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PubMed1987

Calcium-promoted DNA cleavage by eukaryotic topoisomerase II: trapping the covalent enzyme-DNA complex in an active form.

The effects of calcium ions on interactions between Drosophila melanogaster topoisomerase II and DNA were assessed. Although the divalent cation could not support DNA strand passage, it was able to promote high levels of enzyme-mediated DNA cleavage. Moreover, sites of cleavage on plasmid pBR322 generated in calcium-promoted reactions were similar to those obtained in the presence of magnesium. When calcium-containing enzyme-DNA mixtures were treated with ethylenediaminetetraacetic acid, cleaved nucleic acids could be generated in the absence of sodium dodecyl sulfate (SDS) or other denaturing detergents. The product of this SDS-independent calcium-promoted reaction was a covalent topoisomerase II-DNA complex. Enzyme molecules trapped in such complexes were found to be kinetically competent. Therefore, calcium should be a valuable tool for studying the enzymology of topoisomerase II mediated DNA cleavage.

Animals↗
PubMed1988

Adenosine, deoxyadenosine, and deoxyguanosine induce DNA cleavage in mouse thymocytes.

When thymocytes were cultured with adenosine, deoxyadenosine, or deoxyguanosine at 1 mM for 24 h, DNA cleavage at internucleosomal sites with multiples of approximately 180 bp was induced, followed by lactate dehydrogenase release into the medium. In the presence of coformycin, an adenosine deaminase inhibitor, or formycin B, a purine nucleoside phosphorylase inhibitor, DNA cleavage was induced by these nucleosides at concentrations of less than 50 microM. Other purine and pyrimidine ribo- and deoxyribonucleosides did not induce DNA cleavage or LDH release. Because thymocyte nuclei contain a Ca2+,Mg2+-dependent endonuclease, which preferentially cuts DNA in its linker regions, DNA fragmentation induced by the three purine nucleosides was suggested to occur through increased activity of the endonuclease. The DNA cleavage induced by the nucleosides required protein phosphorylation and synthesis, inasmuch as it was inhibited by an inhibitor of protein kinases, H-7, and by an inhibitor of protein synthesis, cycloheximide. The inhibition of DNA cleavage was accompanied by a reduction in lactate dehydrogenase release, suggesting a causal relationship between DNA cleavage and cell death. The DNA cleavage and subsequent cell lysis might be related to the selective thymocyte deletion observed in patients with adenosine deaminase or purine nucleoside phosphorylase deficiency.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Stimulation of topoisomerase II-mediated DNA cleavage by three DNA-intercalating plant alkaloids: cryptolepine, matadine, and serpentine.

Cryptolepine, matadine, and serpentine are three indoloquinoline alkaloids isolated from the roots of African plants: Cryptolepis sanguinolenta, Strychnos gossweileri, and Rauwolfia serpentina, respectively. For a long time, these alkaloids have been used in African folk medicine in the form of plant extracts for the treatment of multiple diseases, in particular as antimalarial drugs. To date, the molecular basis for their diverse biological effects remains poorly understood. To elucidate their mechanism of action, we studied their interaction with DNA and their effects on topoisomerase II. The strength and mode of binding to DNA of the three alkaloids were investigated by spectroscopy. The alkaloids bind tightly to DNA and behave as typical intercalating agents. All three compounds stabilize the topoisomerase II-DNA covalent complex and stimulate the cutting of DNA by topoisomerase II. The poisoning effect is more pronounced with cryptolepine than with matadine and serpentine, but none of the drugs exhibit a preference for cutting at a specific base. Cryptolepine which binds 10-fold more tightly to DNA than the two related alkaloids proves to be much more cytotoxic toward B16 melanoma cells than matadine and serpentine. The cellular consequences of the inhibition of topoisomerase II by cryptolepine were investigated using the HL60 leukemia cell line. The flow cytometry analysis shows that the drug alters the cell cycle distribution, but no sign of drug-induced apoptosis was detected when evaluating the internucleosomal fragmentation of DNA in cells. Cryptolepine-treated cells probably die via necrosis rather than via apoptosis. The results provide evidence that DNA and topoisomerase II are the primary targets of cryptolepine, matadine, and serpentine.

Alkaloids↗
PubMed1990

DNA cleavage during ethanol metabolism: role of superoxide radicals and catalytic iron.

The generation of superoxide and related free radicals and the mobilization of catalytic iron due to ethanol metabolism have been suggested as mechanisms of alcohol-induced liver injury as well as of the increased risk of cancer observed in alcoholics. Cleavage of double stranded DNA is produced by both free radicals as well as by catalytic iron. The effects of ethanol metabolism on DNA cleavage were therefore studied in vitro as well as in vivo in isolated hepatocytes. Intactness of double stranded DNA was studied by measuring ethidium bromide fluorescence after DNA electrophoresis. In vitro, the metabolism of acetaldehyde by aldehyde oxidase caused cleavage of Lambda phage DNA. Cleavage was inhibited by both superoxide dismutase and desferrioxamine indicating the role of superoxide radicals and catalytic iron respectively. Studies with HIND III digests of the Lambda phage indicate a lack of specificity in the breaks with respect to nucleotide sequences. Addition of EDTA greatly enhanced cleavage. In vivo, ethanol metabolism caused minimal breakage in hepatocyte DNA and addition of acetaldehyde (100 microM) markedly enhanced cleavage; all cleavage was inhibited by desferrioxamine. The metabolism of ethanol to acetaldehyde and the further metabolism of acetaldehyde by aldehyde oxidase generates free radicals and mobilizes iron; these may contribute to alcohol-induced injury and carcinogenesis.

Acetaldehyde↗
PubMed1994

Base mutation analysis of topoisomerase II-idarubicin-DNA ternary complex formation. Evidence for enzyme subunit cooperativity in DNA cleavage.

Antitumor drugs, such as anthracyclines, interfere with mammalian DNA topoisomerase II by forming a ternary complex, DNA-drug-enzyme, in which DNA strands are cleaved and covalently linked to the enzyme. In this work, a synthetic 36-bp DNA oligomer derived from SV40 and mutated variants were used to determine the effects of base mutations on DNA cleavage levels produced by murine topoisomerase II with and without idarubicin. Although site competition could affect cleavage levels, mutation effects were rather similar among several cleavage sites. The major sequence determinants of topoisomerase II DNA cleavage without drugs are up to five base pairs apart from the strand cut, suggesting that DNA protein contacts involving these bases are particularly critical for DNA site recognition. Cleavage sites with adenines at positions -1 were detected without idarubicin only under conditions favouring enzyme binding to DNA, showing that these sites are low affinity sites for topoisomerase II DNA cleavage and/or binding. Moreover, the results indicated that the sequence 5'-(A)TA/(A)-3' (the slash indicates the cleaved bond, parenthesis indicate conditioned preference) from -3 to +1 positions constitutes the complete base sequence preferred by anthracyclines. An important finding was that mutations that improve the fit to the above consensus on one strand can also increase cleavage on the opposite strand, suggesting that a drug molecule may effectively interact with one enzyme subunit only and trap the whole dimeric enzyme. These findings documented that DNA recognition by topoisomerase II may occur at one or the other strand, and not necessarily at both of them, and that the two subunits can act cooperatively to cleave a double helix.

Adenine↗
PubMed1986

The production of topoisomerase II-mediated DNA cleavage in human leukemia cells predicts their susceptibility to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA).

Protein-associated DNA cleavage is produced in mammalian cells treated with active antileukemic DNA intercalating agents such as 4'(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). We have examined the ability of m-AMSA to produce DNA cleavage in 3 human myeloid leukemic cell lines with different sensitivities to the cytotoxic actions of m-AMSA to see if the magnitude of DNA cleavage correlated with the degree of m-AMSA sensitivity. DNA alkaline elution was used to quantify DNA cleavage. The amount of m-AMSA-induced DNA cleavage in the two lines sensitive to m-AMSA was 1-2 orders of magnitude greater than that in an m-AMSA-resistant leukemic line. The m-AMSA resistant line had been developed by prolonged exposure of one of the sensitive lines to m-AMSA. This finding was not secondary to a decreased uptake of m-AMSA in the resistant cell line. m-AMSA treatment of the nuclei isolated from the three lines produced DNA cleavage frequencies comparable to the cleavage frequencies produced by m-AMSA treatment of the whole cells from which the nuclei were isolated. The DNA cleaving ability stimulated by m-AMSA is thought to be mediated by drug-induced effects on topoisomerase II, a nuclear enzyme that mediates alterations in DNA conformation. Alterations in the manner in which this enzyme interacts with antineoplastic agents may explain the emergence of resistant cells following initially successful chemotherapy.

Aminoacridines↗
PubMed2005

Chlorella virus Marburg topoisomerase II: high DNA cleavage activity as a characteristic of Chlorella virus type II enzymes.

Although the formation of a covalent enzyme-cleaved DNA complex is a prerequisite for the essential functions of topoisomerase II, this reaction intermediate has the potential to destabilize the genome. Consequently, all known eukaryotic type II enzymes maintain this complex at a low steady-state level. Recently, however, a novel topoisomerase II was discovered in Paramecium bursaria chlorella virus-1 (PBCV-1) that has an exceptionally high DNA cleavage activity [Fortune et al. (2001) J. Biol. Chem. 276, 24401-24408]. If robust DNA cleavage is critical to the physiological functions of chlorella virus topoisomerase II, then this remarkable characteristic should be conserved throughout the viral family. Therefore, topoisomerase II from Chlorella virus Marburg-1 (CVM-1), a distant family member, was expressed in yeast, isolated, and characterized. CVM-1 topoisomerase II is 1058 amino acids in length, making it the smallest known type II enzyme. The viral topoisomerase II displayed a high DNA strand passage activity and a DNA cleavage activity that was approximately 50-fold greater than that of human topoisomerase IIalpha. High DNA cleavage appeared to result from a greater rate of scission rather than promiscuous DNA site utilization, inordinately tight DNA binding, or diminished religation rates. Despite the fact that CVM-1 and PBCV-1 topoisomerase II share approximately 67% amino acid sequence identity, the two enzymes displayed clear differences in their DNA cleavage specificity/site utilization. These findings suggest that robust DNA cleavage is intrinsic to the viral enzyme and imply that chlorella virus topoisomerase II plays a physiological role beyond the control of DNA topology.

Amino Acid Sequence↗
PubMed2004

Flexibility at Gly-194 is required for DNA cleavage and relaxation activity of Escherichia coli DNA topoisomerase I.

The proposed mechanism of type IA DNA topoisomerase I includes conformational changes by the single enzyme polypeptide to allow binding of the G strand of the DNA substrate at the active site, and the opening or closing of the "gate" created on the G strand of DNA to the passing single or double DNA strand(s) through the cleaved G strand DNA. The shifting of an alpha helix upon G strand DNA binding has been observed from the comparison of the type IA DNA topoisomerase crystal structures. Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this alpha helix in Escherichia coli DNA topoisomerase I showed that flexibility around this glycine residue is required for DNA cleavage and relaxation activity and supports a functional role for this hinge region in the enzyme conformational change.

DNA Topoisomerases, Type I↗
PubMed1987

Effect of cell proliferation and chromatin conformation on intercalator-induced, protein-associated DNA cleavage in human brain tumor cells and human fibroblasts.

Antineoplastic intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) stabilize a cleavable complex between topoisomerase II and DNA. The production of protein-associated DNA cleavage in whole cells exposed to m-AMSA is thought to represent the cellular correlate of this topoisomerase II-mediated reaction. Protein-associated DNA cleavage can be quantified in mammalian cells by using alkaline elution technology. In an attempt to understand the impact of phenotypic and biochemical cellular characteristics on protein-associated DNA cleavage, we quantified m-AMSA-induced DNA cleavage in quiescent or proliferative normal human fibroblasts (cell strain 1508) and human glioblastoma cells (line T98G) as well as in asynchronously proliferating HeLa cells. The magnitude of DNA cleavage in quiescent fibroblasts and quiescent glioblastoma cells was identical and low relative to that observed in the HeLa cells. The magnitude of DNA cleavage was enhanced in both cell types following proliferation. This enhancement was greater in the glioblastoma cells than in the fibroblasts. These results were not due to alterations in cellular m-AMSA uptake. Chromatin was more elongated (open) in the quiescent glioblastoma cells than in the quiescent fibroblasts (as visualized by using the premature chromosome condensation assay), suggesting chromatin accessibility to drug per se may not be a critical determinant of the magnitude of m-AMSA-induced DNA cleavage. The onset of the enhanced m-AMSA-induced DNA cleavability that accompanied proliferation closely followed the formation of regions of localized chromatin decondensation, a late G1 event, and coincided with the onset of enhanced thymidine uptake, a marker for the onset of S phase. m-AMSA-induced cytotoxicity was also enhanced in proliferating compared with quiescent cells. The major finding of this study is that the cellular target for m-AMSA, putatively topoisomerase II, is more susceptible to drug action in proliferating cells than in quiescent cells. Effects of chromatin conformation or cellular phenotype upon topoisomerase II-mediated events such as m-AMSA-induced DNA cleavage are less certain.

Amsacrine↗
PubMed2005

Potential-modulated DNA cleavage by (N-salicylideneglycinato)copper(II) complex.

The interaction of aqua (N-salicylideneglycinato)copper(II) (Cu(salgly)2+) complex with calf thymus DNA has been investigated by cyclic voltammetry. Potential-modulated DNA cleavage in the presence of Cu(salgly)2+ complex was performed at a gold electrode in a thin layer cell. DNA can be efficiently cleaved by electrochemically reducing Cu(salgly)2+ complex to Cu(salgly)+ complex at -0.7 V (vs. Ag/AgCl). When the solution was aerated with a small flow of O2 during electrolysis, the extent of DNA cleavage was dramatically enhanced, and hydroxyl radical scavengers inhibited DNA cleavage. These results suggested that O2 and hydroxyl radical were involved in potential-modulated DNA cleavage reaction. The percentage of DNA cleavage was enhanced as the working potential was shifted to more negative values and the electrolysis time was increased. It was also dependent on the ratio of Cu(salgly)2+ complex to DNA concentration. The cleaved DNA fragments were separated by high performance liquid chromatography (HPLC). The experimental results indicated that the method for potential-modulated DNA cleavage by Cu(salgly)2+ complex was simple and efficient.

Chromatography, High Pressure Liquid↗
PubMed2002

Distamycin A, a minor groove binder, changes enediyne-induced DNA cleavage sites and enhances apoptosis.

We examined the effects of a minor groove binder, distamycin A, on DNA cleavage and apoptosis induced by an enediyne antitumor antibiotic, C1027, using 32P-labeled DNA fragments obtained from human genes. C1027 alone induced DNA cleavage particularly at the 5'-TTTT-3'/3'-AAAA-5' sequence (cutting sites are underlined). The addition of distamycin A enhanced the double-strand DNA cleavage at the 5'-CCT-3'/3'-GGA-5' and 5'-CCA-3'/3'-GGT-5' sequences, with a two-nucleotide 3'-stagger of the cleaved residues. These results suggest that distamycin A forms a heterodimer with C1027 to bind to DNA at GC-rich regions, resulting in amplification of DNA cleavage at these regions. Distamycin A enhanced C1027-induced DNA ladder formation and cytotoxicity in HL-60 cells. Therefore, amplification of DNA cleavage at GC-rich regions may result in enhancement of apoptosis. The present study on amplifiers of antitumor agents showed a novel approach to the potentially effective antitumor therapy.

Aminoglycosides↗
PubMed2000

A continuous assay for DNA cleavage: the application of "break lights" to enediynes, iron-dependent agents, and nucleases.

Although extensive effort has been applied toward understanding the mechanism by which enediynes cleave DNA, a continuous assay for this phenomenon is still lacking. In fact, with the exception of assays for DNase, continuous assays for most DNA cleavage events are unavailable. This article describes the application of "molecular break lights" (a single-stranded oligonucleotide that adopts a stem-and-loop structure and carries a 5'-fluorescent moiety, a 3'-nonfluorescent quenching moiety, and an appropriate cleavage site within the stem) to develop the first continuous assay for cleavage of DNA by enediynes. Furthermore, the generality of this approach is demonstrated by using the described assay to directly compare the DNA cleavage by naturally occurring enediynes [calicheamicin and esperamicin), non-enediyne small molecule agents (bleomycin, methidiumpropyl-EDTA-Fe(II), and EDTA-Fe(II]), as well as the restriction endonuclease BamHI. Given the simplicity, speed, and sensitivity of this approach, the described methodology could easily be extended to a high throughput format and become a new method of choice in modern drug discovery to screen for novel protein-based or small molecule-derived DNA cleavage agents.

Aminoglycosides↗
PubMed1995

Abasic sites stimulate double-stranded DNA cleavage mediated by topoisomerase II. DNA lesions as endogenous topoisomerase II poisons.

Several clinically relevant anticancer drugs induce genomic mutations and cell death by increasing topoisomerase II-mediated DNA breakage. To determine whether endogenous DNA damage also affects this cleavage event, the effects of abasic sites (the most commonly formed spontaneous DNA lesion) on topoisomerase II activity were investigated. The presence of 3 abasic sites/plasmid stimulated enzyme-mediated DNA breakage > 6-fold, primarily by enhancing the forward rate of cleavage. This corresponds to a potency that is > 2000-fold higher than that of the anticancer drug, etoposide. These findings suggest that abasic sites represent endogenous topoisomerase II poisons and imply that anticancer drugs mimic the cleavage-enhancing actions of naturally occurring DNA lesions.

Animals↗
PubMed1990

Activation of internucleosomal DNA cleavage in human CEM lymphocytes by glucocorticoid and novobiocin. Evidence for a non-Ca2(+)-requiring mechanism(s).

Internucleosomal DNA cleavage is the key molecular event of the cytolytic phase of glucocorticoid-induced lymphocytolysis. We find that novobiocin, the topoisomerase II inhibitor, is a potent inducer of in vivo internucleosomal DNA cleavage in human CEM lymphocytes. This in vivo effect is very rapid, time- and dose-dependent, requires cellular integrity, and does not require de novo protein synthesis. Recently our data (Alnemri, E. S., and Litwack, G. (1989) J. Biol. Chem. 264, 4104-4111) suggested that activation of DNA cleavage in CEM-C7 lymphocytes by glucocorticoids is independent of calcium uptake. Similarly, the novobiocin effect is also independent of calcium uptake and does not occur in isolated CEM nuclei or in CEM cells treated previously with the divalent cation ionophore A23187. Internucleosomal DNA cleavage induced by novobiocin or glucocorticoid generates blunt-ended double-stranded DNA fragments possessing 3'-hydroxyls and 5'-phosphates. As demonstrated by gel retardation analysis and DNase I footprinting, novobiocin causes the disruption and unfolding of an in vitro reconstituted mononucleosome so that it becomes more susceptible to DNase I cleavage. Our data suggest that 1) novobiocin rapid activation of internucleosomal DNA cleavage and chromatin changes in CEM lymphocytes are molecular features of apoptosis or programmed cell death. 2) CEM lymphocytes apparently do not express a Ca2(+)-dependent endonuclease. 3) The mechanism(s) of glucocorticoid or novobiocin-induced DNA cleavage in CEM lymphocytes involves activation of a constitutive non Ca2(+)-dependent endonuclease. We propose that the majority of nuclear chromatin is maintained in a highly compact and charge-neutralized state and that disruption of this highly ordered structure, directly by novobiocin or indirectly by glucocorticoid, may lead to the exposure and unmasking of internucleosomal linker DNA regions which are substrates for a constitutive non-Ca2(+)-dependent endonuclease.

Amsacrine↗
PubMed1994

Conformational properties of topoisomerase II inhibitors and sequence specificity of DNA cleavage.

The sequence specificity of topoisomerase-II-mediated DNA cleavage, stimulated by 2-methyl-9-hydroxy ellipticinium and 4',5,7-trihydroxyflavone (genistein) was investigated by sequencing analysis of DNA cleavage sites and molecular modeling techniques. The former drug exhibits a marked preference for a T base at the position immediately preceding the cleavage site (-1). The latter shares the preference for the same base, with an additional preference for a thymine at position +1. The cleavage intensity patterns in the presence of the two drugs differ considerably. From a conformational point of view, ellipticinium and genistein exhibit similar overall shape and dimensions. However, the fused ring system in the former generates a planar structure whereas the single bond, connecting the two aromatic portions in the latter, allows internal rotation. The most stable conformation of genistein corresponds to a deviation of about 40 degrees from planarity. A computer-assisted analysis was carried out to compare the steric and electrostatic properties of the two compounds. Two types of preferred (energetically almost degenerate) alignment for the two molecules were found. One corresponds to overlapping of the 9-hydroxyl containing ring of ellipticinium with the 4'-hydroxyphenyl moiety of genistein, the other envisages the same moiety of ellipticine superimposed to the hydroxyl-benzopyrone portion of genistein. The structural similarities of the test drugs might account for the common preference for stimulation of DNA cleavage at position +1, whereas the different possible arrangements of genistein in the cleavable complex could explain both the additional +1 specificity exhibited by this compound and the differences in cleavage intensity patterns observed in comparison to ellipticinium.

Base Sequence↗
PubMed2005

An ab initio quantum mechanics calculation that correlates with ligand orientation and DNA cleavage site selectivity in camptothecin-DNA-topoisomerase I ternary cleavage complexes.

Camptothecin (CPT), a cytotoxic natural alkaloid isolated from Camptotheca acuminata, and its derivatives represent an important class of cancer chemotherapeutic drugs that act by inhibiting topoisomerase I (top1). The mechanism of top1 inhibition by CPT has been determined by X-ray crystallography. Biochemical studies carried out both in vitro and in vivo indicated that CPT has strict DNA sequence preference for -1 T and strong preference for +1 G at the cleavage site. To understand the molecular determinants for the CPT binding orientation and DNA sequence selectivity, we present a quantum mechanics calculation where only pi-pi stacking interactions were included to shed some light on the mechanism of this sequence selectivity. This ab initio calculation can not only reproduce the experimental binding orientation of CPT at the cleavage site but also shows very good correlation between the binding energy for different sequences and the observed frequency of CPT-stabilized sites in the SV40 viral genome. Therefore, it can be concluded that hydrogen bonding of the ligand to the surrounding amino acid residues of the protein is of minor significance. The present method should be applicable to other polycyclic top1 inhibitors.

Antineoplastic Agents, Phytogenic↗
PubMed1989

12-O-tetradecanoylphorbol 13-acetate induces DNA cleavage at linker regions in mouse thymocytes.

A phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), induced the cleavage of nuclear DNA at linker regions in cultured mouse thymocytes. Similar DNA fragmentation was induced by 1-oleoyl-2-acetyl-glycerol, a synthetic diacylglycerol, but not by 4 alpha-phorbol-12,13 didecanoate. The DNA fragmentation was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine dihydrochloride, an inhibitor of protein kinase C, as well as actinomycin D and cycloheximide. It appears that TPA induces DNA cleavage through activation of protein kinase C and synthesis of yet unidentified protein(s). That the inhibition of DNA fragmentation was accompanied by a reduction in cell lysis suggests a causal relationship between DNA fragmentation and cell death.

Animals↗
PubMed1987

Effect of polyamine depletion by alpha-difluoromethylornithine or (2R,5R)-6-heptyne-2,5-diamine on drug-induced topoisomerase II-mediated DNA cleavage and cytotoxicity in human and murine leukemia cells.

The effects of alpha-difluoromethylornithine (DFMO), an ornithine analogue which is an ornithine decarboxylase inhibitor, on the actions of the topoisomerase II-reactive agents 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposide (VP-16) were investigated in 2 murine L1210 leukemia lines and 2 human HL-60 leukemia lines. One of the human lines was resistant to the cytotoxic and DNA cleaving effects of m-AMSA (HL-60/AMSA). In all 4 lines, alpha-DFMO depleted cellular putrescine and spermidine to nondetectable levels. VP-16-induced DNA cleavage (quantified using alkaline elution) was decreased in all lines following alpha-DFMO treatment. The m-AMSA-induced DNA cleavage was decreased in one of the L1210 lines and in the HL-60 line sensitive to m-AMSA; m-AMSA-induced DNA cleavage was increased in the other L1210 line. The low frequency of m-AMSA-induced DNA cleavage produced in HL-60/AMSA was unaffected by alpha-DFMO treatment. Alterations in drug-mediated DNA effects induced by alpha-DFMO could not be uniformly explained by alpha-DFMO-induced alterations in m-AMSA or VP-16 cellular uptake, as indicated by direct measurements of cell-associated drug or results of DNA cleavage assays in nuclei isolated from alpha-DFMO-treated cells. Exogenous putrescine prevented the effects of alpha-DFMO on drug-induced DNA cleavage, substantiating polyamine depletion as the cause of the altered frequency of DNA cleavage. Cytotoxicity assays in 2 of the lines demonstrated that drug-induced reductions in colony-forming ability paralleled drug-induced DNA cleavage. (2R,5R)-6-heptyne-2,5-diamine, a putrescine analogue which is also an ornithine decarboxylase inhibitor, was also used to deplete polyamine levels in HL-60. (2R,5R)-6-heptyne-2,5-diamine was more potent than alpha-DFMO and produced effects on m-AMSA- and VP-16-induced DNA cleavage and cytotoxicity identical to those produced by alpha-DFMO.

Alkynes↗