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Updated: Sep 21, 2026
Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method
Published on: December 16, 2014
[In situ nick translation for detection of DNA damages in glioma cells]
1Department of Neurosurgery, Osaka University, Japan.
Abstract:
DNA damaging agents such as nitrosoureas are widely used for the treatment of malignant gliomas. Therefore, quantitative measurement of DNA damages induced by antineoplastic drugs is useful to judge the efficacy of the drug and understand the pharmacological action of the drug. We have utilized in situ nick translation method to demonstrate "nicks" in DNA of glioma cells treated by various antineoplastic agents. Exponentially growing rat 9 L glioma cells (4 x 10(4] were seeded in the chamber slide. After fourty eight hours, the medium was changed to that containing various concentration of the drug (ACNU, cis-DDP, BLM, ADM and VP-16) and the cell was treated for 1 hour. Then, the cell was fixed for 10 minutes in methanol-acetic acid (v/v 3:1). Following fixation, the cell was incubated in the nick translation mixture containing E. coli DNA polymerase I, 3H-TTP, and 4 dNTP's (ATP, GTP, CTP, CTP and TTP) for 10 minutes at room temperature. The slide was dipped in the autoradiographic emulsion, exposed for 4 days at 4 degrees C, and then developed, the number of the silver grains over nuclei was counted under the microscope. For comparison of the effect of the drug to glioma cells, IC50 (inhibitory concentration of the drug for 50% cell kill) of each drug was determined by treating the cell for 48 hours at the various concentration of the drug. Small number of the silver grains was noted in cells with no treatment. Over IC50 as the concentration of the drug increased, the number of the nick increased in cells treated with bleomycin or adriamycin which are known to produce single strand breaks in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
This study quantifies DNA damage in glioma cells treated with antineoplastic drugs using in situ nick translation. Increased DNA nicks correlate with drug concentration for agents like bleomycin, aiding in evaluating drug efficacy.
Area of Science:
- Molecular Biology
- Cancer Research
- Pharmacology
Context:
- Malignant gliomas are treated with DNA damaging agents like nitrosoureas.
- Quantitative measurement of drug-induced DNA damage is crucial for assessing therapeutic efficacy and understanding drug mechanisms.
Purpose:
- To utilize the in situ nick translation method to detect DNA nicks in glioma cells exposed to various antineoplastic agents.
- To correlate DNA damage levels with drug concentrations and determine drug efficacy.
Summary:
- Rat 9L glioma cells were treated with ACNU, cis-DDP, BLM, ADM, and VP-16 at various concentrations.
- DNA damage was quantified by counting silver grains via autoradiography after in situ nick translation.
- A dose-dependent increase in DNA nicks was observed for bleomycin and adriamycin above their IC50 values.
Impact:
- The in situ nick translation method provides a quantitative measure of DNA damage induced by antineoplastic drugs.
- This technique can aid in evaluating the efficacy of drugs used in malignant glioma treatment.
- Understanding drug-induced DNA damage contributes to the pharmacological understanding of antineoplastic agents.
