Farnesyltransferase FTase Inhibitors Effectively Inhibit Prenylation of HRas

The role ofthe H-Ras oncogene in radiation resistance was initially studied using early passage REF transformed with either H-Ras alone, v-, or c-myc alone, or v-myc together with H-Ras. Using this model, it was established that transfection with H-Ras resulted in increased radioresistance, whereas transfection with v- or c-myc alone did not (5,36) (Fig. 1). The finding that activated H-Ras oncogene expression in REF cells caused an increase in radiation resistance led to the hypothesis that inhibiting H-Ras activity in these cells should reduce their radiation resistance if Ras activation was the necessary factor for the observed increase in radioresistance.

To test this hypothesis, REF cells were treated with the farnesyltransferase (FTase) inhibitor FTI-277. The inhibition ofH-Ras farnesylation was monitored by Western-blot analysis ofRas protein migration on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Prenylated forms ofRas have been shown to migrate more rapidly than unprenylated Ras on SDS-polyacrylamide gels (37,38). Treatment ofH-rasV12 oncogene transformed REF cells (3.7, 4R, and 5R) with various doses of FTI-277 (2.5-10 ^M) for 24 h resulted in the majority of the H-Ras being unprocessed (Fig. 2). However, cells expressing wild-type c-H-Ras were less susceptible to the effects ofthis inhibitor. Treatment ofuntransformed REF resulted in detectable H-Ras in the slower moving, unprocessed form, although the majority of the H-Ras remained in the farnesylated form in spite of the treatment with FTI-277 at doses up to 30 ^M. MR4 cells (REF immortalizedbyv-myc) appearto express very low levels ofH-Ras (we could only detect Ras protein in these cells using a pan-Ras antibody) and little change in the migration of Ras was seen with treatment up to 10 ^M FTI-277 after 24 h.

The targeting of prenyltransferases to a particular Ras protein is in large part dictated by the CAAX recognition sequence found at the carboxyl terminal portion of Ras and other prenylated proteins (39). The cysteine within the carboxyl terminal end of H-Ras, CVLS, is the target for prenylation by FTase. In contrast, CAAX sequences terminating in leucine have a greatly reduced affinity for farnesyltransferase and are geranylgera-nylated by geranylgeranyltransferase I (GGTase I) during processing (40). A chimeric H-rasV12 with CVLL as the CAAX motif is fully transforming in NIH 3T3 cells and Rat-1

Fig. 1. Survival of REF cells transformed with H-Ras plus myc or immortalized with myc alone. Log-phase cultures of rat embryo fibroblasts transformed with H-rasV12 plus v-myc (•), or immortalized with c-myc (▲) were irradiated and plated for clonogenic survival. Cultures were stained and scored for the formation of colonies after 2 wk. Surviving fraction, plotted on the Y-axis is defined as: Number of colonies formed / (Number of cells plated) • (Plating efficiency).

Dose, Gy

Fig. 1. Survival of REF cells transformed with H-Ras plus myc or immortalized with myc alone. Log-phase cultures of rat embryo fibroblasts transformed with H-rasV12 plus v-myc (•), or immortalized with c-myc (▲) were irradiated and plated for clonogenic survival. Cultures were stained and scored for the formation of colonies after 2 wk. Surviving fraction, plotted on the Y-axis is defined as: Number of colonies formed / (Number of cells plated) • (Plating efficiency).

cells. This altered H-Ras also transformed primary REF in co-transfection with v-myc, but at a lower efficiency than the farnesylated form (41). Compared to 3.7 or 5R, cells transformed with H-rasV12 CVLL (REF-GG) adhere poorly to tissue culture dishes and do not form discrete colonies. These cells serve as useful controls in our experiments since the H-rasV12 CVLL should be impervious to the effects of FTI-277, but should be sensitive to GGTase I inhibition of geranylgeranyltransferase. When REF-GG were treated with up to 10 ^M FTI-277, no change in mobility of H-Ras-CVLL was observed (Fig. 2), thus demonstrating the specificity of the FTI-277 inhibitor for FTase over geranylgeranyltransferase. In contrast, the geranylgeranyltransferase inhibitor GGTI-298 (42,43) demonstrated effective inhibition of the geranylgeranylation of the chimeric H-rasV12 with the CVLL recognition sequence for geranylgeranyltransferase at inhibitor doses as low as 4 ^M, whereas H-rasV12 expressed by the 5R cell line that has the FTase recognition sequence CVLS, and is thus farnesylated, was only partially inhibited at a dose of 32 ^M (Fig. 3).

Fig. 2. Effects on Ras farnesylation of 24-h treatment with FTI-277. Cells in log-phase culture were treated with the indicated dose of FTI-277 (p.M). Cell lysates for the indicated cell types were obtained after 24 h and analyzed by Western blotting. H-Ras specific antibody was used for all blots except MR4 where pan Ras-specific antibody results are shown owing to very low levels ofH-Ras expression. Farnesylated (F) and unfarnesylated (UF) Ras are indicated.

Fig. 2. Effects on Ras farnesylation of 24-h treatment with FTI-277. Cells in log-phase culture were treated with the indicated dose of FTI-277 (p.M). Cell lysates for the indicated cell types were obtained after 24 h and analyzed by Western blotting. H-Ras specific antibody was used for all blots except MR4 where pan Ras-specific antibody results are shown owing to very low levels ofH-Ras expression. Farnesylated (F) and unfarnesylated (UF) Ras are indicated.

Fig. 3. Effects on Ras geranylgeranylation of 24-h treatment with GGTI-298. Cells in log phase culture weretreatedwiththe indicated dose ofGGTI-298 (pM) andRas statusmonitoredas above. 5R cells are transformedwith H-rasV12. REF-GG are transformedwith an H-rasV12 chimeric molecule that is prenylatedby geranylgeranyltransferase. Unfarnesylated Ras expressed in5R cells (U-F) and ungeranylgeranylated Ras expressed in REF-GG cells (U-GG) after treatment with GGTI-298 are indicated.

2.2. Effect of FTI-277 and GGTI-298 Treatment on Apoptosis After Irradiation

Fig. 3. Effects on Ras geranylgeranylation of 24-h treatment with GGTI-298. Cells in log phase culture weretreatedwiththe indicated dose ofGGTI-298 (pM) andRas statusmonitoredas above. 5R cells are transformedwith H-rasV12. REF-GG are transformedwith an H-rasV12 chimeric molecule that is prenylatedby geranylgeranyltransferase. Unfarnesylated Ras expressed in5R cells (U-F) and ungeranylgeranylated Ras expressed in REF-GG cells (U-GG) after treatment with GGTI-298 are indicated.

2.2. Effect of FTI-277 and GGTI-298 Treatment on Apoptosis After Irradiation

We have previously shown that exposure of REF cells immortalized by v-myc to ionizing radiation results in high levels ofapoptosis, whereas cells transformed by H-Ras plus v-myc have substantially lower levels of apoptosis. This demonstrated that transformed cells expressing activated H-Ras, which were more resistant to radiation killing, were also more resistant to the induction of apoptosis by radiation than cells that lacked H-Ras expression. It further implied that loss of H-Ras activity in these cells would lead to increased radiation-induced apoptosis and decreased clonogenic survival. As a first test of this prediction, the effect of inhibiting H-Ras farnesylation on apoptosis after irradiation was examined. Cells were irradiated with 10 Gy and concurrently treated with various concentrations of FTI-277. The extent of apoptosis induced by the irradiation of 3.7 cells, which express activated H-Ras and v-myc, was greatly enhanced by treatment with FTI-277 (Fig. 4). The maximum effect was seen at 5 ^M with a significant increase also seen at 2.5 ^M. Thus, at doses of FTI that inhibit H-rasV12 farnesylation, the level

Fig. 4. Apoptosis after irradiation and FTI-277 treatment. Early passage rat embryo fibroblast cells (REF) or myc + Ras transformed REF cells (3.7) were treated with the indicated concentration of FTI-277 at the time of irradiation with 10 Gy. Apoptosis was quantitated 24 h later by scoring for changes in nuclear morphology after staining with propidium iodide.

Fig. 4. Apoptosis after irradiation and FTI-277 treatment. Early passage rat embryo fibroblast cells (REF) or myc + Ras transformed REF cells (3.7) were treated with the indicated concentration of FTI-277 at the time of irradiation with 10 Gy. Apoptosis was quantitated 24 h later by scoring for changes in nuclear morphology after staining with propidium iodide.

Fig. 5. FTI-277-mediated enhancement in radiation-induced apoptosis is specific for cells expressing farnesylated H-Ras. Cells were treated with FTI-277 at the time of irradiation and assessed 24 h laterforapoptosis, as shown inFig. 4.4Rand5Rcells aretransformedwithH-rasV12. REF-GG are transformed with an H-rasV12 chimeric molecule that is prenylated by geranylgeranyltransferase.

Fig. 5. FTI-277-mediated enhancement in radiation-induced apoptosis is specific for cells expressing farnesylated H-Ras. Cells were treated with FTI-277 at the time of irradiation and assessed 24 h laterforapoptosis, as shown inFig. 4.4Rand5Rcells aretransformedwithH-rasV12. REF-GG are transformed with an H-rasV12 chimeric molecule that is prenylated by geranylgeranyltransferase.

of apoptosis after irradiation is increased. This increase is specific for cells expressing activatedH-Ras, as FTI-277 treatment ofREF cells, which are untransformed, causedno increase in apoptosis after irradiation.

The ability of FTI-277 to augment irradiation-induced apoptosis in cells transformed by the H-Ras oncogene was confirmed in 4R and 5R cells (Fig. 5). These are two independent clones of REF cells transformed by H-rasV12 alone. These results demonstrate

Fig. 6. Apoptosis in REF cells expressing geranylgeranylated Ras is only induced by a geranylgera-nyltransferase inhibitor. REF-GG cells were irradiated in the presence of 5 p,M FTI-277 or 8 p,M GGTI-298 and scored for the presence of apoptotic cells 24 h after irradiation, as shown in Fig. 4.

Fig. 7. Inhibition of H-Ras farnesylation in H-Ras transformed murine prostate cells by FTI-277. Mouse prostate tumor cells transformed by retroviral transduction with the H-rasV12 + myc oncogenes were treated with the indicated doses (p.M) of FTI-277. After 24 h samples were harvested for Western-blot analysis with anti-H-Ras antibody. The upper band (arrow) corresponds to unfarne-sylated H-Ras. C, controls.

Fig. 7. Inhibition of H-Ras farnesylation in H-Ras transformed murine prostate cells by FTI-277. Mouse prostate tumor cells transformed by retroviral transduction with the H-rasV12 + myc oncogenes were treated with the indicated doses (p.M) of FTI-277. After 24 h samples were harvested for Western-blot analysis with anti-H-Ras antibody. The upper band (arrow) corresponds to unfarne-sylated H-Ras. C, controls.

that the presence of an activated H-Ras alone is sufficient to cause increased radiation-induced apoptosis after FTI-277 treatment.

A further control for the specificity of apoptosis induction after irradiation was obtained using the REF-GG cell line. Because the H-rasV12 expressed by these cells was not affected by FTI-277 treatment, the level ofapoptosis after irradiation shouldnotbe increased after FTI-277 treatment. These cells had a relatively high baseline level of apoptosis of about 6% (Fig. 5). This was increasedbyirradiation to 12%. Treatmentofthese cells withFTI-277 slightly increased the baseline level of apoptosis, but had no significant effect on enhancing the extent of apoptosis after irradiation. In contrast, treatment of REF-GG cells with the geranylgeranyltransferase inhibitor GGTI-298 significantly increased both the basal level of apoptosis in these cells and the apoptosis observed after irradiation (Fig. 6). Thus, the increase in apoptosis seen after irradiation andprenyltransferase inhibitor treatment appears to correlate with inhibition of oncogenic H-Ras prenylation.

We have extended the observations obtained in the REF model system to mouse prostate tumor cells derived by transduction ofH-Ras and v-myc into mouse embryo urogenital sinus cells (44,45). Treatment of either primary tumor cells or a metastatic clone of this tumor line showed a dose-dependent reduction ofthe farnesylated form and the accumulation of the more slowly migrating, unprocessed form of H-Ras (Fig. 7). Thus, FTI-277 isan effective inhibitor ofRas farnesylation in transformed prostatic epithelial cells.

Fig. 8. Increased radiation-induced apoptosis in Ras transformed mouse prostate tumor cells. Mouse prostate tumor cells transformed by retroviral transduction with the H-rasV12 + myc oncogenes were treated with the indicated concentrations of FTI-277 (pM) and irradiated 24 h later with 10 Gy. Apoptosis was quantitated 24 h after irradiation by scoring for changes in nuclear morphology visualized by epifluorescent microscopy after staining ofcells with propidium iodide. (A) Prostate tumor cells cultured from a primary tumor. (B) Prostate tumor cells cultured from an isolated metastasis derived from the tumor shown in (A).

Fig. 8. Increased radiation-induced apoptosis in Ras transformed mouse prostate tumor cells. Mouse prostate tumor cells transformed by retroviral transduction with the H-rasV12 + myc oncogenes were treated with the indicated concentrations of FTI-277 (pM) and irradiated 24 h later with 10 Gy. Apoptosis was quantitated 24 h after irradiation by scoring for changes in nuclear morphology visualized by epifluorescent microscopy after staining ofcells with propidium iodide. (A) Prostate tumor cells cultured from a primary tumor. (B) Prostate tumor cells cultured from an isolated metastasis derived from the tumor shown in (A).

When these cells were examined for radiation-induced apoptosis after treatment with FTI-277 at the doses shown to inhibit Ras farnesylation, a significant increase in apoptosis was seen (Fig. 8). Thus, inhibition offarnesylation in prostate cells as well as fibroblasts resulted in increased radiation-induced apoptosis.

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