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Neil Osheroff

Publications and source records attributed to Neil Osheroff.

30 records · Page 2Linked to original sources
PubMed2004

Biological characterization of MLN944: a potent DNA binding agent.

MLN944 (XR5944) is a novel bis-phenazine that has demonstrated exceptional efficacy against a number of murine and human tumor models. The drug was reported originally as a dual topoisomerase I/II poison, but a precise mechanism of action for this compound remains to be determined. Several lines of evidence, including the marginal ability of MLN944 to stabilize topoisomerase-dependent cleavage, and the sustained potency of MLN944 in mammalian cells with reduced levels of both topoisomerases, suggest that other activities of the drug exist. In this study, we show that MLN944 intercalates into DNA, but has no effect on the catalytic activity of either topoisomerase I or II. MLN944 displays no significant ability to stimulate DNA scission mediated by either topoisomerase I or II compared with camptothecin or etoposide, respectively. In addition, yeast genetic models also point toward a topoisomerase-independent mechanism of action. To examine cell cycle effects, synchronized human HCT116 cells were treated with MLN944, doxorubicin, camptothecin, or a combination of the latter two to mimic a dual topoisomerase poison. MLN944 treatment was found to induce a G(1) and G(2) arrest in cells that is unlike the typical G(2)-M arrest noted with known topoisomerase poisons. Finally, transcriptional profiling analysis of xenograft tumors treated with MLN944 revealed clusters of regulated genes distinct from those observed in irinotecan hydrochloride (CPT-11)-treated tumors. Taken together, these findings suggest that the primary mechanism of action of MLN944 likely involves DNA binding and intercalation, but does not appear to involve topoisomerase inhibition.

Animals↗
PubMed2003

Reciprocal DNA topoisomerase II cleavage events at 5'-TATTA-3' sequences in MLL and AF-9 create homologous single-stranded overhangs that anneal to form der(11) and der(9) genomic breakpoint junctions in treatment-related AML without further processing.

Few t(9;11) translocations in DNA topoisomerase II inhibitor-related leukemias have been studied in detail and the DNA damage mechanism remains controversial. We characterized the der(11) and der(9) genomic breakpoint junctions in a case of AML following etoposide and doxorubicin. Etoposide-, etoposide metabolite- and doxorubicin-induced DNA topoisomerase II cleavage was examined in normal homologues of the MLL and AF-9 breakpoint sequences using an in vitro assay. Induction of DNA topoisomerase II cleavage complexes in CEM and K562 cell lines was investigated using an in vivo complex of enzyme assay. The translocation occurred between identical 5'-TATTA-3' sequences in MLL intron 8 and AF-9 intron 5 without the gain or loss of bases. The 5'-TATTA-3' sequences were reciprocally cleaved by DNA topoisomerase II in the presence of etoposide, etoposide catechol or etoposide quinone, creating homologous 4-base 5' overhangs that would anneal to form both breakpoint junctions without any processing. der(11) and der(4) translocation breakpoints in a treatment-related ALL at the same site in MLL are consistent with a damage hotspot. Etoposide and both etoposide metabolites induced DNA topoisomerase II cleavage complexes in the hematopoietic cell lines. These results favor the model in which the chromosomal breakage leading to MLL translocations in DNA topoisomerase II inhibitor-related leukemias is a consequence of DNA topoisomerase II cleavage.

Adolescent↗
PubMed2003

Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks.

Topoisomerase II is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating transient double-strand DNA breaks. While the enzyme cannot perform its essential cellular functions without cleaving DNA, this scission activity is inherently dangerous to chromosomal integrity. In fact, etoposide and other clinically important anticancer drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. Cells rely heavily on recombination to repair double-strand DNA breaks, but the specific pathways used to repair topoisomerase II-generated DNA damage have not been defined. Therefore, Saccharomyces cerevisiae was used as a model system to delineate the recombination pathways that repair DNA breaks generated by topoisomerase II. Yeast cells that expressed wild-type or a drug-hypersensitive mutant topoisomerase II or overexpressed the wild-type enzyme were examined. Based on cytotoxicity and recombination induced by etoposide in different repair-deficient genetic backgrounds, double-strand DNA breaks generated by topoisomerase II appear to be repaired primarily by the single-strand invasion pathway of homologous recombination. Non-homologous end joining also was triggered by etoposide treatment, but this pathway was considerably less active than single-strand invasion and did not contribute significantly to cell survival in S.cerevisiae.

Cell Cycle↗
PubMed2003

Quinolone action against human topoisomerase IIalpha: stimulation of enzyme-mediated double-stranded DNA cleavage.

Several important antineoplastic drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. These compounds act by two distinct mechanisms. Agents such as etoposide inhibit the ability of topoisomerase II to ligate enzyme-linked DNA breaks. Conversely, compounds such as quinolones have little effect on ligation and are believed to stimulate the forward rate of topoisomerase II-mediated DNA cleavage. The fact that there are two scissile bonds per double-stranded DNA break implies that there are two sites for drug action in every enzyme-DNA cleavage complex. However, since agents in the latter group are believed to act by locally perturbing DNA structure, it is possible that quinolone interactions at a single scissile bond are sufficient to distort both strands of the double helix and generate an enzyme-mediated double-stranded DNA break. Therefore, an oligonucleotide system was established to further define the actions of topoisomerase II-targeted drugs that stimulate the forward rate of DNA cleavage. Results indicate that the presence of the quinolone CP-115,953 at one scissile bond increased the extent of enzyme-mediated scission at the opposite scissile bond and was sufficient to stimulate the formation of a double-stranded DNA break by human topoisomerase IIalpha. These findings stand in marked contrast to those for etoposide, which must be present at both scissile bonds to stabilize a double-stranded DNA break [Bromberg, K. D., et al. (2003) J. Biol. Chem. 278, 7406-7412]. Moreover, they underscore important mechanistic differences between drugs that enhance DNA cleavage and those that inhibit ligation.

Amino Acid Substitution↗
PubMed2003

Activation of the DNA-dependent protein kinase by drug-induced and radiation-induced DNA strand breaks.

The DNA-dependent protein kinase (DNA-PK) is a DNA-end activated protein kinase that is required for efficient repair of DNA double-strand breaks (DSBs) and for normal resistance to ionizing radiation. DNA-PK is composed of a DNA-binding subunit, Ku, and a catalytic subunit, DNA-PKcs (PRKDC). We have previously shown that PRKDC is activated when the enzyme interacts with the terminal nucleotides of a DSB. These nucleotides are often damaged when DSBs are introduced by anticancer agents and could therefore prevent recognition by DNA-PK. To determine whether DNA-PK could recognize DNA strand breaks generated by agents used in the treatment of cancer, we damaged plasmid DNA with anticancer drugs and ionizing radiation. The DNA breaks were tested for the ability to activate purified DNA-PK. The data indicate that DSBs produced by bleomycin, calicheamicin and two types of ionizing radiation ((137)Cs gamma rays and N(7+) ions: high and low linear energy transfer, respectively) activate DNA-PK to levels matching the kinase activation obtained with simple restriction endonuclease-induced DSBs. In contrast, the protein-linked DSBs produced by etoposide and topoisomerase II failed to bind and activate DNA-PK. Our findings indicate that DNA-PK recognizes DSBs regardless of chemical complexity but cannot recognize the protein-linked DSBs produced by etoposide and topoisomerase II.

Aminoglycosides↗
PubMed2003

Stabilization of eukaryotic topoisomerase II-DNA cleavage complexes.

Topoisomerase II is an essential enzyme that plays critical roles in many DNA processes, including chromosome segregation. In order to carry out its important physiological functions, topoisomerase II creates and rejoins double-stranded breaks in the genetic material. Thus, while the enzyme is necessary for cell survival, it also has the capacity to fragment the genome. Topoisomerase II-mediated DNA breaks are sequestered within a covalent enzyme-DNA complex. Normally, these "cleavage complexes" are present at low levels and are tolerated by the cell. However, conditions that significantly increase the physiological concentration or life-time of topoisomerase II-DNA cleavage complexes lead to chromosomal translocations and other mutagenic events, and can induce cell death pathways. The potentially lethal aspect of enzyme mechanism has been exploited by a number of highly successful anticancer agents. Since drugs that increase levels of topoisomerase II-DNA cleavage complexes transform the enzyme into a potent cellular toxin, they are referred to as topoisomerase II "poisons" to distinguish them from compounds that inhibit the catalytic activity of the enzyme. Recent evidence indicates that many DNA lesions also act as topoisomerase II poisons. This finding has provided tremendous insight into enzyme and drug action and raises important questions regarding the physiological interactions of topoisomerase II with DNA damage. Since the DNA cleavage and ligation reactions of topoisomerase II are fundamental to its physiological and pharmacological functions, this review will focus on how the enzyme cuts and rejoins the double helix and how these reactions are altered by topoisomerase II poisons.

Antineoplastic Agents↗
PubMed2002

A two-drug model for etoposide action against human topoisomerase IIalpha.

The widely used anticancer drug etoposide kills cells by increasing levels of topoisomerase II-mediated DNA breaks. While it is known that the drug acts by inhibiting the ability of topoisomerase II to ligate cleaved DNA molecules, the precise mechanism by which it accomplishes this action is not well understood. Because there are two scissile bonds per enzyme-mediated double-stranded DNA break, it has been assumed that there are two sites for etoposide in every cleavage complex. However, it is not known whether the action of etoposide at only one scissile bond is sufficient to stabilize a double-stranded DNA break or whether both drug sites need to be occupied. An oligonucleotide system was utilized to address this important issue. Results of DNA cleavage and ligation assays support a two-drug model for the action of etoposide against human topoisomerase IIalpha. This model postulates that drug interactions at both scissile bonds are required in order to increase enzyme-mediated double-stranded DNA breaks. Etoposide actions at either of the two scissile bonds appear to be independent of one another, with each individual drug molecule stabilizing a strand-specific nick rather than a double-stranded DNA break. This finding suggests (at least in the presence of drug) that there is little or no communication between the two promoter active sites of topoisomerase II. The two-drug model has implications for cancer chemotherapy, the cellular processing of etoposide-stabilized enzyme-DNA cleavage complexes, and the catalytic mechanism of eukaryotic topoisomerase II.

Antigens, Neoplasm↗
PubMed2002

Proteomic analysis of human metaphase chromosomes reveals topoisomerase II alpha as an Aurora B substrate.

The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few substrates have been identified for Aurora B, so that the precise role it plays in controlling mitosis remains to be elucidated. To identify potential novel mitotic substrates of Aurora B, extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those phosphorylated by Aurora B revealed topoisomerase II alpha (topo IIalpha) as a potential Aurora B substrate. Purified recombinant human topo IIalpha was phosphorylated by Aurora B in vitro, confirming this proteomic approach as a valid method for the initial definition of candidate substrates of key mitotic kinases.

Antigens, Neoplasm↗
PubMed2002

Site-specific DNA cleavage by Chlorella virus topoisomerase II.

The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.

Animals↗
PubMed2002

MLL-SEPTIN6 fusion recurs in novel translocation of chromosomes 3, X, and 11 in infant acute myelomonocytic leukaemia and in t(X;11) in infant acute myeloid leukaemia, and MLL genomic breakpoint in complex MLL-SEPTIN6 rearrangement is a DNA topoisomerase II cleavage site.

We examined the MLL translocation in two cases of infant AML with X chromosome disruption. The G-banded karyotype in the first case suggested t(X;3)(q22;p21)ins(X;11)(q22;q13q25). Southern blot analysis showed one MLL rearrangement. Panhandle PCR approaches were used to identify the MLL fusion transcript and MLL genomic breakpoint junction. SEPTIN6 from chromosome band Xq24 was the partner gene of MLL. MLL exon 7 was joined in-frame to SEPTIN6 exon 2 in the fusion transcript. The MLL genomic breakpoint was in intron 7; the SEPTIN6 genomic breakpoint was in intron 1. Spectral karyotyping revealed a complex rearrangement disrupting band 11q23. FISH with a probe for MLL confirmed MLL involvement and showed that the MLL-SEPTIN6 junction was on the der(X). The MLL genomic breakpoint was a functional DNA topoisomerase II cleavage site in an in vitro assay. In the second case, the karyotype revealed t(X;11)(q22;q23). Southern blot analysis showed two MLL rearrangements. cDNA panhandle PCR detected a transcript fusing MLL exon 8 in-frame to SEPTIN6 exon 2. MLL and SEPTIN6 are vulnerable to damage to form recurrent translocations in infant AML. Identification of SEPTIN6 and the SEPTIN family members hCDCrel and MSF as partner genes of MLL suggests a common pathway to leukaemogenesis.

Acute Disease↗
PubMed2002

Human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for DNA ligation. Use of 5' covalently activated oligonucleotide substrates to study enzyme mechanism.

Despite the importance of topoisomerase II-mediated DNA ligation to the essential physiological functions of the enzyme, the mechanistic details of this important reaction are poorly understood. Because topoisomerase II normally does not release cleaved DNA molecules prior to ligation, it is not known whether all of the nucleic acid specificity of its cleavage/ligation cycle is embodied in DNA cleavage or whether ligation also contributes specificity to the enzyme. All currently available ligation assays require that topoisomerase II cleave the initial DNA substrate before rejoining can be monitored. Consequently, it has been impossible to examine the specificity of DNA ligation separately from that of scission. To address this issue, a cleavage-independent topoisomerase II DNA ligation assay was developed. This assay utilizes a nicked oligonucleotide whose 5'-phosphate terminus at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. Human topoisomerase IIalpha and enzymes with active-site mutations that abrogated cleavage activity ligated the activated nick by catalyzing the direct attack of the terminal 3'-OH on the activated 5'-phosphate. Results with different DNA sequences indicate that human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for ligation that parallels its specificity for DNA cleavage.

Antigens, Neoplasm↗