Radiosensitization of 24kDa bFGF Expressing Cells by FTI277

As discussed previously, FTI was shown to radiosensitize human cells expressing activated Ras. Given the possibility that bFGF radiation resistance was also mediated by a prenylated protein, we analyzed the effect ofthe specific inhibitors FTI-277 on the radio-sensitivity ofthe radioresistant HeLacells expressing the 24-kDabFGF isoform. We first compared the effect ofinhibiting protein farnesylation on the ability ofradioresistant 24-kDa bFGF transfected HeLa cells (HeLa 3A) and control cells (HeLa PINA) to survive irradiation. Cells were treated with the specific FTase inhibitor, FTI-277 (20 pM) for 48 h prior to irradiation. At these concentrations, no inhibition of cellular proliferation was detected. After 48 h of treatment with FTI-277, Ha-Ras processing was inhibited as evidenced by the appearance of the slowly migrating unprocessed form as documented by Western-blot analysis (Fig. 19A), whereas no effect was seen on the processing of the geranylgeranylated RaplA protein (Fig. 19B).

We next determined the effect of FTase inhibition on radiation survival of HeLa 3A and HeLa PINA cell lines in clonogenic assays (Fig. 20). FTI-277 did not affect the sensitivity of HeLa PINA to radiation treatment. In contrast, the radioresistance of HeLa 3A was dramatically reduced in the presence of 20 pM FTI-277 prior to irradiation. Thus, FTI-277 increases the sensitivity of HeLa 3A but not HeLa PINA cells to ionizing radiation.

To be certain that the radiosensitization of cells expressing the 24-kDa bFGF isoform by FTI-277 was not owing to a change in the distribution ofthe different isoforms, particularly to a switch from the 24-kDa to the 18-kDa isoform, cells were treated as described previously with FTI-277 and the cellular content of the various bFGF isoforms was analyzed for HeLa PINA and HeLa 3A. FTI-277 treatment did not alter bFGF distribution in HeLa 3A and in HeLa PINA (not shown). Thus, the radiosensitizer effect of FTI-277 on HeLa 3A could not be attributed to changes in bFGF isoforms expression.

We next asked whether the radiosensitizer effect of FTI-277 was caused by induction of apoptosis as we had shown in Ha-Ras transfected REF (see ref. 41 and Fig. 5). No radiation-induced apoptosis was detected after FTI-277 treatment of either HeLa 3A or HeLa PINA as assessed at 4, 24, and 48 h after irradiation. However, morphological examination of irradiated HeLa PINA and HeLa3A cells treated with FTI-277 prior to irradiation revealed a significant increase in the percentage of giant cells in the HeLa3A cells, whereas no effect was noticed for radiosensitive HeLa PINA cells (not shown). This finding is consistent with FTI-277 radiosensitization of HeLa cells expressing the 24-kDa isoform through increased postmitotic cell death rather than by induction of apoptosis.

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