CP248, a Derivative of Exisulind, Causes Growth Inhibition, Mitotic Arrest, and Abnormalities in Microtubule Polymerization in Glioma Cells 1 Supported by awards from Cell Pathways, Inc., the T. J. Martell Foundation, and the National Foundation for Cancer Research (to I. B. W.). Additional grant support was from the American Cancer Society and NIH (to G. G. G.). A. F. P. was supported by a fellowship from the Fonds de la Recherche en Santé du Québec.1

Effects of CP248 on mitotic 9L cells. A, a mitotic 9L cell after treatment with 0.1% DMSO (solvent control). B, a 9L cell treated with 50 ng/ml Colcemid (positive control). C, a mitotic 9L cell after treatment with 0.075 μm CP248. Cells were treated for 12 h and fixed with methanol. Chromosomal DNA was stained blue with DAPI. MTs were stained green and MAD2 proteins were stained red with the respective antibodies and were visualized by indirect immunofluorescence, as described in “Materials and Methods.” Abnormal spindle apparatus is notable in the CP248-treated cells. White bar, 10 μm.

Effects of CP248 on interphase 9L cells. The 9L cells were treated with 0.075 μm CP248 (A, C) or 0.1% DMSO (B, D) for 6 h, fixed with methanol; and DNA, MTs, and centrosomes were visualized by indirect immunofluorescence, as described in Fig. 2 and “Materials and Methods.” In B, DMSO-treated 9L cells show a tight correlation of centrosomes (stained red) with the MT array (white arrow). In A, CP248-treated 9L cells show a dissociation of the centrosome (white arrow) from the MT array (yellow arrow). In C, CP248-treated 9L cells were stained for stable Glu MTs (red) and DNA (blue). In D, DMSO-treated 9L cells were stained for stable Glu MT (red) and for DNA (blue). White bars, 20 μm.

Effects of CP248 on MTs in interphase 9L cells. 9L cells were treated with 0.1% DMSO (A), 0.2 μm CP248 (B), or 0.4 μm CP248 (C) for 2 h. The cells were fixed with methanol, and MTs were visualized by immunofluorescence, as described in “Materials and Methods.” The 9L cells were treated with 0.4 μm CP248 for 2 h, washed three times with fresh medium, and then incubated for an additional 15 (D), 30 (E), or 60 (F) min, before fixation and immunostaining. White bars, 20 μm.

Effects of CP248 on MTs in NIH 3T3 fibroblasts. The cells were grown in medium in the absence of serum for 48 h. They were then incubated in medium minus serum containing: 0.1% DMSO (A); 0.1 μm CP248 (B); 200 μm exisulind (C); 1.5 μm CP461 (D); 0.1 μm CP248 plus 10 μm KT5823 (E); 0.1 μm CP248 plus 10 μm Rpt-8-pCPT0cGMP (F); 170 nm YC-1 (G); or 4 mm dibutyryl cGMP (H). After 2 h of incubation with these agents, the cells were fixed with methanol and the MTs visualized by immunofluorescence, as described in “Materials and Methods.” White bars, 20 μm.