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Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay
Published on: December 19, 2012
Tirapazamine-induced DNA damage measured using the comet assay correlates with cytotoxicity towards hypoxic tumour
B G Siim1, P L van Zijl, J M Brown
1Department of Radiation Oncology, Stanford University School of Medicine, CA 94305-5468, USA.
Abstract:
Tirapazamine (SR 4233), a bioreductive drug selectively toxic towards hypoxic cells, is presently in phase II clinical trials. Since it would not be expected that all tumours would respond equally to the drug, we are exploring ways of predicting the response of individual tumours. In this study we have tested whether the comet assay, which measures DNA damage in individual cells, can provide a simple, surrogate end point for cell killing by tirapazamine. We examined the relationship between the cytotoxicity of tirapazamine under hypoxic conditions and tirapazamine-induced DNA strand breaks in murine (SCCVII, EMT6, RIF-1) and human (HT1080, A549, HT29) tumour cell lines. These results were compared with the relationship between tirapazamine cytotoxicity and another measure of the ability of cells to metabolise tirapazamine; high-performance liquid chromatography (HPLC) analysis of tirapazamine loss or formation of the two electron reduction product SR 4317. The correlation between the hypoxic cytotoxic potency of tirapazamine and DNA damage was highly significant (r = 0.905, P = 0.013). A similar correlation was observed for hypoxic potency and tirapazamine loss (r = 0.812, P = 0.050), while the correlation between hypoxic potency and SR 4317 formation was not significant (r = 0.634, P = 0.171). The hypoxic cytotoxicity of tirapazamine in vitro can therefore be predicted by measuring tirapazamine-induced DNA damage using the comet assay. This approach holds promise for predicting the response of individual tumours to tirapazamine in the clinic.
Insights
The comet assay can predict how well tumors will respond to tirapazamine, a hypoxia-targeting drug. Measuring DNA damage using the comet assay shows a strong correlation with tirapazamine
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Tirapazamine (SR 4233) is a bioreductive drug effective against hypoxic tumor cells.
- Predicting individual tumor response to tirapazamine is crucial for clinical application.
- The comet assay measures DNA damage, potentially serving as a surrogate endpoint for drug efficacy.
Purpose of the Study:
- To evaluate the comet assay's ability to predict tirapazamine's hypoxic cytotoxicity.
- To correlate tirapazamine-induced DNA damage with its cytotoxic effects in various tumor cell lines.
- To compare the comet assay with HPLC analysis of tirapazamine metabolism for predicting drug response.
Main Methods:
- Assessed tirapazamine cytotoxicity under hypoxic conditions in murine and human tumor cell lines.
- Measured tirapazamine-induced DNA strand breaks using the comet assay.
- Analyzed tirapazamine loss and SR 4317 formation via HPLC for comparison.
Main Results:
- A highly significant correlation was found between hypoxic cytotoxicity and tirapazamine-induced DNA damage (r = 0.905, P = 0.013).
- A significant correlation was also observed between hypoxic potency and tirapazamine loss (r = 0.812, P = 0.050).
- Correlation between hypoxic potency and SR 4317 formation was not significant (r = 0.634, P = 0.171).
Conclusions:
- The comet assay provides a reliable method for predicting tirapazamine's hypoxic cytotoxicity.
- Measuring DNA damage with the comet assay is a promising approach for predicting individual tumor response to tirapazamine in clinical settings.
