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The TdT-mediated dUTP nick end labeling assay precisely assesses the DNA damage in human tumor xenografts
H Kikuchi1, S Ujiie, R Kanamaru
1Division of Pharmacotherapy, Miyagi Cancer Center, Natori.
Abstract:
Cultured HL-60, HeLa S3 and WiDr cells grown in male BALB/c nu/nu mice were studied by conventional and field-inversion DNA gel electrophoresis (FIGE), as well as by means of cytomorphological approaches, including TdT-mediated dUTP nick end labeling (TUNEL) assay. Chemosensitivity tests revealed HL-60 to be sensitive to vindesine (VDS), and HeLa S3 and WiDr to mitomycin C (MMC). Although VDS-treated HL-60 exhibited condensation of chromatin and a DNA ladder, MMC-exposed HL-60 cells showed apoptotic figures without typical DNA ladders. With MMC-treated WiDr cells, neither DNA ladders nor apoptotic figures were observed. Cells characterized by chromatin condensation were TUNEL-positive in both treated and untreated cases with the exception of the MMC-treated WiDr case, in which many TUNEL-positive cells were observed without cytomorphological changes. On FIGE, DNA fragments of approximately 50, 300 and 400 kbp were detected in groups treated with both effective and ineffective drugs, as well as in untreated controls. Furthermore, change of the time parameters in FIGE resulted in different sizes (550 and 850 kbp) of DNA fragments. These findings indicate that i) cell death is not always detectable in terms of apoptotic figures or DNA oligonucleosomal fragmentation, ii) only the TUNEL assay is a reliable tool to detect DNA damage and, iii) FIGE does not provide accurate size profiles of macromolecular DNA fragments.
Insights
The TdT-mediated dUTP nick end labeling (TUNEL) assay reliably detects DNA damage in cancer cells, even when apoptotic figures or DNA ladders are absent. Field-inversion DNA gel electrophoresis (FIGE) is less accurate for sizing DNA fragments.
Area of Science:
- Cell biology
- Molecular biology
- Cancer research
Background:
- Assessing drug-induced cell death in cancer models is crucial for therapeutic development.
- Conventional methods for detecting DNA damage, such as DNA laddering and apoptotic morphology, may not always be sensitive or accurate.
- Field-inversion DNA gel electrophoresis (FIGE) is a technique used to analyze large DNA fragments.
Purpose of the Study:
- To evaluate the reliability of different methods for detecting DNA damage and cell death in cultured cancer cells treated with chemotherapeutic agents.
- To compare the efficacy of TdT-mediated dUTP nick end labeling (TUNEL) assay, cytomorphology, and FIGE in identifying drug-induced cellular changes.
Main Methods:
- Cultured HL-60, HeLa S3, and WiDr cells were grown in immunocompromised mice.
- Cells were treated with vindesine (VDS) or mitomycin C (MMC).
- Conventional and field-inversion DNA gel electrophoresis (FIGE), cytomorphological analysis, and TUNEL assay were performed.
Main Results:
- VDS-treated HL-60 cells showed chromatin condensation and DNA ladders. MMC-treated HL-60 cells displayed apoptotic figures without DNA ladders. MMC-treated WiDr cells showed neither.
- TUNEL assay detected DNA damage in cells with chromatin condensation, including cases lacking typical apoptotic features or DNA ladders.
- FIGE detected DNA fragments but did not provide accurate size profiles, with fragment sizes varying based on parameter adjustments.
Conclusions:
- Cell death and DNA damage are not always accompanied by observable apoptotic figures or DNA oligonucleosomal fragmentation.
- The TUNEL assay is a more reliable method for detecting DNA damage compared to cytomorphology and DNA laddering.
- FIGE is not suitable for accurately determining the size of macromolecular DNA fragments in this context.