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Effects of Luzopeptins on Protein B23 Translocation and Ribosomal RNA Synthesis in HeLa Cells

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030

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> luzopeptin A> luzopeptin B>> 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.

¹ These studies were supported by Cancer Research Center Grant CA-10893, P1, awarded by National Cancer Institute, Department of Health and Human Services, USPHS; the DeBakey Medical Foundation; the Davidson Fund; the Pauline Sterne Wolff Memorial Foundation; the H. Leland Kaplan Cancer Research Endowment; the Linda and Ronny Finger Cancer Research Endowment Fund; and the William S. Farish Fund.

Received 9/30/85. Accepted 11/ 4/85.

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