Chen K G, Jaffrézou J P, Fleming W H, Durán G E, Sikic B I
Department of Medicine, Stanford University School of Medicine, California 94305-5306.
Cancer Res. 1994 Sep 15;54(18):4980-7.
Fluctuation analysis experiments were performed in the human sarcoma cell line MES-SA to assess whether selection or induction mechanisms determine resistance to doxorubicin (DOX), mutation rates, and the nature of the surviving clones. Thirteen flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 3.3 x 10(6) cells. After reseeding in 96-well plates, each population was treated with 40 nM DOX for 2 weeks. Surviving colonies were scored and harvested. Clones were propagated and analyzed for drug resistance phenotype. Expression of the mdr1, mrp, and topoisomerase II alpha and II beta genes was analyzed by reverse transcription-polymerase chain reaction. Accumulation of the P-glycoprotein substrate rhodamine-123 was measured by flow cytometry, with and without the cyclosporin D analogue SDZ PSC 833. Cellular glutathione levels were measured by flow cytometry, and M(r) 110,000 vesicular protein (p110) expression was detected by immunohistochemistry. Analysis of variance supported the hypothesis of spontaneous mutations rather than induction conferring DOX resistance. At this stringent level (5-6 log cell killing) of drug exposure, the mutation rate was estimated at 1.8 x 10(-6) per cell generation. All 30 propagated clones demonstrated cross-resistance to vinblastine, etoposide, and paclitaxel (Taxol), but not to cisplatin or bleomycin. Increased mRNA levels of mdr1 were observed in all 27 clones tested, including at least 1 from each of the 13 populations. No alterations were found in expression or level of topoisomerase II alpha or II beta, mrp, glutathione, and p110. Expression of P-glycoprotein was confirmed by flow cytometry using the monoclonal antibody UIC2. In almost all tested clones, decreased intracellular rhodamine-123 accumulation was modulated by 2 microM SDZ PSC 833, and the vinblastine resistance in all examined clones was completely reversed by SDZ PSC 833 and verapamil. Our study demonstrates that survival of cells exposed to DOX in a single step occurs as a result of a stochastic process consistent with mutational events. Activation of the mdr1 gene is the predominant mechanism selected by DOX in these resistant clones.
在人肉瘤细胞系MES-SA中进行波动分析实验，以评估选择或诱导机制是否决定对多柔比星（DOX）的耐药性、突变率以及存活克隆的性质。将13个培养瓶中各接种2000个细胞/瓶，培养至汇合群体，约为3.3×10⁶个细胞。在接种到96孔板后，每个群体用40 nM DOX处理2周。对存活菌落进行计数并收获。对克隆进行传代培养并分析耐药表型。通过逆转录-聚合酶链反应分析mdr1、mrp以及拓扑异构酶IIα和IIβ基因的表达。使用和不使用环孢素D类似物SDZ PSC 833，通过流式细胞术测量P-糖蛋白底物罗丹明-123的蓄积。通过流式细胞术测量细胞内谷胱甘肽水平，并通过免疫组织化学检测分子量为110,000的囊泡蛋白（p110）的表达。方差分析支持自发突变而非诱导赋予DOX耐药性的假设。在这种严格的药物暴露水平（5 - 6个对数级别的细胞杀伤）下，估计突变率为每细胞世代1.8×10⁻⁶。所有30个传代克隆均表现出对长春碱、依托泊苷和紫杉醇（泰素）的交叉耐药性，但对顺铂或博莱霉素无交叉耐药性。在所有27个测试克隆中均观察到mdr1的mRNA水平升高，包括来自13个群体中的至少1个群体的克隆。未发现拓扑异构酶IIα或IIβ、mrp、谷胱甘肽和p110的表达或水平有改变。使用单克隆抗体UIC2通过流式细胞术证实了P-糖蛋白的表达。在几乎所有测试克隆中，2 μM SDZ PSC 833可调节细胞内罗丹明-123蓄积的减少，并且SDZ PSC 833和维拉帕米可完全逆转所有检测克隆中的长春碱耐药性。我们的研究表明，一步暴露于DOX的细胞存活是由与突变事件一致的随机过程导致的。mdr1基因的激活是这些耐药克隆中DOX选择的主要机制。
