Compositions and methods for the detection of DNA topoisomerase II complexes with DNA

a technology of dna topoisomerase and complexes, applied in the field of molecular biology and oncology, can solve problems such as cell death, and achieve the effects of preventing leukemogenic events and increasing the expression of dna topoisomerase ii

Inactive Publication Date: 2006-03-23
THE CHILDRENS HOSPITAL OF PHILADELPHIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0008] In addition, it is known that cytotoxic chemotherapy may cause cell death that is followed by bone marrow progenitor cell proliferation with accompanying increased native DNA topoisomerase II expression. A patient has recently bee identified with leukemia with an MLL translocation which emerged after chemotherapy that did not include a DNA topoisomerase II poison (Langer, 2003). Accordingly, it is another objective of the present invention to provide compositions and methods for detecting topoisomerase II / DNA complexes that are formed by the native enzyme at elevated levels, and as a means to prevent leukemogenic events associated with chemotherapy.

Problems solved by technology

In addition, it is known that cytotoxic chemotherapy may cause cell death that is followed by bone marrow progenitor cell proliferation with accompanying increased native DNA topoisomerase II expression.

Method used

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  • Compositions and methods for the detection of DNA topoisomerase II complexes with DNA
  • Compositions and methods for the detection of DNA topoisomerase II complexes with DNA
  • Compositions and methods for the detection of DNA topoisomerase II complexes with DNA

Examples

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example i

Identification and Tracing of Translocation Breakpoint Sequences in Leukemias in Patients, and Evidence from In Vitro Assays to Suggest that Translocation Breakpoints are Topoisomerase II Cleavage Sites

Therapy, Clinical Course and Detection of MLL-AF-4 Translocation in Sequential Bone Marrow Specimens of Patient t-120

[0056] In patient t-120, rearrangements consistent with both derivative chromosomes from the t(4;11) translocation were detected by Southern blot analysis in the bone marrow from ALL diagnosis, and both breakpoint junctions were characterized by panhandle PCR approaches (Raffini et al., 2002). The clinical course, primary neuroblastoma therapy and molecular analyses are summarized in FIG. 1A. All available sequential bone marrows were examined for the presence of the translocation. The translocation also was detectable by Southern blot at 9 months from the start of treatment. The translocation was not detectable by Southern blot analysis in the cells used for autolog...

example ii

Hematopoietic Progenitor Cells—a Model Cellular System for Analysis of Chromosomal Translocations Mediated by Elevated Expression of Topoisomerase II

Characterization of Native DNA Topoisomerase IIα mRNA and Protein Expression in Human CD34+ Cells

[0067] By quantitative real-time PCR analysis, DNA topoisomerase IIα mRNA expression in the cell lines RS4:11, SEM-K2 and K562 cells was 1.54(1.16+2.03)-fold, 1.55(1.14-2.10)-fold and 2.75(2.53-2.99)-fold relative to ex vivo-expanded human CD34+ cells (Table 1), indicating that DNA topoisomerase IIα mRNA in proliferating bone marrow stem cells is in the range of that in hematopoietic cell lines. Western blot analysis suggested that DNA topoisomerase IIα protein expression in the ex vivo-expanded human CD34+ cells was also high and comparable to that in hematopoietic cell lines (FIG. 6).

TABLE 1Relative DNA topoisomerase IIα mRNA expression in leukemia celllines compared to CD34+ cellsΔCTNormalized DNADNA(Avg. DNAtopoisomerase IIαtopoisom...

example iii

Ice Bioassay Detects Topoisomerase—DNA and Topoisomerase-DNA-Drug Covalent Complexes in Total Genomic DNA of Hematopoietic Cell Lines and CD34 Cells

DNA Topoisomerase II Covalent Complexes are Detected in Hematopoietic Cell Lines Treated with Chemotherapy

[0068] Although etoposide has been previously studied in ICE assays of, eg., the hematopoietic cell line CEM, induction of DNA topoisomerase II cleavage complexes by etoposide metabolites had not been previously studied in a cellular context. A modified in vivo complex of enzyme (ICE) bioassy was employed to determine whether etoposide metabolites induce DNA topoisomerase II covalent complexes in the chromatin context of hematopoietic cell lines (11). The assay entails trapping the DNA covalently bound to DNA topoisomerase II by phosphotyrosine linkage using protein denaturants and detection on a Western blot. Isolation of the protein-bound DNA using a CsCl cushion, as elaborated by Topogene, streamlines the methodology compared t...

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Abstract

Compositions, methods, and kits for detecting DNA topoisomerase II-DNA complexes are disclosed.

Description

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 60 / 608,331, filed on Sep. 9, 2004. The foregoing application is incorporated by reference herein.[0002] Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S. Government has certain rights in the invention described herein, which was made in part with funds from the National Institutes of Health, Grant Numbers RO1 CA77683, CA85469, and CA80175.FIELD OF THE INVENTION [0003] The present invention relates to the fields of molecular biology and oncology. More specifically, the invention provides compositions and methods for detection of DNA topoisomerase II complexes with genomic DNA. BACKGROUND OF THE INVENTION [0004] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full. [0005] Epipo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/6876C12Q2600/142C12Q2600/156
Inventor FELIX, CAROLYNBALDWIN, DONALD
Owner THE CHILDRENS HOSPITAL OF PHILADELPHIA
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