Analysis of Restriction Enzyme-Induced DNA Double-Strand Breaks in Chinese Hamster Ovary Cells by Pulsed-Field Gel Electrophoresis: Implications for Chromosome Damage

Ager, D.D.; Phillips, John W.; Columna, Elma Abella; Winegar, Richard A.; Morgan, William F.

doi:10.2307/3578132

Cited by 31 publications

(9 citation statements)

References 21 publications

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“…Induction of DSBs was observed beginning at 30 min, peaking around 1 h, and being undetectable by 2 h using a TUNEL assay (Nelms et al, 1998) (Figure 1a). These results are consistent with the previous work demonstrating that PvuII-induced DSBs peak around 30-60 min post-electroporation and become undetectable within the next 2 h (Ager et al, 1991). The PvuIIelectroporated cells also exhibited NF-kB activation as detected by NF-kB-dependent GFP expression, similar to TNFa stimulated conditions (Figure 1b).…”

Section: Electroporation Of Restriction Enzymes Cause Ikk and Nf-kb Asupporting

confidence: 92%

“…Previous studies have shown that restriction enzymes could efficiently create DSBs in the mammalian genomic DNA following electroporation (Ager et al, 1991;Kinashi et al, 1995). Induction of DSBs was observed beginning at 30 min, peaking around 1 h, and being undetectable by 2 h using a TUNEL assay (Nelms et al, 1998) (Figure 1a).…”

Section: Electroporation Of Restriction Enzymes Cause Ikk and Nf-kb Amentioning

confidence: 80%

“…Electroporation was performed with 300 V and 950 mF in a Bio-Rad Gene Pulser apparatus with capacitance extender as previously described (Ager et al, 1991;Kinashi et al, 1995). PvuII heat inactivation was performed at 651C for 20 min.…”

Section: Transfection Of Restriction Enzymesmentioning

confidence: 99%

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NF-κB activation by combinations of NEMO SUMOylation and ATM activation stresses in the absence of DNA damage

et al. 2006

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The inactive transcription factor NF-jB is localized in the cytoplasm and rapidly responds to a variety of extracellular factors and intracellular stress conditions to initiate multiple cellular responses. While the knowledge regarding NF-jB signaling pathways initiated by extracellular ligands is rapidly expanding, the mechanisms of activation by intracellular stress conditions are not well understood. We recently described a critical role for a small ubiquitin-like modifier (SUMO) modification of NF-jB essential modulator (NEMO), the regulatory subunit of the IjB kinase, in response to certain genotoxic stress conditions. One important unanswered question is whether the role of this modification is limited to the genotoxic agents or some other signaling pathways also employ SUMOylation of NEMO to regulate NF-jB activation. Here, we report that a variety of other stress conditions, including oxidative stress, ethanol exposure, heat shock and electric shock, also induce NEMO SUMOylation, thus demonstrating that DNA damage per se is not necessary for this NEMO modification to occur. Moreover, combinations of certain SUMO stress and ATM (ataxia telangiectasia mutated) activation conditions lead to NF-jB activation without inducing DNA damage. Our study helps to conceptualize how individual or a combination of different stress conditions may funnel into this previously unappreciated signal transduction mechanism to regulate the activity of the ubiquitous NF-jB transcription factor.

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Section: Electroporation Of Restriction Enzymes Cause Ikk and Nf-kb Asupporting

confidence: 92%

Section: Electroporation Of Restriction Enzymes Cause Ikk and Nf-kb Amentioning

confidence: 80%

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NF-κB activation by combinations of NEMO SUMOylation and ATM activation stresses in the absence of DNA damage

et al. 2006

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“…If considerable rejoining and capping occur before all restitution or breakage has finished, then the pool of loose ends does not build up as much at large concentrations compared with small concentrations . This would be easy to imagine when cells are treated with restriction enzymes, which may be active in cells for prolonged periods of time (Costa and Bryant 1990, Ager et al 1991 . Now, by the way the data are being analysed, this leads to a smaller estimate for ),*/K* and to a less drastic discrepancy in Figure 5 at small concentrations .…”

Section: Discussionmentioning

confidence: 99%

Modelling the Formation of Polycentric Chromosome Aberrations

Sachs

Yates

Tarver

et al. 1992

International Journal of Radiation Biology

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Exchange-type chromosome aberrations produced by ionizing radiation or restriction enzymes are believed to result from pairwise interaction of DNA double-strand breaks (dsb). In addition to dicentrics, such aberrations may include higher-order polycentrics (tricentrics, tetracentrics, etc.). We have developed computer programs that calculate the probability of the various polycentrics for a given average number of pairwise interactions. Two models are used. Model I incorporates kinetic competition between restitution, complete exchanges (illegitimate recombination events), and incomplete exchanges. Model II allows unrestituted breaks even if there is no recombination. The models were applied to experimental observations of aberrations produced in G1 Chinese hamster ovary cells after electroporation with the restriction enzyme PvuII, which produces blunt-end dsb. We found, experimentally and theoretically, that there was a maximum in the number and multiplicity of polycentrics per cell: beyond a certain PvuII concentration no additional or higher-order polycentrics were produced. Computer-generated relationships, which were remarkably similar for both models and for all values of the adjustable parameters, were found between dicentrics per cell and higher-order polycentrics per cell. Excellent agreement was found between the experimental observations and the consensus theoretical curve relating tricentrics per cell to dicentrics per cell. The observed number of higher polycentrics per cell for a given number of dicentrics per cell was somewhat larger than the consensus theoretical prediction. The observed number of centric rings per cell was markedly larger than the consensus theoretical value, presumably owing to intrachromosomal localization ('proximity effects'). The computer models also provided estimates for the adjustable parameters; for example, in model I the fraction of incomplete exchanges was found to be about 35%.

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“…By contrast, restriction endonucleases (REs), which can be introduced into mammalian cells by electroporation, cause DSBs at defined chromosomal locations (Bryant 1988, Ager et al . 1991, Obe et al .…”

Section: Introductionmentioning

confidence: 99%

Formation of carcinogenic chromosomal rearrangements in human thyroid cells after induction of double-strand DNA breaks by restriction endonucleases

Evdokimova¹,

Gandhi²,

Rayapureddi³

et al. 2012

Endocrine-Related Cancer

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Ionizing radiation (IR) exposure increases the risk of thyroid cancer and other cancer types. Chromosomal rearrangements, such as RET/PTC, are characteristic features of radiation-associated thyroid cancer and can be induced by radiation in vitro. IR causes double-strand breaks (DSBs), suggesting that such damage leads to RET/PTC, but the rearrangement mechanism has not been established. To study the mechanism, we explored the possibility of inducing RET/PTC by electroporation of restriction endonucleases (REs) into HTori-3 human thyroid cells. We used five REs, which induced DSB in a dose-dependent manner similar to that seen with IR. Although all but one RE caused DSB in one or more of the three genes involved in RET/PTC, rearrangement was detected only in cells electroporated with either PvuII (25 and 100 U) or StuI (100 and 250 U). The predominant rearrangement type was RET/PTC3, which is characteristic of human thyroid cancer arising early after Chernobyl-related radioactive iodine exposure. Both enzymes that produced RET/PTC had restriction sites only in one of the two fusion partner genes. Moreover, the two enzymes that produced RET/PTC had restriction sites present in clusters, which was not the case for RE that failed to induce RET/PTC. In summary, we establish a model of DSB induction by RE and report for the first time the formation of carcinogenic chromosomal rearrangements, predominantly RET/PTC3, as a result of DSB produced by RE. Our data also raise a possibility that RET/PTC rearrangement can be initiated by a complex DSB that is induced in one of the fusion partner genes.

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Analysis of Restriction Enzyme-Induced DNA Double-Strand Breaks in Chinese Hamster Ovary Cells by Pulsed-Field Gel Electrophoresis: Implications for Chromosome Damage

Cited by 31 publications

References 21 publications

NF-κB activation by combinations of NEMO SUMOylation and ATM activation stresses in the absence of DNA damage

NF-κB activation by combinations of NEMO SUMOylation and ATM activation stresses in the absence of DNA damage

Modelling the Formation of Polycentric Chromosome Aberrations

Formation of carcinogenic chromosomal rearrangements in human thyroid cells after induction of double-strand DNA breaks by restriction endonucleases

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