Yung B Y, Busch H, Chan P K
Cancer Res. 1986 Feb;46(2):922-5.
Localization of protein B23 in HeLa cells after treatment with luzopeptin A and its analogues was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with luzopeptin A (50 ng/ml), luzopeptin B (500 ng/ml), or luzopeptin D (10 ng/ml) for 2 h, uniform nucleoplasmic rather than specific nucleolar fluorescence was observed. Luzopeptin C had no effect on protein B23 translocation. Luzopeptin D, A, and B inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with 50% inhibitory concentration values of 3.7 +/- 1.1 (SD), 10.8 +/- 2.1, and 122.0 +/- 34.0 ng/ml, respectively. Less than 10% inhibition of [3H]uridine incorporation was found with luzopeptin C (500 ng/ml and 2 h incubation). Ribosomal RNAs (28 and 18S) were isolated from HeLa cells treated with luzopeptin D (50 ng/ml; 2 h). They were then separated and analyzed in 1% agarose gel electrophoresis. There were 90.1 +/- 1.38 and 95.0 +/- 1.04% inhibition of [3H]uridine incorporation into 28 and 18S ribosomal RNA, respectively. The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was luzopeptin D greater than luzopeptin A greater than luzopeptin B much greater than luzopeptin C, which correlates with the order of their 50% inhibitory concentration values for inhibition of [3H]uridine incorporation. With 34-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. With 70-85% inhibition of RNA synthesis, a uniform nucleoplasmic fluorescence was observed. These results indicate that translocation of protein B23 as observed by indirect immunofluorescence may be a rapid and simple screening test for the selection of antitumor agents which inhibit ribosomal RNA synthesis.
采用间接免疫荧光法研究了经鲁佐肽A及其类似物处理后，蛋白质B23在HeLa细胞中的定位。在对照HeLa细胞中观察到明亮的核仁荧光。用鲁佐肽A（50 ng/ml）、鲁佐肽B（500 ng/ml）或鲁佐肽D（10 ng/ml）处理2小时后，观察到均匀的核质荧光而非特异性的核仁荧光。鲁佐肽C对蛋白质B23的转位没有影响。鲁佐肽D、A和B抑制[3H]尿苷掺入HeLa细胞的三氯乙酸不溶性部分，其50%抑制浓度值分别为3.7±1.1（标准差）、10.8±2.1和122.0±34.0 ng/ml。用鲁佐肽C（500 ng/ml，孵育2小时）发现对[3H]尿苷掺入抑制不到10%。从用鲁佐肽D（五十 ng/ml；2小时）处理的HeLa细胞中分离出核糖体RNA（28S和18S）。然后将它们在1%琼脂糖凝胶电泳中分离并分析。[3H]尿苷掺入28S和18S核糖体RNA的抑制率分别为90.1±1.38%和95.0±1.04%。核仁荧光丧失和核质荧光同时增加的效力顺序为鲁佐肽D大于鲁佐肽A大于鲁佐肽B远大于鲁佐肽C，这与其抑制[3H]尿苷掺入的50%抑制浓度值顺序相关。当RNA合成抑制34 - 55%时，观察到核和核仁的B23免疫荧光。当RNA合成抑制70 - 85%时，观察到均匀的核质荧光。这些结果表明，通过间接免疫荧光观察到的蛋白质B23转位可能是一种快速简单的筛选试验，用于选择抑制核糖体RNA合成的抗肿瘤药物。
