From Reversal of the Ras-induced transformed phenotype by HR12, a novel ras farnesylation inhibitor, is mediated by the Mek/Erk pathway.
The Journal of cell biology.
The Journal of cell biology.
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Figure 2. HR12-treatment induces stress-fiber formation in Rat1/ras cells. Rat1/ras cells were treated as in Fig 1 and stained with TRITC-labeled phalloidin. (a) No treatment, (b) 24 h, (c) 48 h, (d) 72 h, and (e) 48 h exposure to HR12, followed by 24 h without the inhibitor. (f) Untransformed Rat1 cells. Bar, 10 µm.
- We examined the effect of the Ras-FTI, HR12, on Rat1/ras cells (fibroblasts stably transformed with Ha-rasV12). Fig 1 Fig 2 Fig 3 show a comparison of nontransformed Rat1 cells, Rat1/ras cells, and HR12-treated Rat1/ras cells (48 h, 20 µM).
- Stress fibers, labeled by TRITC-phalloidin, were also disrupted (Fig 2).
- In Rat1 cells, F-actin was engaged as a dense web of conspicuous stress-fibers (Fig 2 f).
- Rat1/ras cells appeared elongated, with numerous F-actin–containing protrusions and ruffles, but essentially no actin bundles (Fig 2 a).
- At 48 h and later, circumferential bundles also became apparent (Fig 2c and Fig d).
- Removal of HR12 for 24 h, after a 48-h treatment, resulted in the loss of organized actin bundles and the appearance of a more diffuse pattern (Fig 2 e).
- Actin exists in a dynamic equilibrium between a Triton-soluble pool and Triton-insoluble cytoskeletal filaments. Fig 2 shows that HR12 triggers dramatic assembly of actin into stress fiber networks in Rat1/ras cells.
- In contrast to the reports cited above in support of the RhoB theory, we show here: (a) by 15 h, most of Ras population is unprocessed (Fig 6), which corresponds to the kinetics of the morphological changes (Fig 1 and Fig 2); (b) NIH3T3myr-ras cells fail to form adhesions in response to HR12, unlike NIH3T3 cells transformed by farnesylation-dependent oncogenic ras (Fig 9); and (c) HR12 treatment leads to the accumulation of high levels of oncogenic Ras in the cytoplasm, followed by potent inhibition of Mek/Erk activation (Fig 5 and Fig 6).