| 1. |
- Mathew, Sherin T, et al.
(författare)
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A flow cytometry assay that measures cellular sensitivity to DNA-damaging agents, customized for clinical routine laboratories.
- 2016
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Ingår i: Clinical biochemistry. - : Elsevier BV. - 1873-2933 .- 0009-9120. ; 49:7-8, s. 566-572
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Tidskriftsartikel (refereegranskat)abstract
- The clonogenic assay examines cell sensitivity to toxic agents and has been shown to correlate with normal tissue sensitivity to radiotherapy in cancer patients. The clonogenic assay is not clinically applicable due to its intra-individual variability and the time frame of the protocol. We aimed to develop a clinically applicable assay that correlated with the clonogenic assay.
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| 2. |
- Mathew, Sherin T
(författare)
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Measurement of sensitivity to DNA damaging agents
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- There is a large inter-individual variation in intrinsic sensitivity in patients receiving treatment with DNA damaging agents. Cancer therapy exemplifies this problem where patients experience varying degree of normal tissue side effects in response to radiation or chemotherapy. For this reason, it is necessary to develop an assay to predict sensitivity of a patient prior to treatment with DNA damaging agents. This may allow more individualized treatment and improve the therapeutic index. In paper I and II we focused on developing and validating a flow cytometry - based cell division assay (CD) that uses the thymidine analogue EdU (5-ethynyl-2’-deoxyuridine) to measure the proliferative ability after DNA damaging treatment. In paper I, the CD assay measured sensitivity to radiation of human skin fibroblasts with a correlation similar to the standard clonogenic survival assay in a relatively short time frame. Using the easily sampled peripheral blood lymphocytes, the CD assay found variation in intrinsic sensitivity to radiation and detected increased sensitivity in patients with DNA repair defects. In paper II, the CD assay was further validated for measurement of cell sensitivity to DNA damaging drugs. The results indicated that the assay can be used to identify sensitive patients. Exposure to ionizing radiation generates free radicals that carry out most part of the toxic effects. The cellular antioxidant system regulated by the Nrf2 transcription factor plays a key role in protecting cells against radical induced damage; hence in paper III we have investigated if pretreating cells with Nrf2 activators influence the sensitivity to radiation. Results from paper III demonstrated that repeated treatment using the isothiocyanate sulforaphane protected human skin fibroblasts from toxic effects of ionizing radiation in an Nrf2-dependent manner. In paper IV we found that repeated pretreatment of cells with Nrf2 activators, sulforaphane or synthetic triterpenoid bardoxolone methyl trained the cells to acquire resistance against higher toxic concentrations of both drugs. Together these results indicate that repeated stimulation of Nrf2 system can enhance cytoprotection and that adaptation to stress may be a general feature of the Nrf2 response mechanism.
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| 3. |
- Mathew, Sherin T, et al.
(författare)
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Repeated Nrf2 stimulation using sulforaphane protects fibroblasts from ionizing radiation
- 2014
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Ingår i: Toxicology and Applied Pharmacology. - : Elsevier BV. - 0041-008X. ; 276:3, s. 188-194
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Tidskriftsartikel (refereegranskat)abstract
- Most of the cytotoxicity induced by ionizing radiation is mediated by radical-induced DNA double-strand breaks. Cellular protection from free radicals can be stimulated several fold by sulforaphane-mediated activation of the transcription factor Nrf2 that regulates more than 50 genes involved in the detoxification of reactive substances and radicals. Here, we report that repeated sulforaphane treatment increases radioresistance in primary human skin fibroblasts. Cells were either treated with sulforaphane for four hours once or with four-hour treatments repeatedly for three consecutive days prior to radiation exposure. Fibroblasts exposed to repeated-sulforaphane treatment showed a more pronounced dose-dependent induction of Nrf2-regulated mRNA and reduced amount of radiation-induced free radicals compared with cells treated once with sulforaphane. In addition, radiation- induced DNA double-strand breaks measured by gamma-H2AX foci were attenuated following repeated sulforaphane treatment. As a result, cellular protection from ionizing radiation measured by the 5-ethynyl-2'-deoxyuridine (EdU) assay was increased, specifically in cells exposed to repeated sulforaphane treatment. Sulforaphane treatment was unable to protect Nrf2 knockout mouse embryonic fibroblasts, indicating that the sulforaphane-induced radioprotection was Nrf2-dependent. Moreover, radioprotection by repeated sulforaphane treatment was dose-dependent with an optimal effect at 10 uM, whereas both lower and higher concentrations resulted in lower levels of radioprotection. Our data indicate that the Nrf2 system can be trained to provide further protection from radical damage. (c) 2014 Elsevier Inc. All rights reserved.
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| 4. |
- Viktorisson, Adam, et al.
(författare)
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A control for the day-to-day normalization of the flow cytometry γ-H2AX assay for clinical routine.
- 2018
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Ingår i: Cytometry. Part B, Clinical cytometry. - : Wiley. - 1552-4957 .- 1552-4949. ; 94:6, s. 946-949
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Tidskriftsartikel (refereegranskat)abstract
- The phosphorylation of histone H2AX (γ-H2AX) at the DNA double-strand break (DSB) site is frequently used for quantifying DSBs and may be useful as a biomarker for clinical applications. We have previously reported a flow cytometry-based quantification of γ-H2AX for clinical routine. One major challenge, however, is the lack of a control sample for normalization of the day-to-day variation of the flow cytometry γ-H2AX assay.Here, we report development of a mix-control sample containing peripheral blood mononuclear cells (PBMC) from 10 control individuals, for normalization of day-to-day variation of the flow cytometry-γ-H2AX assay.We showed that control individuals sampled on different days show an average day-to-day variation (CV) of 34%, which was reduced to 12% after normalization to the control sample. The normalization allowed detection of radiosensitivity of lymphoblastoid cell lines from ataxia telangiectasia patients, sampled over three days.The mix-control sample, consisting of 10 control individuals' PBMC, can be used as a control sample to normalize for day-to-day variation of the γ-H2AX assay. The use of this sample will facilitate integration of the γ-H2AX assay into clinical routine. © 2018 International Clinical Cytometry Society.
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