FTI resulted in increased Ras activity and activated ERK and p38 MAPK pathways. Inhibiting GGTβ in 293T cells resulted in decreased Ras activity. This finding suggests that GG’d Ras has a higher affinity for activating Raf and other effectors which have Raf-like binding domains such as TIAM1. TIAM1 functions as a Ras-controlled GEF activator of Rac (56). Rac is an identified activator of Mirk which in turn can activate MKK3/6, the upstream activator of p38 MAPK (57). Raf and TIAM1 have similar Ras binding domains which explains why ERK and p38 MAPK can both be activated in tumor cells in response to increased Ras activation. Raf and/orTIAM1 specifically may be responsible for the growth- inhibiting and toxic effects of GG’d K-Ras. Future experiments, beyond the scope of this thesis, could elucidate these functions.
To confirm direct activation by Ras of Raf-1 and TIAM1, immunoprecipitations could be performed. MAPKs have intricate networks and are often activated in response to variable signals within a cell. Increased Ras activity could be activating either ERK or p38 MAPKs alone or both pathways. To determine whether the activity of these two MAPK pathways are responsible for the effects of increased GG’d Ras, cells could be treated with tipifarnib, and separate immunoprecipitations could be performed with either Raf-1 or TIAM1 conjugated to agarose beads. This experiment would allow for direct analysis of how much GG’d Ras is signaling though these respective pathways. This study would also determine if these two pathways are directly activated through increased Ras activation or if the activation of these MAPKs is through integrated signaling within the cells. Activation of downstream targets and immunoprecipitations could also be analyzed in FTβ shRNA transduced cells. We hypothesize that both of these pathways are directly contributing to reduced cell viability and cell cycle arrest from increased GG’d K-Ras activity induced by FTI.
Transduction of WT K-Ras also resulted in an increased percentage of dead and/or dying 293T cells. We hypothesize this is due to increased GG’d K-Ras from the FTase enzymatic activity being fully saturated in K-Ras WT cells. To determine the activity levels of each enzyme, positively transduced cells could be sorted, collected, and then made into lysates. The lysates could be run on an immunoblot and probed for GGTase, FTase, HDJ-2, and Rap-1. HDJ-2 would be a marker for FTase enzyme activity being saturated. If the FTase enzymatic activity is saturated, unprenylated HDJ-2 would be present. The same would be true for Rap-1 and GGTase. If GGTase were saturated, increased levels of Rap-1 would not be GG’d. We
77 predict that cells transduced with WT K-Ras would have increased GGTase expression and an increase in unfarnesylated HDJ-2. These findings would confirm that FTase is saturated and K-Ras is increasingly GG’d in WT K-Ras transduced cells. This would imply over expression of K-Ras in cells that are not already dependent on K-Ras signaling results in increased levels of active GG’d K-Ras even in the absence of an FTI.
To determine if the level of expression of GG’d K-Ras influences cell viability, cells transduced with the Ras-mKate fusion constructs could be sorted based on their fluorescence intensity, which would correspond directly with exogenous Ras activity. GG’d K-Ras cells could be separated by low, medium, and high-expressing GG’d K-Ras cells 48 hours post transduction to determine if lower expression of this protein would have the same cytotoxic effects observed in cells with high-level expression. As signal intensity is dependent on protein expression, we hypothesize cells with less GG’d K-Ras would be able to overcome the toxicities associated with increased GG’d K-Ras more quickly than cells with higher expression.
It would be important to confirm that GG’d K-Ras is resulting in reduced cell viability and cell cycle arrest in tumorigenic cells. Transductions and transfections of K-CAIL (GG’d) could be done in the tumor cell lines SaOS2, COL, and OS187. Transduced/transfected cells could be sorted into negative and positive populations. From these sorted populations lysates could be made and cell cycle analysis could be performed. From analysis during the sort, we predict inducing GG’d K-Ras in tumor cell lines will result in a high percentage of dead and/or dying cells. We also predict that inducing expression of GG’d K-Ras would result in increased ERK and p38 MAPK signaling, an increased percentage of cells with sub-diploid DNA, and an increase of cells arrested in the G2/M portions of the cell cycle.
The results of these experiments would confirm that over expression of active GG’d K- Ras is mediating cytotoxic and antiproliferative effects. These results would further support that FTI increases GG’d K-Ras to mediate these effects.
78