Radiosensitization Of Human Tumors Expressing Activated Ras Oncogenes

Fig. 10. Inhibition of H-Ras farnesylation in human tumor cells by FTI-277. Cells with mutated (T24 and HS578T) or wild-type H-Ras (HeLa, HT-29, and SKBr-3) were treated with the indicated concentration (pM) ofFTI-277 for 24 h or with an equal volume of10 mM dithiothreitol (DTT) in DMSO carrier (C, control). Cell lysates were then obtained for Western-blot analysis. Blots were probed with monoclonal antibody (MAb) to H-Ras. Arrows indicate migration of unfarnesylated H-Ras.

Fig. 10. Inhibition of H-Ras farnesylation in human tumor cells by FTI-277. Cells with mutated (T24 and HS578T) or wild-type H-Ras (HeLa, HT-29, and SKBr-3) were treated with the indicated concentration (pM) ofFTI-277 for 24 h or with an equal volume of10 mM dithiothreitol (DTT) in DMSO carrier (C, control). Cell lysates were then obtained for Western-blot analysis. Blots were probed with monoclonal antibody (MAb) to H-Ras. Arrows indicate migration of unfarnesylated H-Ras.

used both assays because in some cell lines pretreatment with FTI-277 inhibited cell attachment after plating ofcells. In limiting dilution analysis, the effect ofinhibitor treatment was determined by comparing the frequency of colonies arising in microtiter wells inoculated with varying cell numbers in the range calculated to yield an average of one cell per well. In contrast to clonogenic assays carried out by plating cells in culture dishes and scoring for colony formation, the limiting dilution analysis of cell clonogenicity is not influenced by loose attachment ofcells immediately after plating, because individual microtiter wells are scored simply as positive or negative for clonogens, not for colony number (46-48). Thus, loose adherence at plating and secondary colony formation could not influence this measurement. The data are presented as the natural log of negative wells vs the number of cells plated. The effect of treatment on clonogenic survival was determined by obtaining the linear regression for the data from each treatment group and comparing the slopes of the resulting lines. Presented in this way, the steeper the slope of the linear regression, the greater the clonogenic survival.

Both methods of determining radiation survival were used to determine the effects of treating T24 cells with 5 ^M FTI-277 for 24 h prior to irradiation and 24 h after irradiation. Five ^M FTI-277 was the dose ofinhibitor chosen as inhibition offarnesylation was seen in all cells at this dose, and the treatment was not significantly cytotoxic (not shown). The clonogenic survival ofT24 cells after irradiation from 1 to 4 Gy in the presence ofinhibitor was reduced at all radiation doses (Fig. 11A). The surviving fraction after 2 Gy (SF2) measured by clonogenic survival was reduced from 0.68 to 0.45 by treatment with FTI-277. By extrapolatingfromthelimitingdilutionanalysis (Fig. 11B), the SF2 wasreduced from 0.86 to 0.5. Thus, both methods detected an equivalent reduction in surviving fraction of T24 cells after FTI-277 treatment.

The effect of inhibiting farnesylation was also tested on HS578T, a breast tumor cell line with an H-RasV12 mutation. FTI-277 reducedthe survival ofHS578T after 2 Gy from 0.79 to 0.63 when radiation survival was adjusted for the cytotoxicity of the inhibitor treatment.

In orderto ensure that the radiosensitization seen after FTI-277 treatment was aprop-erty common to FTase inhibitors, and not a unique characteristic of FTI-277, we determined the effects of treating T24 cells with another FTase inhibitor, L744,832 (Merck Pharmaceuticals). This inhibitor was independently designed and synthesized and has

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Fig. 11. Radiosensitization of human tumor cells expressing activated H-Ras. (A) T24 cells were pretreated with 5 p.MFTI-277 (■) or with an equal volume of10 mMDTT in DMSO carrier (□) prior to plating for clonogenic survival determination. Dishes were then irradiated with 1-4 Gy as indicated and cultured an additional 24 h. The culture medium was then removed, and cultures were then refed with drug-free medium and allowed to grow for an additional 8 d (controls) or 2 wk (FTI-277 treated) prior to staining for colony formation. The points shown are the mean and standard deviations obtained from at least three plates. The plating efficiency of unirradiated cultures treated with inhibitor was 0.77; the plating efficiency of control cells was 0.85.

T24 cells (B,C) orSKBr-3 cells (D) were platedatthe indicatedcellnumberperwellin96-well microtiter plates after 24 h pretreatment with inhibitor (O ,•) or with an equal concentration of DTT/ DMSO carrier (□,■). Dishes were then irradiated with 2 Gy (solid symbols) and cultured an additional 24 h. All cultures were then re-fedwith drug-free medium to obtain a 10-fold dilution of the inhibitor and allowed to grow for 3 wk. Inhibitor treatment was 5 p,M FTI-277 in (B) and (D), and 5 p,M L744,832 in (C). The points shown are the mean and standard deviations obtained for duplicate plates. Linear regression in all instances had an r2 value above 0.95.

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Fig. 11. Radiosensitization of human tumor cells expressing activated H-Ras. (A) T24 cells were pretreated with 5 p.MFTI-277 (■) or with an equal volume of10 mMDTT in DMSO carrier (□) prior to plating for clonogenic survival determination. Dishes were then irradiated with 1-4 Gy as indicated and cultured an additional 24 h. The culture medium was then removed, and cultures were then refed with drug-free medium and allowed to grow for an additional 8 d (controls) or 2 wk (FTI-277 treated) prior to staining for colony formation. The points shown are the mean and standard deviations obtained from at least three plates. The plating efficiency of unirradiated cultures treated with inhibitor was 0.77; the plating efficiency of control cells was 0.85.

T24 cells (B,C) orSKBr-3 cells (D) were platedatthe indicatedcellnumberperwellin96-well microtiter plates after 24 h pretreatment with inhibitor (O ,•) or with an equal concentration of DTT/ DMSO carrier (□,■). Dishes were then irradiated with 2 Gy (solid symbols) and cultured an additional 24 h. All cultures were then re-fedwith drug-free medium to obtain a 10-fold dilution of the inhibitor and allowed to grow for 3 wk. Inhibitor treatment was 5 p,M FTI-277 in (B) and (D), and 5 p,M L744,832 in (C). The points shown are the mean and standard deviations obtained for duplicate plates. Linear regression in all instances had an r2 value above 0.95.

significant structural and pharmacological differences from FTI-277. L744,832 also effectively blocked H-Ras farnesylation within 24 h of treatment (Fig. 12). L744,832 by itself reduced colony formation by 73%. It also caused a significant reduction in clonogenic survival after 2 Gy irradiation in T24 cells (Fig. 11C). The surviving fraction of irradiated cells treated with this inhibitor was nearly half that of cells irradiated without inhibitor treatment (0.37 vs 0.62) when corrected for decreased clonogenicity. The cumu-

Fig. 12. Inhibition ofH-Ras farnesylation by L744,832. T24 cells were treatedwith 5 pMFTI-277 (277), 2 pML744,832 (744), or DTT/DMSO carrier (C, Control) for 24 hpriorto lysis forprotein analysis by Western blotting. Blots were probed with MAb to H-Ras. Arrow indicates migration of unfarnesylated H-Ras.

Fig. 12. Inhibition ofH-Ras farnesylation by L744,832. T24 cells were treatedwith 5 pMFTI-277 (277), 2 pML744,832 (744), or DTT/DMSO carrier (C, Control) for 24 hpriorto lysis forprotein analysis by Western blotting. Blots were probed with MAb to H-Ras. Arrow indicates migration of unfarnesylated H-Ras.

Fig. 13. Inhibition of K-Ras prenylation by FTI-277. SW480 cells were treated with the indicated concentrations ofFTI-277 (pM) for 48 h. Cell lysates were analyzed by Western blotting with either an H-Ras MAb (top) or a K-Ras MAb (bottom). Arrow indicated unfarnesylated Ras bands.

lative effect was a reduction in clonogenic survival in cells receiving both inhibitor and radiation to 10% of untreated control cells.

To assess whether FTI-277 radiosensitization was specific to cells with activated H-Ras, we examined survival of cells expressing wild-type Ras after FTI-277 treatment. The HT-29 colon carcinoma and SKBr-3 breast cancer cell lines were assessed for survival after 2 Gy irradiation in the presence of 5 pM FTI-277, a dose of inhibitor that was documented to inhibit wild-type H-Ras prenylation in these cells (Fig. 10). The limiting dilution analysis is shown in Fig. 11D for SKBr-3. The SF2 obtained in this analysis was 0.47 for controls and 0.45 in FTI-277-treated cells, showing that inhibition of far-nesylation had no effect on radiation survival in these cells. FTI-277 treatment alone showed some toxicity to HT-29 cells reducing clonogenicity by 35%; however, no significant change in the SF2 ofHT-29 cells was seen after correcting forthe toxicity ofthe drug (SF2 = 0.78 in controls and 0.82 in treated cells). Thus, FTI-277 treatment of cells under conditions that inhibited wild-type H-Ras prenylation did not increase radiation-induced cell death in cells that do not have activated H-Ras oncogenes. These findings are consistent with a correlation between radiation sensitization by FTI-277 and the presence of activated H-Ras oncogenes.

Because the effect of FTI-277 treatment is largely specific for H-Ras over K-Ras, we next asked whether K-Ras prenylation could be blocked by FTI-277. As shown in Fig. 13, the SW480 colon carcinoma cell line expressing H-Ras and activated K-Ras showed altered migration of H-Ras with as little as 2.5 pM FTI-277 treatment, whereas altered migration of K-Ras became evident only at 30 pM FTI-277. At this dose, FTI-277 inhibits both farnesylation and geranylgeranylation (38). Thus, although FTI-277 specifically inhibits farnesylation ofH-Ras and K-Ras remains prenylated at doses ofFTI-277 below 30 pM, at 30 pM some inhibition of K-Ras prenylation was seen. We then used the dose of 30 pM FTI-277 to determine whether SW480 cells could be radiosensitized. The results

Fig. 14. Inhibition of K-Ras prenylation by combined FTI + GGTI treatment. Log phase cultures ofhuman colon (SW480, HT29) or lung (A549) carcinomas were treated with 5 pM FTI-277, 8 pM GGTI-298, orwith both 5 pM FTI-277 plus 8 pM GGTI-298 for 48 h afterwhich cell lysates were obtainedand analyzedby WesternblottingwithMAbs to K-Ras. Control (C) cultures were treated with carrier alone.

Fig. 14. Inhibition of K-Ras prenylation by combined FTI + GGTI treatment. Log phase cultures ofhuman colon (SW480, HT29) or lung (A549) carcinomas were treated with 5 pM FTI-277, 8 pM GGTI-298, orwith both 5 pM FTI-277 plus 8 pM GGTI-298 for 48 h afterwhich cell lysates were obtainedand analyzedby WesternblottingwithMAbs to K-Ras. Control (C) cultures were treated with carrier alone.

of clonogenic survival assays demonstrated the possibility of radiosensitizing human tumor cells expressing activated K-Ras using FTI-277 alone, although the degree of radiosensitization was modest (not shown).

Because the dose of FTI-277 required to inhibit K-Ras prenylation was a dose where inhibition ofGGTase I would be expected, we investigated the possibility that combined FTI plus GGTI treatment could be used as a more effective means of inhibiting K-Ras prenylation and increasing radiation sensitivity. Our findings demonstrate that combining FTI-277 with GGTI-298 results in increased inhibition of K-Ras prenylation in cells with either mutant (SW480 and A549) or wild-type K-Ras (HT-29) (Fig. 14).

Because combined prenyltransferase inhibitor treatment was effective in inhibiting K-Ras prenylation, this combination ofinhibitors was used in clonogenic survival assays to test the effect ofthis treatment on radiation survival in cells with either mutant or wildtype K-Ras (Fig. 15). The radiation survival of the SW480 cell line expressing mutant K-Ras was reduced by the treatment with combined FTI-277 + GGTI-298. In contrast, the radiation survival ofthe HT-29 cell line expressing wild-type Ras was not decreased. HeLa cell radiation survival was similarly unaffected by inhibitor treatment.

This observation was extended to the A549 lung carcinoma cell line expressing activating mutations in K-Ras. As shown in Fig. 16, A549 cells also demonstrated significant radiosensitization at 2 Gy after FTI + GGTI treatment. The SF2 derived from the limiting dilution analysis showedareduction from 0.53 for controls to0.15for inhibitor-treated cells.

These findings demonstrate that combined treatment with FTI + GGTI acts syner-gistically to inhibit prenylation of K-Ras and that this treatment is also effective in radiosensitizing human tumor cells expressing an activated K-Ras oncogene product. In contrast to the increase in radiation-induced cell death caused by prenyltransferase inhibitors in cells with activated Ras, the radiation survival of tumor cell lines that do not express activated Ras was not altered after prenyltransferase inhibitor treatment. The HT29 colon carcinoma (Fig. 14), the SKBr-3 breast carcinoma cells and HeLa cervical carcinoma cells that express wild-type Ras demonstrated no significant decrease in radiation survival after treatment with FTI-277 plus GGTI-298 at doses shown to inhibit K-Ras prenylation (Fig. 14). The inhibitors themselves did, however, reduce clonogenic-ity in these cells to a variable extent that showed no correlation with Ras status. Thus, radiosensitization again correlated with the expression of oncogenic Ras, whereas inhibition of clonogenicity did not.

Fig. 15. Radiation survival ofcells with mutant but not wild-type K-Ras is reduced after inhibition ofK-Ras prenylation by FTI + GGTI. SW480, HT-29 and HeLa cells were treated for 24 h with 5 pM FTI-277 + 8 pMGGTI-298 before irradiation for clonogenic survival determination. Inhibitor treatment was maintained for 24 h after irradiation at which time medium was replaced with inhibitor, free medium. Control cells were treated in the same way as inhibitor-treated cells, but with an equal amount of drug-free diluent. (□), control cells; (■), FTI + GGTI-treated cells.

Fig. 15. Radiation survival ofcells with mutant but not wild-type K-Ras is reduced after inhibition ofK-Ras prenylation by FTI + GGTI. SW480, HT-29 and HeLa cells were treated for 24 h with 5 pM FTI-277 + 8 pMGGTI-298 before irradiation for clonogenic survival determination. Inhibitor treatment was maintained for 24 h after irradiation at which time medium was replaced with inhibitor, free medium. Control cells were treated in the same way as inhibitor-treated cells, but with an equal amount of drug-free diluent. (□), control cells; (■), FTI + GGTI-treated cells.

These findings are in agreement with the results obtained in the REF studies where radiosensitization was only seen in cells with activated Ras, and support the hypothesis that radiosensitization obtained after prenyltransferase treatment may result from inhibition of Ras directly. Two potential alternative explanations for these results presented here are that some other pathway is required in addition to signaling by activated Ras for radiation resistance, and that a prenylated protein in such a pathway is the true target of inhibition. Another possibility is that a component of one of the Ras signaling pathways is the critical target of the prenyltransferase inhibitors where radiation resistance is concerned. A potential candidate couldbe the rho B protein, which is also prenylated. In the latter case, inhibition of signaling at rho B would block the signal imparting radiation resistance to cells, even in the presence of Ras activation.

Although the degree of radiosensitization after prenyltransferase treatment of cells with activated Ras was not large, fractionated doses such as those used in clinical radiotherapy can cause even small increases in cell killing to appreciably improve outcome. This is because clinical radiotherapy involves the delivery of small daily doses of radiation over many weeks of treatment. This has the effect of amplifying small differences in radiosensitivity to the power of the number of treatments delivered (typically 30 or

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