Broxterman H J, Schuurhuis G J, Lankelma J, Oberink J W, Eekman C A, Claessen A M, Hoekman K, Poot M, Pinedo H M
Department of Medical Oncology, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands.
Br J Cancer. 1997;76(8):1029-34. doi: 10.1038/bjc.1997.503.
Progress in our understanding of the contribution of P-glycoprotein (P-gp)-mediated resistance to chemotherapy failure in haematological as well as solid tumours has been hampered by the lack of highly sensitive, reliable methods for the detection of P-gp function in fresh human tumour cells. The present study identifies the novel nucleic acid stain SYTO16 as a highly sensitive and specific dye to assess P-gp function. The effect of P-gp is expressed here as the ratio of dye fluorescence (RF) from cells incubated with dye with or without 2 microM of the P-gp inhibitor PSC 833. Using flow cytometric analysis, an RF of 0.9 was found for SYTO16 in the KB3-1 (P-gp-) and 1.6 in KB8 (P-gp+) cells. Three types of patients' cells were studied: (1) in haematopoietic CD34+ cells, which are known to express P-gp, the RF was 6.0 for SYTO16 compared with 2.5 for rhodamine 123 and 1.3 for daunorubicin (mean of five individuals); (2) in acute myeloid leukaemia cells, the RF for SYTO16 was 1.0 in P-gp- and 4.5 in P-gp+ samples; (3) for the first time, we have quantitated P-gp function in fresh human solid tumour (sarcoma) cells. We found, in a P-gp+ leiomyosarcoma, an RF of 16 for SYTO16 and 2.7 for daunorubicin. This means that complete inhibition of P-gp function in these sarcoma cells would lead to an increase of daunorubicin accumulation with 170% compared with 30% in the CD34+ cells. Next, we showed that SYTO16 could be fixed in nuclei by 3.6% formaldehyde treatment, allowing quantification of the nuclear fluorescence on cytospins by laser scanning microscopy. In conclusion, SYTO16 proved to have a combination of favourable properties: it can be excited at 488 nm and has large fluorescence enhancement upon binding to nucleic acids, allowing the use of low, nontoxic (< 10 nM) concentrations. Because the RF for SYTO16 is much higher than for daunorubicin, it can be applied for the determination of P-gp function in relatively small numbers of low-P-gp-expressing tumour cells by laser scanning microscopy. Individual sarcomas were found to have high P-gp function compared with CD34+ cells. This assay may be used to select patients for P-gp modulation protocols.
由于缺乏高灵敏度、可靠的方法来检测新鲜人肿瘤细胞中P-糖蛋白（P-gp）介导的化疗耐药性对血液系统肿瘤及实体瘤化疗失败的影响，我们对这方面的理解一直受到阻碍。本研究确定了新型核酸染料SYTO16是一种评估P-gp功能的高灵敏度和特异性染料。P-gp的作用在此表示为用染料孵育的细胞在添加或不添加2 microM P-gp抑制剂PSC 833时的染料荧光比值（RF）。通过流式细胞术分析，发现SYTO16在KB3-1（P-gp阴性）细胞中的RF为0.9，在KB8（P-gp阳性）细胞中的RF为1.6。研究了三种类型的患者细胞：（1）在已知表达P-gp的造血CD34+细胞中，SYTO16的RF为6.0，而罗丹明123为2.5，柔红霉素为1.3（五名个体的平均值）；（2）在急性髓性白血病细胞中，SYTO16在P-gp阴性样本中的RF为1.0，在P-gp阳性样本中的RF为4.5；（3）我们首次对新鲜人实体瘤（肉瘤）细胞中的P-gp功能进行了定量。我们发现，在一个P-gp阳性的平滑肌肉瘤中，SYTO16的RF为16，柔红霉素的RF为2.7。这意味着这些肉瘤细胞中P-gp功能的完全抑制将导致柔红霉素积累增加170%，而CD34+细胞中为30%。接下来，我们表明SYTO16可以通过3.6%甲醛处理固定在细胞核中，从而通过激光扫描显微镜对细胞涂片上的核荧光进行定量。总之，SYTO16被证明具有多种优良特性：它可以在488 nm处激发，与核酸结合后荧光增强显著，允许使用低浓度、无毒（<10 nM）的染料。由于SYTO16的RF比柔红霉素高得多，它可以通过激光扫描显微镜用于测定相对少量低P-gp表达肿瘤细胞中的P-gp功能。与CD34+细胞相比，发现个别肉瘤具有较高的P-gp功能。该检测方法可用于选择适合P-gp调节方案的患者。
