High-throughput analysis of DNA interstrand crosslinks in human peripheral blood mononuclear cells by automated reverse FADU assay

Debiak, Malgorzata; Panas, Alicja; Steinritz, Dirk; Kehe, Kai; Bürkle, Alexander

High-throughput analysis of DNA interstrand crosslinks in human peripheral blood mononuclear cells by automated reverse FADU assay

Dateien

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Datum

2011

Autor:innen

Debiak, Malgorzata

Panas, Alicja

Steinritz, Dirk

Kehe, Kai

Bürkle, Alexander

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

Toxicology. 2011, 280(1-2), pp. 53-60. ISSN 0300-483X. eISSN 1879-3185. Available under: doi: 10.1016/j.tox.2010.11.007

Zusammenfassung

DNA interstrand crosslinks (ICL) are induced both by several cytotoxic anti-cancer drugs as well as by the chemical warfare agent sulphur mustard (SM). Although measurement of ICL formation could be used in risk assessment or provide valuable predictive information on the response of malignant cells to crosslinking chemotherapeutic agents, respectively, it is currently not applied due to lack of appropriate standardized methodology. Here we describe a fast and convenient procedure for detection of ICL in human peripheral blood mononuclear cells (PBMC) as high-throughput method, termed ‘reverse FADU assay’. This assay detects ICL based on the prevention of time-dependent alkaline unwinding of double-stranded DNA in a cell lysate that starts from single or double strand breaks. We have successfully established and optimized the reverse FADU assay by using human PBMC exposed to the model compounds mitomycin C, melphalan and SM. Our fully automated assay version is faster than currently used methods and possesses similar sensitivity. It operates in a 96-well format, thus allowing parallel analysis of multiple samples. Furthermore, we describe optimized protocols for sample preparation, with sample volume minimized to 100 μl of blood, storage and shipment conditions. We conclude that the reverse FADU assay is an attractive candidate method for monitoring DNA damage induced by DNA crosslinking agents.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Schlagwörter

DNA interstrand crosslinks, FADU assay, comet assay, sulphur mustard, melphalan

Zitieren

ISO 690

DEBIAK, Malgorzata, Alicja PANAS, Dirk STEINRITZ, Kai KEHE, Alexander BÜRKLE, 2011. High-throughput analysis of DNA interstrand crosslinks in human peripheral blood mononuclear cells by automated reverse FADU assay. In: Toxicology. 2011, 280(1-2), pp. 53-60. ISSN 0300-483X. eISSN 1879-3185. Available under: doi: 10.1016/j.tox.2010.11.007

BibTex

@article{Debiak2011-02-04Hight-15140,
  year={2011},
  doi={10.1016/j.tox.2010.11.007},
  title={High-throughput analysis of DNA interstrand crosslinks in human peripheral blood mononuclear cells by automated reverse FADU assay},
  number={1-2},
  volume={280},
  issn={0300-483X},
  journal={Toxicology},
  pages={53--60},
  author={Debiak, Malgorzata and Panas, Alicja and Steinritz, Dirk and Kehe, Kai and Bürkle, Alexander}
}

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Universitätsbibliographie

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