Smith P J, Lacy M, Debenham P G, Watson J V
MRC Clinical Oncology and Radiotherapeutics Unit, MRC Centre, Cambridge, UK.
Carcinogenesis. 1988 Mar;9(3):485-90. doi: 10.1093/carcin/9.3.485.
The bis-benzimidazole dyes (specifically Hoechst 33258 and the more lipophilic derivative Hoechst 33342) are non-intercalating AT base pair-specific ligands which bind to cellular DNA by non-covalent association with the minor groove. The interaction of dye with cellular DNA is thought to be the principal pathway for the cytotoxic, mutagenic and DNA-damaging properties of such agents. Upon binding and near UV light excitation, dye molecules exhibit fluorescence enhancement such that dye/DNA association and dissociation in individual cells can be monitored by flow cytometry. We have studied dye uptake and the DNA--dye dissociation characteristics of a Hoechst 33258-resistant mouse cell line (HoeR415) compared to the response of the parental cell line Ltk-. HoeR415 was found to show similar levels of cross resistance (approximately 10-fold) to Hoechst 33258 and Hoechst 33342 compared to parental responses except that the more lipophilic ligand was approximately 30-fold more toxic. Estimates of Hoechst 33342 uptake using flow cytometry or radiolabelling methods indicated that resistance could not be attributed to reduced cellular uptake, low initial levels or different modes of DNA binding. Both cell lines showed similar initial levels of dye-induced DNA strand-breakage. However, Hoechst 33342 resistance did correlate with an enhanced capacity (10-fold) of HoeR415 to remove dye from cellular DNA compared with the relatively long retention (T 1/2 300 min) of ligand by the parental cell line. Our results are consistent with the view that ligand persistence rather than indirect DNA damage is a more important factor in the cytotoxicity of non-intercalating DNA-binding ligands. A model is presented of the cellular processes of DNA damage recognition and surveillance for ligands which interact with the minor groove of DNA.
双苯并咪唑染料（特别是Hoechst 33258和脂溶性更强的衍生物Hoechst 33342）是一类非嵌入性的、对AT碱基对具有特异性的配体，它们通过与小沟进行非共价结合而与细胞DNA相结合。染料与细胞DNA的相互作用被认为是这类试剂产生细胞毒性、致突变性和DNA损伤特性的主要途径。在结合并受到近紫外光激发后，染料分子会表现出荧光增强，从而可以通过流式细胞术监测单个细胞中染料/DNA的结合和解离情况。我们研究了一种对Hoechst 33258具有抗性的小鼠细胞系（HoeR415）与亲代细胞系Ltk-相比的染料摄取情况以及DNA-染料解离特性。结果发现，与亲代细胞相比，HoeR415对Hoechst 33258和Hoechst 33342表现出相似水平的交叉抗性（约10倍），只是脂溶性更强的配体毒性大约高30倍。使用流式细胞术或放射性标记方法对Hoechst 33342摄取量的估计表明，抗性不能归因于细胞摄取减少、初始水平低或DNA结合方式不同。两种细胞系都表现出相似的染料诱导DNA链断裂的初始水平。然而，与亲代细胞系中配体相对较长的保留时间（半衰期300分钟）相比，HoeR415对Hoechst 33342的抗性确实与从细胞DNA中去除染料的能力增强（10倍）相关。我们的结果与以下观点一致，即配体的持久性而非间接的DNA损伤是影响非嵌入性DNA结合配体细胞毒性的更重要因素。本文提出了一个关于与DNA小沟相互作用的配体的DNA损伤识别和监测细胞过程的模型。
