A significant decrease of S phase and concomitant increase of G0 G1 phase cells was seen in the presence ref 3 of AEE788 accompanied by distinct modifications of cell cycle regulating proteins. The data were more concise in the synchronous than in the asynchronous cell culture model, which is not surprising because specific Inhibitors,Modulators,Libraries effects of AEE788 on mitotic events may become more obvious in a homogeneous cell population. Indeed, Peng and cowork ers reported that the activity of a particular drug limited to certain cell cycle phases may be diluted under asynchro nous conditions. Based on the synchronous cell cul ture model, cdk2, cdk4, cyclin D1 and cyclin E were all found to be reduced, whereas p27 was up regulated by AEE788 in the RCC cell lines.

These findings are important since disturbances of cell cycle control in the tumorigenesis of RCC have recently Inhibitors,Modulators,Libraries been shown to be paralleled by elevation of cyclin D1 and cdk4, accompanied by the attenuation of p27 expression. Inline with the in vitro data, analysis of tumor speci men taken from RCC patients revealed a correlation between cyclin D1 and cyclin E protein level and the tumor proliferation index. Vice versa, an inverse cor relation was seen between p27 expression and tumor size, and RCC patients with p27 low tumors had poorer sur vival than patients with p27 high tumors. Obviously, cyclin D1, cyclin E, cdk4 and p27 represent pivotal elements in RCC cells and targeting these proteins may become an intriguing option Inhibitors,Modulators,Libraries to stop RCC progres sion.

In fact, incubation of RCC cells with thiazolidinedi one decreased the protein levels of cyclin D1 and cdk4, and increased the levels of p27 which altogether led to G0 G1 arrest and massive tumor cell apoptosis. A similar phenomenon has been observed by others treating RCC cells with the short chain Inhibitors,Modulators,Libraries fatty acid sodium butyrate or phenylacetate. The data presented here point to a powerful anti tumoral activity of AEE788. Nevertheless, AEE788 did not reduce cyclin D1, cyclin E, cdk2 and cdk4 at all time points ana lyzed. Cdk1 became even enhanced in synchro nized KTC 26 and A498 cells after 1 h. Therefore, it may be assumed that AEE788 does not com pletely suppress cell mitosis but rather slows down the mitotic cycle. In line with this speculation, the prolifera tive activity of RCC cells presented in figure 4 was drasti cally down regulated, though not totally blocked by AEE788.

Western blot analysis of cell cycle proteins, listed in methods Western blot analysis of cell cycle proteins, listed in methods. Asynchronous A498, Caki 1 or KTC 26 cells were treated either with 1 M or 5 M AEE788 or with 1 nM or 5 nM RAD001, or with a 1 M AEE788 1nM RAD001 combination. Controls remained untreated. Drugs were applied for 6 Inhibitors,Modulators,Libraries or 24 h. Cell lysates were then subjected to SDS PAGE and blotted on the membrane incubated with the respective inhibitor licensed monoclonal antibodies. Beta actin served as the internal control.