Comet assay as a tool to screen for mouse models with inherited radiation sensitivity

Schindewolf, Catherine; Lobenwein, Kurt; Trinczek, Korinna; Gomolka, Maria; Soewarto, Dian; Fella, Christiane; Pargent, Walter; Singh, Narendra P.; Jung, Thomas; Angelis, Martin Hrabé de

doi:10.1007/s003350010106

Cited by 29 publications

(10 citation statements)

References 8 publications

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“…The DNA damage was analyzed by alkaline single cell gel electrophoresis (comet assay), as previously described, 33 with some modifications. Briefly, an aliquot of 1 3 10 5 cells was suspended in 0.75% low melting point agarose and spread on microscopic slides pre-coated with 0.5% NMP agarose (Sigma).…”

Section: Comet Assaymentioning

confidence: 99%

IRS‐1–Rad51 nuclear interaction sensitizes JCV T‐antigen positive medulloblastoma cells to genotoxic treatment

Trojanek

Croul

et al. 2006

Intl Journal of Cancer

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The large T-antigen from human polyomavirus JC (JCV T-antigen) is suspected to play a role in malignant transformation. Previously, we reported that JCV T-antigen requires the presence of a functional insulin-like growth factor I receptor (IGF-IR) for transformation of fibroblasts and for survival of medulloblastoma cell lines; that IGF-IR is phosphorylated in medulloblastoma biopsies and that JCV T-antigen inhibits homologous recombinationdirected DNA repair, causing accumulation of mutations. Here we are evaluating whether JCV T-antigen positive and negative mouse medulloblastoma cell lines, which significantly differ in their tumorigenic properties, are also different in their abilities to repair double strand breaks of DNA (DSBs). Our results show that despite much stronger tumorigenic potential, JCV T-antigen positive medulloblastoma cells are more sensitive to genotoxic agents (cisplatin and c-irradiation). Subsequent analysis of DNA repair of DSBs indicated that homologous recombination-directed DNA repair (HRR) was selectively attenuated in JCV T-antigen positive medulloblastoma cells. JCV T-antigen did not affect HRR directly. In the presence of JCV T-antigen, insulin receptor substrate 1 (IRS-1) translocated to the nucleus where it co-localized with Rad51, possibly attenuating HRR. ' 2006 Wiley-Liss, Inc.

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Section: Comet Assaymentioning

confidence: 99%

IRS‐1–Rad51 nuclear interaction sensitizes JCV T‐antigen positive medulloblastoma cells to genotoxic treatment

Trojanek

Croul

et al. 2006

Intl Journal of Cancer

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“…A number of methods have been identified for assaying DNA damage, including DNA filter elution, constant field gel electrophoresis, pulsed-field gel electrophoresis and comet assay (2,10,11). Comet assay, also called single cell gel electrophoresis (SCGE), is considered to be a sensitive, fast and convenient technique for detecting DNA damage and repair in a single cell (12,13) and it is, therefore, a prospective radiosensitivity prediction method for clinical use (2). …”

Section: Introductionmentioning

confidence: 99%

Nested PCR for mtDNA-4977-bp deletion and comet assay for DNA damage - a combined method for radiosensitivity evaluation of tumor cells

et al. 2014

Oncology Letters

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To identify an effective method of evaluating the radiosensitivity of human tumor cell lines in vitro, the present study adopted mtDNA-4977-bp deletion coupled with comet assay. The three human tumor cell lines applied were HepG2, EC-9706 and MCF-7. The surviving fraction (SF), ratio of the mtDNA-4977-bp deletion and DNA damage were detected by MTT assay, nested polymerase chain reaction (PCR) technique and comet assay, respectively. Clearly, lower SFs were found for the HepG2 and EC-9706 cells as compared with the MCF-7 cells following irradiation at doses of 2, 4 and 8 Gy, indicating a higher radiosensitivity for the HepG2 and EC-9706 cells. Additionally, no significant differences were identified in the mtDNA-4977-bp deletions found among HepG2, EC-9706 and MCF-7 cells by PCR following 1- or 4-Gy γ-ray irradiation, while increased deletion ratios of mtDNA-4977 bp were observed in HepG2 and EC-9706 cells following 8-Gy irradiation, in contrast to decreases in MCF-7 cells. The most notable differences among these three tumor cell lines were observed by comet assay following 8–Gy γ-ray irradiation. A combined method of nested PCR and comet assay, therefore, is the most effective and accurate method in evaluating the radiosensitivity of tumor cells.

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“…In this assay, a small number of cells are required to evaluate the DNA damage and it is possible to measure the DNA damage in individual cells within the cell population. Therefore, the comet assay seems to be a valuable tool for the detection of radiation-induced DNA damage and the DNA repair capability of cells [34]. In addition, due to the many advantages of this assay, it is expected that the comet assay can be applied as a method for the evaluation of cellular radiosensitivity in the clinical setting.…”

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confidence: 99%

The Relationship between Cellular Radiosensitivity and Radiation-Induced DNA Damage Measured by the Comet Assay

Wada

Kurahayashi

Kobayashi

et al. 2003

The Journal of Veterinary Medical Science

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ABSTRACT. The relationship between deoxyribonucleic acid (DNA) damage and the cell death induced by γ-irradiation was examined in three kinds of cells, Chinese hamster ovary fibroblast CHO-K1, human melanoma HMV-II and mouse leukemia L5178Y. Cell survival was determined by a clonogenic assay. The induction and rejoining of DNA strand breaks induced by radiation were measured by the alkaline and neutral comet assays. L5178Y cells were the most radiosensitive, while CHO-K1 cells and HMV-II cells were radioresistant. There was an inverse relationship between the survival fraction at 2 Gy (SF2) and the yield of initial DNA strand breaks per unit dose under the alkaline condition for the comet assay, and also a relationship between SF2 and the residual DNA strand breaks (for 4 hr after irradiation) under the neutral condition for the comet assay, the latter being generally considered to be relative to cellular radiosensitivity. In the present analysis, it was considered that the alkaline condition for the comet assay was optimal for evaluating the initial DNA strand breaks, while the neutral condition was optimal for evaluating the residual DNA strand breaks. Since the comet assay is simpler and more rapid than other methods for detecting radiation-induced DNA damage, this assay appears to be a useful predictive assay for evaluating cellular clonogenic radiosensitivity of tumor cells. KEY WORDS: comet assay, DNA strand break, γ-irradiation, predictive assay, radiosensitivity.J. Vet. Med. Sci. 65(4): 471-477, 2003 Different types of cell lines show different radiosensitivities. In addition, even a tumor of the same origin and pathology, can show radiosensitivity over quite a wide range [4], and therefore, understanding the radiosensitivity of an individual tumor is of great importance in the clinical situation. Knowledge of the intrinsic radiosensitivities of cells from a tumor biopsy can help produce an optimized radiotherapy protocol for individual patients.Reproductive death measured by a clonogenic assay is an important index for the cell death induced by radiation, and the cellular radiosensitivities of fibroblasts and tumor cells have been evaluated by a clonogenic assay [13,17]. A good correlation was found between the reproductive death of fibroblasts measured by the clonogenic assay and the severity of late normal tissue effects, such as fibrosis and erythema, after radiotherapy [7,14]. However, this clonogenic assay consumes a lot of time, and the cells must be proliferated in vitro [4,14]. Hence this assay is not optimal for routine clinical use, and thus there is now much interest in exploiting a more rapid and simple assay to evaluate cellular radiosensitivity [8,25].It is said that the cell death caused by radiation is due to deoxyribonucleic acid (DNA) damage [29]. DNA double strand breaks (dsbs) are considered to be closely related to cell death because it is suggested that they can lead directly to chromosome aberration and the loss of genetic material [22]. To date, the correlation between radiose...

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Comet assay as a tool to screen for mouse models with inherited radiation sensitivity

Cited by 29 publications

References 8 publications

IRS‐1–Rad51 nuclear interaction sensitizes JCV T‐antigen positive medulloblastoma cells to genotoxic treatment

IRS‐1–Rad51 nuclear interaction sensitizes JCV T‐antigen positive medulloblastoma cells to genotoxic treatment

Nested PCR for mtDNA-4977-bp deletion and comet assay for DNA damage - a combined method for radiosensitivity evaluation of tumor cells

The Relationship between Cellular Radiosensitivity and Radiation-Induced DNA Damage Measured by the Comet Assay

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