Correction: DNA Damage, Homology-Directed Repair, and DNA Methylation.

[This corrects the article DOI: 10.1371/journal.pgen.0030110.].

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.

New therapeutic perspectives in CCDC6 deficient lung cancer cells.

Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC.

Gold(III) macrocycles: nucleotide-specific unconventional catalytic inhibitors of human topoisomerase I.

Topoisomerase IB (Top1) is a key eukaryotic nuclear enzyme that regulates the topology of DNA during replication and gene transcription. Anticancer drugs that block Top1 are either well-characterized interfacial poisons or lesser-known catalytic inhibitor compounds. Here we describe a new class of cytotoxic redox-stable cationic Au(3+) macrocycles which, through hierarchical cluster analysis of cytotoxicity data for the lead compound, 3, were identified as either poisons or inhibitors of Top1. Two pivotal enzyme inhibition assays prove that the compounds are true catalytic inhibitors of Top1. Inhibition of human topoisomerase IIα (Top2α) by 3 was 2 orders of magnitude weaker than its inhibition of Top1, confirming that 3 is a type I-specific catalytic inhibitor. Importantly, Au(3+) is essential for both DNA intercalation and enzyme inhibition. Macromolecular simulations show that 3 intercalates directly at the 5'-TA-3' dinucleotide sequence targeted by Top1 via crucial electrostatic interactions, which include π-π stacking and an Au···O contact involving a thymine carbonyl group, resolving the ambiguity of conventional (drug binds protein) vs unconventional (drug binds substrate) catalytic inhibition of the enzyme. Surface plasmon resonance studies confirm the molecular mechanism of action elucidated by the simulations.

GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair.

In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair.

Gold(III) complexes of pyridyl- and isoquinolylamido ligands: structural, spectroscopic, and biological studies of a new class of dual topoisomerase I and II inhibitors.

The structures, spectroscopy, and cytotoxicity of four novel nominally square-planar gold(III) chelates 1-4 with the general formula cis-AuCl2(X), where the ligand X is an anionic bidentate pyridyl- or isoquinolylamido chelating agent, are described. The Au-N(amido), Au-N(pyridyl), and Au-N(isoquinolyl) distances are 2.002(9)-2.016(3), 2.01(1)-2.037(3), and 2.037(3) Å, respectively. Density functional theory simulations afforded accurate gold(III) coordination geometries for 1-4 (bond distances and angles to within 5% of the X-ray values), while accurate transition energies were limited to those calculated in the UV spectral region. The complexes had variable stability in dimethyl sulfoxide: compound 3 (relatively rigid) was indefinitely stable, compounds 1 and 2 (conformationally flexible) slowly demetalated over 30 days, and 4 (extensively aromatic) formed an insoluble precipitate after 10 days (72 h in an aqueous buffer). The isoquinolylamido derivative 4 was sufficiently cytotoxic in the NCI-60 screen to undergo full five-dose testing. Notably low GI50 (1.8, 2.3, and 3.2 µM) and IC50 (4.0, 9.8, and 15 µM) values were recorded for the OVCAR-3, IGROV1, and SW-620 cell lines, respectively. Hierarchical cluster analysis employing the National Cancer Institute (NCI) data for known anticancer drugs and 4 revealed that compound 4 is mechanistically identical with the topoisomerase IIα (Top2) poison zorubicin and statistically similar to the topoisomerase IB (Top1) poisons camptothecin and 9-methoxycamptothecin. The Top2-catalyzed decatenation reaction of kinetoplast DNA was studied as a function of the concentration of 4: the compound acts as an interfacial poison of Top2 at low concentrations (<1 µM) and a catalytic inhibitor of the enzyme above 5 µM. Gel mobility shift assays (plasmid DNA substrate) showed that the catalytic inhibition of Top2 likely correlates with DNA binding by 4 at concentrations >5 µM. Compound 4 is also a catalytic inhibitor of Top1 at higher concentrations, consistent with DNA binding by the complex.

A solid phase assay for topoisomerase I interfacial poisons and catalytic inhibitors.

We report a mechanism-based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells, and retained DNA is detected by Pico Green fluorescence. Compounds that result in an increase in Pico Green staining represent potential topoisomerase interfacial poisons, whereas those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a "bimodal" readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. This method is rapid, robust, economical, and scalable for large library screens.

DNA methyltransferase 1-associated protein (DMAP1) is a co-repressor that stimulates DNA methylation globally and locally at sites of double strand break repair.

Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability.

Differentiation linked regulation of telomerase activity by Makorin-1.

To understand telomere homeostasis, a significant aspect of cancer and growth control, it is important to examine telomerase induction as well as mechanisms of regulated elimination. Makorin-1 (MKRN1) was previously shown to be an E3 ubiquitin ligase that targets the telomerase catalytic subunit (hTERT) for proteasome processing (Kim et al., Genes Dev 19:776-781, 2005). In this study we examined expression and regulation of endogenous MKRN1 during the cell cycle and terminal differentiation. When WI-38 cells transition from active growth into a resting G1 state, basal levels of MKRN1 were found to increase by sixfold. In contrast, cancer cells typically contained low or in some cases undetectable levels of MKRN1 protein. HL-60 cells growing exponentially in culture contain no detectable MKRN1; however, following terminal differentiation, MKRN1 mRNA and protein levels are strongly up-regulated while hTERT mRNA, hTERC, and telomerase are shut down. The initial decrease in telomerase activity is due to a gradual reduction in transcription of the hTERT gene that occurs during the first 12 h of terminal differentiation. MKRN1 protein appears between 12 and 24 h and is attended by a more rapid loss of telomerase activity. As more MKRN1 protein accumulates, significantly less telomerase activity is seen. Addition of the proteasome inhibitor, MG132, reverses the loss of telomerase activity; therefore, reductions in telomerase activity are dynamic, ongoing, and correlated with robust up-regulation of MKRN1 as the cells terminally differentiate. The data are consistent with the idea that MKRN1 represents a telomerase elimination pathway to rapidly draw down the activity during differentiation or cell cycle arrest when telomerase action at chromosome ends is no longer necessary.

SUMOylation enhances DNA methyltransferase 1 activity.

DNA methylation regulates gene expression through a complex network of protein-protein and protein-DNA interactions in chromatin. The maintenance methylase, DNMT1 (DNA methyltransferase 1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications. The mechanistic details that explain how DNMT1 catalytic action is directed and regulated in chromatin are important in our overall understanding of gene control. In this work, we show that DNMT1 is modified by SUMOylation and we have mapped these SUMOylation sites by defined mutations. SUMOylated DNMT1 is catalytically active on genomic DNA in vivo and we find that SUMOylation significantly enhances the methylase activity of DNMT1 both in vitro and in chromatin. These data suggest that SUMOylation modulates the endogenous activity of a prominent epigenetic maintenance pathway in somatic cells.

DNA damage, homology-directed repair, and DNA methylation.

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching.

DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes (Escherichia coli and Mycobacterium smegmatis DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes (E. coli DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, K M, V max, and k cat. We found that k cat for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5'-triphosphate binding to E. coli DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors.

Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo.

While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.

Non-homologous end joining induced alterations in DNA methylation: A source of permanent epigenetic change.

In addition to genetic mutations, epigenetic revision plays a major role in the development and progression of cancer; specifically, inappropriate DNA methylation or demethylation of CpG residues may alter the expression of genes that promote tumorigenesis. We hypothesize that DNA repair, specifically the repair of DNA double strand breaks (DSB) by Non-Homologous End Joining (NHEJ) may play a role in this process. Using a GFP reporter system inserted into the genome of HeLa cells, we are able to induce targeted DNA damage that enables the cells, after successfully undergoing NHEJ repair, to express WT GFP. These GFP+ cells were segregated into two expression classes, one with robust expression (Bright) and the other with reduced expression (Dim). Using a DNA hypomethylating drug (AzadC) we demonstrated that the different GFP expression levels was due to differential methylation statuses of CpGs in regions on either side of the break site. Deep sequencing analysis of this area in sorted Bright and Dim populations revealed a collection of different epi-alleles that display patterns of DNA methylation following repair by NHEJ. These patterns differ between Bright and Dim cells which are hypo- and hypermethylated, respectively, and between the post-repair populations and the original, uncut cells. These data suggest that NHEJ repair facilitates a rewrite of the methylation landscape in repaired genes, elucidating a potential source for the altered methylation patterns seen in cancer cells, and understanding the mechanism by which this occurs could provide new therapeutic targets for preventing this process from contributing to tumorigenesis.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair.

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

Histone deacetylase inhibitors activate p21(WAF1) expression via ATM.

Histone deacetylase (HDAC) inhibitors are known to induce expression of genes such as p21(WAF1), thereby, leading to cell cycle arrest. In this work, we show that p21(WAF1) induction by HDAC inhibitors (depsipeptide and trichostatin A) is defective in Ataxia telangiectasia (AT) cells but normal in matched wild-type (WT) cells (human diploid fibroblasts). To verify the role of ATM in this effect, we show that ectopic expression of the WT ATM gene in an AT cell line fully restores p21(WAF1) induction by the HDAC inhibitors. Furthermore, because caffeine and wortmannin attenuate p21(WAF1) induction in WT cells, it is probable that the phosphatidylinositol 3'-kinase activity is essential for this process. Besides the p21(WAF1) promoter, activation of topoisomerase IIIalpha and SV40 promoters by the HDAC inhibitors are also decreased in the AT cell lines relative to WT cells; thus, these findings pertain to other promoters. Finally, despite the obvious induction deficiency of gene expression, the overall levels of H3 and H4 histone acetylation appear to be the same between AT and normal cells in response to HDAC inhibitor treatments. Taken together, the data indicate that ATM is involved in histone acetylation-mediated gene regulation.

DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism.

We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells.

An "Unlikely" Pair: The Antimicrobial Synergy of Polymyxin B in Combination with the Cystic Fibrosis Transmembrane Conductance Regulator Drugs KALYDECO and ORKAMBI.

Novel combination therapies are desperately needed for combating lung infections caused by bacterial "superbugs". This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the cystic fibrosis (CF) drugs KALYDECO (ivacaftor) and ORKAMBI (ivacaftor + lumacaftor) against Gram-negative pathogens that commonly colonize the CF lung, in particular, the problematic Pseudomonas aeruginosa. The in vitro synergistic activity of polymyxin B combined with ivacaftor or lumacaftor was assessed using checkerboard and static time-kill assays against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa isolates from the lungs of CF patients. Polymyxin B, ivacaftor, and lumacaftor were ineffective when used individually against polymyxin-resistant (MIC ≥ 4 mg/L) isolates. However, when used together, the combination of clinically relevant concentrations of polymyxin B (2 mg/L) combined with ivacaftor (8 mg/L) or ivacaftor (8 mg/L) + lumacaftor (8 mg/L) displayed synergistic killing activity against polymyxin-resistant P. aeruginosa isolates as demonstrated by a 100-fold decrease in the bacterial count (CFU/mL) even after 24 h. The combinations also displayed excellent antibacterial activity against P. aeruginosa under CF relevant conditions in a sputum medium assay. The combination of lumacaftor (alone) with polymyxin B showed additivity against P. aeruginosa. The potential antimicrobial mode of action of the combinations against P. aeruginosa was investigated using different methods. Treatment with the combinations induced cytosolic GFP release from P. aeruginosa cells and showed permeabilizing activity in the nitrocefin assay, indicating damage to both the outer and inner Gram-negative cell membranes. Moreover, scanning and transmission electron micrographs revealed that the combinations produce outer membrane damage to P. aeruginosa cells that is distinct from the effect of each compound per se. Ivacaftor was also shown to be a weak inhibitor of the bacterial DNA gyrase and topoisomerase IV with no effect on either human type I or type IIα topoisomerases. Lumacaftor displayed the ability to increase the cellular production of damaging reactive oxygen species. In summary, the combination of polymyxin B with KALYDECO or ORKAMBI exhibited synergistic activity against highly polymyxin-resistant P. aeruginosa CF isolates and can be potentially useful for otherwise untreatable CF lung infections.

Down modulation of topoisomerase I affects DNA repair efficiency.

Topoisomerase I (topo I) relaxes supercoiled DNA through a breakage/rejoining reaction which involves a transient covalent bond between topo I and the 3' end of the cleaved DNA strand. Topo I activity is now shown to be involved in DNA damage/repair pathway in vivo. Down regulating topo I levels using anti-sense RNA approach inhibits repair of UV-induced DNA lesions, negatively affects clonogenic survival following UV-irradiation, and reduces the formation of repair patches at the cytological level. Finally, topo I is actively recruited onto genomic DNA following DNA damage by UV light without inducing ubiquitin-dependent degradation of topo I. Thus, topo I activity is important, possibly required, for pre- or post-DNA damage processing in nucleotide excision repair (NER).

Syntheses and biological activities of disaccharide daunorubicins.

Carbohydrate moiety is found in many anticancer nature products. To explore the carbohydrate moiety of daunorubicin in enhancing anticancer efficacy, several daunorubicin derivatives bearing disaccharide (1-8) have been synthesized. Their cytotoxicities were tested in leukemia K562 and colon cancer SW620 cells. Topoisomerase II (topo II) poisoning was performed with the in vivo complex of topoisomerase bioassay. In both cell lines, compounds with various terminal 2,6-dideoxy sugars (compounds 1, 3, 5, and 8) showed 30- to 60-fold higher anticancer activity than compounds with 2-deoxy- or 6-deoxy sugar (compounds 6 and 7). Compounds with an alpha-linkage between two sugar units (compound 3) showed 35-fold higher anticancer activity than compounds with a beta-linkage (compound 4). In addition, the anticancer activities of these compounds correlated with their ability to target topo II mediated genomic DNA damage in vivo. Compounds 1 and 3 with 2,6-dideoxy sugars produced more covalent topo-DNA complex than compounds with 2-deoxy sugar (6) and 6-deoxy sugar (7). Compounds with an alpha-configuration of terminal 2,6-dideoxy sugar (compounds 1 and 3) showed higher topo II poisoning than their counterparts with the beta-configuration (compounds 2 and 4). These results indicate that sugar moieties in daunorubicin play a significant role in its anticancer activity and topo II inhibition. The second sugar of disaccharide daunorubicin should possess 2,6-dideoxy with alpha-linkage to the first sugar to exhibit better anticancer activity.