Janssen Hilde L K, Hoebers Frank J, Sprong Debbie, Goethals Laurence, Williams Kaye J, Stratford Ian J, Haustermans Karin M, Balm Alfons J, Begg Adrian C
Division of Experimental Therapy, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Radiother Oncol. 2004 Jan;70(1):91-7. doi: 10.1016/j.radonc.2003.09.012.
Hypoxia is a strong negative prognostic factor for all three major treatment modalities for cancer. The bioreductive drug pimonidazole is currently under clinical investigation as a hypoxia marker. In human head and neck tumors, in addition to staining patterns typical of chronic hypoxia, staining was seen specifically around areas of keratinization, raising the question of whether this is hypoxia-related. This could influence quantitative hypoxia estimates using this marker. We investigated here whether the differentiation-related staining was caused by locally high reductive enzyme levels.
The nitrotetrazolium compound NBT was used, which is reduced by nitroreductases to yield a blue color. The assay was validated on three genetically related MDA231 human mammary carcinoma cell lines: wildtype, overexpressing DT-diaphorase (DT1), and overexpressing cytochrome p450 reductase (R4). Increased NBT staining under normoxia was indeed seen for both R4 and DT1 lines. Pimonidazole staining under normoxia was only seen in the R4 line.
Frozen tumor sections from 20 patients with head and neck cancer injected with pimonidazole were incubated with NBT. Parallel sections were stained for pimonidazole. Staining patterns were then compared on matched images, and areas of keratinization scored for the presence or absence of pimonidazole and NBT. Pimonidazole staining was seen in 56% of keratinized areas, and of these, 78% showed increased NBT staining, indicating that high reductase levels are not a necessary requirement for differentiation-associated pimonidazole staining. In a second series, frozen sections of tumors from 15 patients not receiving pimonidazole were incubated with NBT and compared with staining after incubation with pimonidazole under both oxic and hypoxic conditions. Pimonidazole staining of some keratinizing areas under oxic conditions was seen. Of these areas, only a proportion (70%) showed increased NBT staining, confirming the lack of correspondence between keratin-associated pimonidazole staining and reductase levels.
Hypoxia-independent pimonidazole staining can occur in more differentiated head and neck tumors, necessitating caution in hypoxia quantification. These data argue against a causative role for locally high reductase levels in differentiation-associated staining. DT-diaphorase appears to play no role in pimonidazole reduction.
缺氧是癌症三大主要治疗方式的一个强有力的负面预后因素。生物还原药物匹莫硝唑目前正作为一种缺氧标志物进行临床研究。在人类头颈部肿瘤中，除了典型的慢性缺氧染色模式外，在角化区域周围还特别观察到染色，这就引发了这是否与缺氧相关的问题。这可能会影响使用该标志物进行的定量缺氧评估。我们在此研究与分化相关的染色是否由局部高还原酶水平引起。
使用了硝基四氮唑化合物NBT，它被硝基还原酶还原后会产生蓝色。该检测方法在三种基因相关的MDA231人乳腺癌细胞系上进行了验证：野生型、过表达DT - 黄递酶（DT1）和过表达细胞色素P450还原酶（R4）。在常氧条件下，R4和DT1细胞系确实都观察到NBT染色增加。在常氧条件下，仅在R4细胞系中观察到匹莫硝唑染色。
将20例注射了匹莫硝唑的头颈部癌患者的冰冻肿瘤切片与NBT一起孵育。平行切片进行匹莫硝唑染色。然后在匹配图像上比较染色模式，并对角化区域是否存在匹莫硝唑和NBT进行评分。在角化区域的56%中观察到匹莫硝唑染色，其中78%显示NBT染色增加，这表明高还原酶水平不是与分化相关的匹莫硝唑染色的必要条件。在第二个系列中，将15例未接受匹莫硝唑的患者的肿瘤冰冻切片与NBT一起孵育，并与在有氧和缺氧条件下与匹莫硝唑孵育后的染色进行比较。在有氧条件下，在一些角化区域观察到匹莫硝唑染色。在这些区域中，只有一部分（70%）显示NBT染色增加，证实了与角蛋白相关的匹莫硝唑染色和还原酶水平之间缺乏对应关系。
在分化程度较高的头颈部肿瘤中可能会出现与缺氧无关的匹莫硝唑染色，这在缺氧定量时需要谨慎。这些数据表明局部高还原酶水平在与分化相关的染色中不起因果作用。DT - 黄递酶似乎在匹莫硝唑还原中不起作用。
