A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses

Hasegawa, Mayu; Iwai, Shigenori; Kuraoka, Isao

doi:10.1016/j.mrgentox.2010.04.002

Cited by 7 publications

(11 citation statements)

References 22 publications

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“…2B shows that EU signal was observed in nucleoli of nonirradiated cells but not in UV-irradiated cells, indicating that RNA synthesis is inhibited in response to UV damage. Results of the click reaction were consistent with our previous results (Hasegawa et al, 2010), which show that BrU nuclei staining disappears in response to UV irradiation under our experimental conditions. It is worth noting for routine bioassay that the duration of the click reaction is shorter than that of the antigen-antibody reaction, because no second antibody reaction is needed.…”

Section: Resultssupporting

confidence: 92%

An RNA synthesis inhibition assay for detecting toxic substances using click chemistry

2014

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-Biological risk assessment studies of chemical substances that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication, but also with transcription. There is no simple method for the detection of the DNA lesion-induced inhibition of transcription. Here, we report an assay for estimating the toxicity of chemical substances by visualizing transcription in mammalian cells using nucleotide analog 5-ethynyluridine (EU) and its click chemistry reaction. Ultraviolet light and representative chemical substances (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) of DNAdamaging agents show toxicity, as indicated by RNA synthesis inhibition in response to DNA damage in HeLa cells. Using titanium dioxide, we observed RNA synthesis inhibition in response to the rutile form, but not the anatase form, indicating that rutile titanium dioxide is a toxic substance. Because this method is based on the transcriptional response to DNA lesions, we can use terminally differentiated neuronlike PC12 cells, the differentiation of which can be induced by nerve growth factors, for evaluating chemical substances. Ultraviolet light and some chemicals (camptothecin, 4-nitroquinoline-1-oxide, mitomycin C, and cisplatin, but not etoposide) inhibited RNA synthesis in non-differentiated PC12 cells. Conversely, camptothecin and cisplatin did not inhibit RNA synthesis in differentiated PC12 cells, but 4-nitroquinoline-1-oxide, mitomycin C, and etoposide did. And using titanium dioxide, we did not observed any RNA synthesis inhibition. These data suggest that this method might be used to estimate the potential risk of chemical substances in differentiated mammalian cells, which are the most common cell type found in the human body.

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Section: Resultssupporting

confidence: 92%

An RNA synthesis inhibition assay for detecting toxic substances using click chemistry

2014

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“…In addition, we have previously shown similar results using NER-and TC-NER-deficient cells (Hasegawa et al, 2010). These results suggest that our technique can use various types of cells, for example, mouse embryonic cells, non-divid- ing cells, and stem cells.…”

Section: Discussionsupporting

confidence: 76%

“…Previously, we demonstrated that our simple and rapid method using the visualization of transcription in a mammalian cell system is able to detect the recovery of RNA synthesis after the repair of UV-induced DNA lesions, which involves a step of RNA synthesis inhibition during TC-NER (Hasegawa et al, 2010). The experimental design is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

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A method for detecting genetic toxicity using the RNA synthesis response to DNA damage

2011

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-To date, biological risk assessment studies of chemicals that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication but also with transcription. Therefore, detecting the damaging effects of DNA lesions during transcription might be important for estimating the safety of chemical mutagens and carcinogens. However, methods to address these effects have not been developed. Here, we report a simple, non-isotopic method for determining the toxicity of chemical agents by visualizing transcription in a mammalian cell system. The method is based on the measurement of the incorporation of bromouridine (as the uridine analogue) into the nascent RNA during RNA synthesis inhibition (RSI) induced by the stalling of RNA polymerases at DNA lesions on the transcribed DNA strand, which triggers transcriptioncoupled nucleotide excision repair (TC-NER). When we tested chemical agents (camptothecin, etoposide, 4-nitroquinoline-1-oxide, mitomycin C, methyl methanesulfonate, and cisplatin) in HeLa cells by the method, RSI indicative of genomic toxicity was observed in the nucleoli of the tested cells. This procedure provides the following advantages: 1) it uses common, affordable mammalian cells (HeLa cells, WI38VA13 cells, human dermal fibroblasts, or Chinese hamster ovary cells) rather than genetically modified microorganisms; 2) it can be completed within approximately 8 hr after the cells are prepared because RNA polymerase responses during TC-NER are faster than other DNA damage responses (replication, recombination, and apoptosis); and 3) it is safe because it uses non-radioactive bromouridine and antibodies to detect RNA synthesis on undamaged transcribed DNA strands.

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“…RRS was generally thought to correspond to TC-NER, until studies by Mullenders and colleagues demonstrated that defective RRS actually represents a defect in transcription initiation after UV [135]. Interestingly, a recent study [136] observed that the RRS after UV is primarily nucleolar RNA synthesis, consistent with transcription by RNA polymerase I.…”

Section: A New Look At An Old Hypothesis: Cs As a Rnapi And Rnapiimentioning

confidence: 99%

Blinded by the UV light: How the focus on transcription-coupled NER has distracted from understanding the mechanisms of Cockayne syndrome neurologic disease

Brooks

2013

DNA Repair

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Cockayne syndrome (CS) is a devastating neurodevelopmental disorder, with growth abnormalities, progeriod features, and sun sensitivity. CS is typically considered to be a DNA repair disorder, since cells from CS patients have a defect in transcription-coupled nucleotide excision repair (TC-NER). However, cells from UV-sensitive syndrome patients also lack TC-NER, but these patients do not suffer from the neurologic and other abnormalities that CS patients do. Also, the neurologic abnormalities that affect CS patients (CS neurologic disease) are qualitatively different from those seen in NER-deficient XP patients. Therefore, the TC-NER defect explains the sun sensitive phenotype common to both CS and UVsS, but cannot explain CS neurologic disease. However, as CS neurologic disease is of much greater clinical significance than the sun sensitivity, there is a pressing need to understand its molecular basis. While there is evidence for defective repair of oxidative DNA damage and mitochondrial abnormalities in CS cells, here I propose that the defects in transcription by both RNA polymerases I and II that have been documented in CS cells provide a better explanation for many of the severe growth and neurodevelopmental defects in CS patients than defective DNA repair. The implications of these ideas for interpreting results from mouse models of CS, and for the development of treatments and therapies for CS patients are discussed.

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A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses

Cited by 7 publications

References 22 publications

An RNA synthesis inhibition assay for detecting toxic substances using click chemistry

An RNA synthesis inhibition assay for detecting toxic substances using click chemistry

A method for detecting genetic toxicity using the RNA synthesis response to DNA damage

Blinded by the UV light: How the focus on transcription-coupled NER has distracted from understanding the mechanisms of Cockayne syndrome neurologic disease

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