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Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors
1Biochemical and Molecular Pharmacology Section, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Abstract:
Previous studies with the multidrug-resistant human HL60 cell line have shown a 3-4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative. Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells. Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells. This decrease in DNA cleavage activity in the presence of VP-16 was, in part, related to a 2-3-fold decrease in both the amount and activity of topoisomerase II in the resistant cell line compared to the sensitive cells. Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei. Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells. Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei. In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line. These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16. Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.
Insights
Multidrug-resistant cells show decreased DNA cleavage by etoposide (VP-16), linked to lower topoisomerase II levels. Cytosolic factors in sensitive cells enhance VP-16 DNA damage, suggesting a novel resistance mechanism.
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) in cancer cells complicates treatment.
- Etoposide (VP-16) is a topoisomerase II inhibitor used in chemotherapy.
- HL60 cell line exhibits significant resistance to VP-16, suggesting multiple resistance mechanisms.
Purpose of the Study:
- To investigate the role of topoisomerase II activity and DNA cleavage in VP-16 resistance in HL60 cells.
- To compare VP-16-induced DNA damage in drug-sensitive and multidrug-resistant HL60 cells.
- To explore the influence of cellular fractions on VP-16's DNA-damaging effects.
Main Methods:
- Assessed VP-16-dependent DNA strand break formation in sensitive and resistant HL60 cells.
- Quantified topoisomerase II amount and activity in both cell lines.
- Evaluated VP-16-induced DNA cleavage in isolated nuclei with and without cytosolic proteins.
Main Results:
- Resistant HL60 cells showed significantly reduced VP-16-dependent DNA cleavage compared to sensitive cells.
- A 2-3 fold decrease in topoisomerase II amount and activity was observed in resistant cells.
- Cytosolic proteins from sensitive cells enhanced VP-16-induced DNA cleavage in nuclei, unlike those from resistant cells.
Conclusions:
- Reduced topoisomerase II levels and activity in resistant HL60 cells contribute to etoposide resistance.
- A cytosolic factor present in sensitive cells, but absent in resistant cells, is crucial for maximal VP-16-induced DNA damage.