Frankfurt O S, Seckinger D, Sugarbaker E V
Oncology Laboratory, Cedars Medical Center, Miami, Florida 33136.
Cytometry. 1990;11(8):894-900. doi: 10.1002/cyto.990110807.
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.
通过对烷化剂敏感和耐药细胞进行流式细胞术分析来测量DNA损伤。用药物处理人卵巢癌细胞系A2780及其对L-苯丙氨酸氮芥（L-PAM）耐药性高7倍的亚系，固定后用检测烷基化DNA中单链区域的单克隆抗体（MOAB）F7-26染色。在流式细胞仪上测量平均荧光强度。染色前对细胞进行加热以增强烷基化DNA中的单链化。与耐药细胞相比，敏感细胞中与DNA结合的MOAB量明显更多。每微克L-PAM处理后，不敏感细胞的荧光增加80个通道，而耐药细胞仅增加17个通道。用L-PAM处理敏感和耐药细胞，以不同比例混合，并用MOAB染色。在荧光直方图上，敏感和耐药细胞群体通过荧光强度超过一个数量级的差异清晰分开。在敏感细胞中检测到2-5%的耐药细胞作为一个单独的细胞亚群。我们得出结论，用MOAB F7-26染色可作为细胞对烷化剂敏感或耐药的指标。通过流式细胞术分析检测异质群体中耐药细胞的小亚群可能有助于监测新出现的耐药性。
