DNA Repair-Profiler consists of a unique collection of mammalian cell lines, each with a deficiency in one of the major DNA repair pathways. The cell lines are applied to assess which DNA repair pathways are involved in the repair of DNA damage induced by genotoxic compounds. This unique collection of cell lines can be applied to study the impact of DNA repair on cell viability after exposure to genotoxic substances as well as the induction and repair of DNA damage and induction of gene mutation. The DNA Repair-Profiler assay is ideally suited as a tool to investigate the genotoxic mode-of-action of compounds to support AOP approaches or to further characterise novel oncology treatments.

Key Features

            • Unique collection of mammalian DNA repair-deficient cell lines to investigate the impact of DNA repair on the toxicity of genotoxic substances
            • Gain insight into the type of DNA damage induced by genotoxic compounds
            • Assess induction of DNA damage or mutations by compounds in presence and absence of DNA repair

DNA Repair-Profiler consists of 7 mammalian cell lines, each with a deficiency in one of the major DNA repair pathways. The cell lines are deficient in base excision repair (BER, nucleotide excision repair (NER), homologous recombination (HR), non-homologous end-joining (NHEJ), mismatch repair (MMR) and translesion synthesis (TLS). Together, these repair pathways cover the repair of base damages (BER), bulky DNA adducts (NER), DNA double-strand breaks (HR, NHEJ), DNA mismatches (MMR) and the bypass of DNA replication-blocking lesions (TLS). The cell lines can be applied to assess which DNA repair pathways are involved in the repair of DNA damage induced by genotoxic compounds. These repair deficient cell lines can provide insight into the genotoxic mode-of-action. The DNA Repair-Profiler assay is ideally suited as a tool to further investigate the mechanisms of genotoxicity as part of an AOP approach.

 

DNA Repair-Profiler cell lines can be applied to assess which DNA repair pathways are involved in repair of DNA damage induced by genotoxic compounds

Highlights of DNA Repair-Profiler

  • Investigate the cytotoxicity of genotoxic compounds in the presence and absence of the major DNA repair pathways
  • Investigate induction and repair of DNA damage in the presence and absence of DNA repair
  • Investigate the impact of the different DNA repair pathways to protect against the mutagenic effect of genotoxic compounds

Typical applications of DNA Repair-Profiler

  • Investigation into the types of DNA damage that are induced by genotoxic compounds
  • Characterisation of novel oncology drugs with a genotoxic mode-of-action and exploration of the susceptibility of tumors cells for an oncology drug

DNA Repair-Profiler experimental design

The sensitivity of DNA repair-proficient and the various DNA repair-deficient cell lines to genotoxic compounds is determined using the clonogenic survival assay. First, a broad dose range finding is performed in DNA repair-proficient Chinese hamster ovary cells (CHO), mouse embryonic fibroblasts (MEF) and mouse embryonic stem cells (mES) using the Alamar Blue cell viability staining.

DNA Repair-Profiler protocol concentration range finding
DNA repair profiling

Next, the cytotoxicity of genotoxic compounds will be investigated using a clonogenic survival assay in DNA repair-proficient and the panel of DNA repair-deficient cell lines. Typically, three compound concentrations that induce 10%, 25% and 50% cytotoxicity in DNA repair proficient cells are applied. The cells are seeded at clonal density and exposed to the compounds for 24h. After 10 days of culture in the absence of the compound to allow the formation of cell colonies, the colonies are stained with methylene blue and counted. Cell survival is expressed as the number of cell clones from exposed cultures relative to unexposed cultures. The impact of DNA repair deficiency on the cytotoxicity of a genotoxic compound is determined.

DNA Repair-Profiler protocol survival assay and data analysis
DNA Repair-Profiler extensions: Assessment of DNA damage induction and induction of gene mutation in absence of DNA repair

 The investigate the induction and repair of DNA damage, DNA strand breaks can be visualised using the comet assay. DNA strand breaks can be caused directly by a genotoxic substance or might be the result of DNA repair intermediates. The DNA Repair-Profiler panel of cell lines can be used to assess the induction of DNA strand breaks as well as their repair.

Unrepaired DNA lesions can ultimately lead to gene mutations. Mutagenesis following exposure to genotoxic compounds can be studied in the presence and absence of DNA repair using the HPRT mutation assay.  DNA Repair-Profiler can be applied to explore the impact of DNA repair on the induction of gene mutation.

Typical output generated from the DNA Repair-Profiler assay, depending on the selection of assays, can include:

  • Cytotoxicity of genotoxic substances in presence and absence of DNA repair from the clonogenic survival assay
  • DNA damage induction in presence and absence of DNA repair from the Comet assay
  • Mutation induction in presence and absence of DNA repair following exposure to genotoxic substances from the Hprt mutation assay

DNA repair and cytotoxicity

The DNA Repair-Profiler cell panel can be applied to investigate the cytotoxicity of genotoxic compounds in the presence and absence of specific DNA repair systems. Cytotoxicity is determined by using the clonogenic survival assay. Identification of the DNA repair pathway that protects cells against the cytotoxicity of a genotoxic compound can provide insight into the type of DNA damage that is induced by the compound.

The cytotoxicity of MMS and Cisplatin is significantly increased in NER-deficient cells and HR-deficient cells in a survival assay

 

The cytotoxicity of MMS and Cisplatin is significantly increased in NER-deficient cells and HR-deficient cells.

DNA damage induction

The induction and repair of DNA damage can be analysed using the comet assay. The induction and repair of DNA damage can be analysed in the presence and absence of specific DNA repair pathways. The Comet assay can be modified by applying specific enzymes that recognize and excise the DNA lesions. This combination will provide a further understanding of the type of DNA damage that is induced by the genotoxic compound.

Exposure to KBrO3 leads to an increase in tail DNA percentage that is higher in the BER-deficient cells with a comet assay

 

In the example above, exposure to KBrO3 leads to an increase in tail DNA percentage that is higher in the BER-deficient cells.

Mutagenesis

Unrepaired DNA can ultimately lead to gene mutations. The DNA Repair-Profiler cell panel can be applied to investigate the impact of the different DNA repair pathways in protection against the mutagenic effect of genotoxic compounds. Mutation induction is measured using the Hprt assay and is plotted as the absolute mutant frequency.

Exposure of repair-proficient cells to ENU leads to an increase in the absolute mutant frequency that is higher in the NER-deficient cells with a HPRT mutation assay.
In the example above, exposure of repair-proficient cells to ENU leads to an increase in the absolute mutant frequency that is higher in the NER-deficient cells.

Service

If you are interested in testing compounds in the DNA Repair-Profiler assay you will need to indicate which cell lines you would like to include and which analysis should be performed. Based on this selection a unique study proposal and quote will be drafted. Are you interested in receiving a quote or do you have any questions? Please reach out!

Photos of our sales team, whom you can reach out to receive a quote or with any questions.

Practical information

Compound requirement

The amount of compound that is required for analysis in the DNA RepairProfiler assay depends on the maximum concentration that will be applied in the project.

 

Turn around time

  • 3-6 weeks, depending on the selection of assays

Type of solvents compatible with the assay

  • DMSO
  • PBS
  • Water
  • Other solvents can be discussed with our Study Director

A standard DNA Repair-Profiler report includes

  • Cytotoxicity testing/dose range finding results
  • Biomarker expression with test compounds

DNA Repair-Profiler is available as a service from Toxys or via scientist.com. Please click here to request for a dummy report.

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