Topoisomerase IIα inhibition following DNA transfection greatly enhances random integration in a human pre-B lymphocyte cell line

Toyoda, Eiji; Kurosawa, Aya; Kamekawa, Haruna; Adachi, Noritaka

doi:10.1016/j.bbrc.2009.03.047

Cited by 5 publications

(6 citation statements)

References 42 publications

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“…These results suggest that random integration frequency is reduced when Top2α expression is strongly suppressed. This is in sharp contrast to our previous finding that absence of Top2β had little or no effect on the frequency of random integration [19].…”

Section: Top2acontrasting

confidence: 99%

“…Earlier work has shown that Top2 inhibitors can enhance random integration via nonhomologous recombination in mammalian cells [17,18]. Intriguingly, we have recently shown that inhibition of Top2α, not Top2β, is critical for the enhancement of random integration [19]. It remains to be elucidated, however, whether Top2α protein is actually involved in spontaneously occurring (i.e., Top2 inhibitor-independent) random integration events under physiological conditions, namely in the absence of forced poisoning of Top2 protein.…”

Section: Introductionmentioning

confidence: 86%

“…Random integration assays were carried out using Nalm-6 cells, essentially as described [7,19]. Briefly, 4×10…”

Section: Transfection and Integration Assaysmentioning

confidence: 99%

“…Luciferase assays were performed as described previously [19]. Briefly, after electroporation with pLucPuro, cells were cultured in growth medium for 5 hr.…”

Section: Luciferase Assaysmentioning

confidence: 99%

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Endogenous Factors Causative of Spontaneous DNA Damage that Leads to Random Integration in Human Cells

Kamekawa

Kurosawa²,

Umehara³

et al. 2013

Gene Technology

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Random integration is a phenomenon in which transfected DNA molecules integrate into (random sites of) the host genome via non-homologous recombination. Although it is assumed that repair of DNA double-strand breaks leads to random integration events, how these endogenous DNA lesions are generated in living cells is poorly understood. In this study, we present evidence that DNA topoisomerase IIα (Top2α) and reactive oxygen species (ROS) are responsible for causing genomic DNA damage that leads to random integration. Specifically, we employed a human pre-B lymphocyte cell line to examine the effects of cellular Top2 expression levels and oxygen concentrations during cell culture. We find that treating cells with Top2α siRNA significantly reduces random integration frequency, while the absence of Top2β had little or no impact. We also show that cells continuously cultured under low (3%) oxygen culture conditions after electroporation display reduced random integration frequency compared to that under normal (21%) oxygen conditions. These findings support the notion that Top2α protein and ROS are endogenous factors that can produce DNA damage leading to random integration of transfected DNA in human cells.

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Section: Top2acontrasting

confidence: 99%

Section: Introductionmentioning

confidence: 86%

“…Random integration assays were carried out using Nalm-6 cells, essentially as described [7,19]. Briefly, 4×10…”

Section: Transfection and Integration Assaysmentioning

confidence: 99%

“…Luciferase assays were performed as described previously [19]. Briefly, after electroporation with pLucPuro, cells were cultured in growth medium for 5 hr.…”

Section: Luciferase Assaysmentioning

confidence: 99%

See 2 more Smart Citations

Endogenous Factors Causative of Spontaneous DNA Damage that Leads to Random Integration in Human Cells

Kamekawa

Kurosawa²,

Umehara³

et al. 2013

Gene Technology

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“…Indeed, we have recently shown that DNA topoisomerase IIα and reactive oxygen species (ROS) are such endogenous factors responsible for causing DNA damage that leads to random integration of transfected DNA in human cells [37]. Transient inhibition of topoisomerase IIα significantly increases random integration [38]; conversely, siRNA-mediated knockdown of topoisomerase IIα reduces random integration [37]. Cells continuously cultured under 3% oxygen conditions after DNA transfection display reduced random-integration frequency compared to that under 21% oxygen conditions [37], although the gene-targeting efficiency was little affected by the low-oxygen culture condition (our unpublished observations).…”

Section: Impact Of Dsb Repair Deficiency On Targeted and Random Integmentioning

confidence: 99%

Repair of Accidental DNA Double-Strand Breaks in the Human Genome and Its Relevance to Vector DNA Integration

Adachi

Saito²,

Kurosawa³

2014

Gene Technology

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Efficient repair of chromosomal DNA damage is crucial for cells to maintain genome integrity. DNA double-strand breaks (DSBs) are the most severe type of DNA lesions that can be caused by various exogenous and endogenous mechanisms, such as ionizing radiation, reactive oxygen species, topoisomerase poisons, or replication errors [1]. DSBs, if left unrepaired or mis-repaired, lead to cell death or chromosomal aberrations [2,3]. Human cells have evolved two fundamentally different mechanisms for repairing chromosomal DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ) [4]. NHEJ not only repairs accidental (non-physiological) DSBs, but is also essential for rejoining physiological DSBs that arise in the process of V(D)J recombination in B and T lymphocytes and class switch recombination in mature B cells [3].A wide variety of proteins have been identified thus far that contribute to the HR and NHEJ machineries [5]. HR is a highly complicated process of DNA transaction, in which Rad51 protein plays an essential role in DNA strand exchange with the aid of several other proteins such as Rad54, Brca2, Rad52, and Rad51 paralogs [6,7]. For HR to occur, DSBs should be processed (i.e., end-resected) to produce a long 3'-overhang single-stranded DNA [8,9], and recent studies have identified a number of proteins involved in end resection or its regulation; among these, Mre11 and CtIP play essential roles in the initial step of end resection [9][10][11]. In contrast to HR, NHEJ is thought to be a rather simpler process that requires, at least biochemically, only four proteins (two protein complexes); specifically, Ku, a heterodimer of Ku70 and Ku80, initiates an NHEJ reaction by binding to the ends of a DSB, and the DNA ligase complex composed of Xrcc4 and Ligase IV (Lig4) seals the ends to complete repair [3]. In most cases, however, many other proteins do participate in NHEJ-mediated repair to trim the DSB ends, which are typically non-ligatable or non-compatible. These additional NHEJ factors involve DNA-PKcs, Artemis, XLF, and DNA polymerase µ/λ; DNA-PKcs and Artemis have evolved in higher eukaryotes and do not exist in yeasts [3,12]. In addition to the classical pathway of NHEJ, recent evidence indicates the existence of a more error-prone mechanism of NHEJ called alternative endjoining that plays a role in DSB repair [3,13]. Alternative end-joining is Ku/Lig4 independent and the precise mechanism remains largely unclear, although PARP1, Ligase III, and several factors involved in end resection (to initiate HR) have been implicated in DSB repair via alternative end-joining [14][15][16].Which DSB repair pathway is beneficial for cells to preserve genome integrity? NHEJ (the classical NHEJ pathway) repairs broken DNA ends with little or no homology and is often associated with nucleotide loss, whereas HR allows for accurate repair of DSBs with the use of homologous DNA sequence, usually located on a sister chromatid [3,4,12]. Such difference in accuracy between the two pathways, however, does not mean...

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piggyBac-mediated generation of stable transfectants with surface human leukocyte antigen expression from a small number of cells

Miura

Inoko

Inoue

et al. 2013

Analytical Biochemistry

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Topoisomerase IIα inhibition following DNA transfection greatly enhances random integration in a human pre-B lymphocyte cell line

Cited by 5 publications

References 42 publications

Endogenous Factors Causative of Spontaneous DNA Damage that Leads to Random Integration in Human Cells

Endogenous Factors Causative of Spontaneous DNA Damage that Leads to Random Integration in Human Cells

Repair of Accidental DNA Double-Strand Breaks in the Human Genome and Its Relevance to Vector DNA Integration

piggyBac-mediated generation of stable transfectants with surface human leukocyte antigen expression from a small number of cells

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