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Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage
O S Frankfurt1, D Seckinger, E V Sugarbaker
1Oncology Laboratory, Cedars Medical Center, Miami, Florida 33136.
Abstract:
DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L-phenylalanine mustard (L-PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7-26 which detects single-stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single-strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per micrograms L-PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L-PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2-5% resistant cells was detected among sensitive cells as a separate cell subset. We conclude that staining with MOAB F7-26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.
Insights
Flow cytometry using monoclonal antibody F7-26 can detect DNA damage from alkylating agents. This method distinguishes sensitive from resistant cancer cells, aiding in monitoring drug resistance.
Area of Science:
- Molecular Biology
- Cancer Research
- Biotechnology
Background:
- Alkylating agents are crucial in cancer chemotherapy.
- Monitoring cellular sensitivity and resistance to these agents is vital for effective treatment.
- Existing methods for assessing drug resistance can be complex and time-consuming.
Purpose of the Study:
- To evaluate the utility of monoclonal antibody (MOAB) F7-26 for detecting DNA damage induced by alkylating agents.
- To assess the capability of MOAB F7-26 in differentiating between drug-sensitive and drug-resistant cancer cells.
- To explore the potential of flow cytometric analysis with MOAB F7-26 for identifying minor subsets of resistant cells in heterogeneous populations.
Main Methods:
- Human ovarian carcinoma cell line (A2780) and a drug-resistant subline were treated with L-phenylalanine mustard (L-PAM).
- Cells were fixed and stained with MOAB F7-26, which targets single-stranded regions in alkylated DNA.
- Flow cytometry was used to measure mean fluorescent intensity, with heating applied to enhance single-strandedness detection.
Main Results:
- Significantly higher MOAB binding was observed in drug-sensitive cells compared to resistant cells.
- Fluorescence intensity increased substantially more in sensitive cells (80 channels/µg L-PAM) than in resistant cells (17 channels/µg L-PAM).
- Flow cytometry successfully separated sensitive and resistant cell populations, with a detection limit of 2-5% resistant cells in mixed populations.
Conclusions:
- Staining with MOAB F7-26 serves as a reliable indicator of cellular sensitivity or resistance to alkylating agents.
- Flow cytometric analysis using MOAB F7-26 can effectively detect and quantify drug-resistant cell subsets within heterogeneous cell populations.
- This method holds promise for monitoring the emergence of drug resistance during cancer therapy.