1 (also known as CRM1) (5% 8 h after irradiation), whose protein is involved in nuclear export of mRNA molecules. Differential expression of these genes after UV irradiation has not been reported. Because of their potential involvement in cell cycle control and differentiation, these proteins could be important for understanding the reaction of keratinocytes after exposure to UV radiation.

Human DNA topoisomerase IIbeta binds and cleaves four-way junction DNA in vitro.

We have used gel retardation analysis to show that human DNA topoisomerase IIbeta can bind a 40 bp linear duplex containing a single DNA topoisomerase IIbeta cleavage site. Furthermore, we demonstrate for the first time that human DNA topoisomerase IIbeta binds to four-way junction DNA. This supports previous suggestions that topoisomerase II may be targeted to supercoiled DNA through the recognition of DNA cruciforms, helix-helix crossovers and hairpins. DNA topoisomerase IIbeta had a 4-fold higher affinity for the four-way junction than for the linear duplex, as demonstrated by protein titration and competition analysis. Furthermore, the DNA topoisomerase IIbeta:four-way junction complex was significantly more salt stable than the complex with linear DNA. The four-way junction contained potential topoisomerase IIbeta cleavage sites straddling the points of strand exchange, and indeed, topoisomerase IIbeta was able to cleave three of these four predicted sites. This indicates that topoiso-merase IIbeta can bind to the centre of the junction. Topoisomerase II has to bind both the transported and the gated DNA helices prior to strand passage, and it is possible that both helices are provided by the four-way junction in this case. The stable complex of DNA topoisomerase IIbeta with four-way junction DNA may provide an ideal substrate for further studies into the mechanism of substrate recognition and binding by DNA topoisomerase II.

Structural organization of the human TOP2A and TOP2B genes.

Eucaryotic topoisomerase II is an essential nuclear enzyme involved in processes such as chromosome condensation, chromatid separation, and in the relief of torsional stress that occurs during DNA transcription and replication. In cells from vertebrate species, there are two forms of the enzyme, designated alpha and beta. Human topoisomerase IIalpha (TOP2A) is encoded by the TOP2A gene on chromosome 17q21-22, and human topoisomerase IIbeta (TOP2B) is encoded by the TOP2B gene on chromosome 3p24. The protein products of these two genes are important cellular targets of several drugs widely used in the treatment of many human cancers, and a variety of mutations in TOP2A have been associated with the development of drug resistance. In the present study, we have defined the intron-exon structures of TOP2A and TOP2B. TOP2A is approx. 30kb whereas TOP2B is at least 49kb. TOP2A and TOP2B contain 35 and 36 exons, respectively, and both genes contain a high proportion of class 0 introns. Alignment of the amino-acid sequences of the two proteins indicates that the intron-exon organization of the two genes is highly conserved, except for the regions encoding the extreme NH2 and COOH termini of the proteins. These findings suggest strongly that the vertebrate isoforms evolved by duplication of an ancestral gene. Mutations in TOP2A associated with drug resistance show clustering in exons 12, 13, 19-21 and 34-35. Knowledge of the genomic organization of TOP2A and TOP2B will be useful for detection of mutations in clinical samples from patients with drug-resistant malignant disease.