Induction of Radiation Resistance by the 24kDa Form of bFGF

In order to examine the relative contributions of the CUG- or AUG-initiated forms in the radioprotective effect attributed to bFGF, cDNA encoding each form were trans-fected in HeLa cells. HeLa cells were transfected with retroviral vector PINA encoding the 24-kDa form (HeLa 3A cells), or the 18-kDa bFGF form (HeLa 5A cells), or the vector alone (HeLa PINA cells). Figure 17 shows the bFGF protein expression profile of the selected clones. Four bands were detected in HeLa and HeLa PINA cells corresponding to the 24-, 21.5-, 21-, and 18-kDa bFGF isoforms. In contrast, HeLa 3A cells only expressed the 24-kDa form. Overexpression of the 24-kDa form was associated with downregulation of the expression of the other parental forms as already described in bovine aortic cells (78). HeLa 5A cells mainly produced the 18-kDa form of bFGF with apparent downregulation of the endogenous high molecular weight forms.

The radiation survival ofthese cell lines was then compared in order to assess the contribution of the 18- and 24-kDa forms to bFGF-mediated radioresistance. As shown in Fig. 18,a significant increase in radioresistance was seen in the 24-kDabFGF transfected cell line compared to wild-type or HeLa PINA cells. In contrast, HeLa 5A cells did not display any significant increase in clonogenic survival compared to the HeLa PINA.

Because exogenous bFGF was reported to inhibit apoptosis in BAEC (61,62), we compared the extent of apoptosis induction 24 h after 5 and 10 Gy irradiation between radioresistant HeLa 3A and HeLa wild-type. No apoptosis was detected in either HeLa wild-type or in HeLa 3A (not shown), suggesting that apoptosis was not involved in irradiation-induced death in these cells.

These findings demonstrate that both high and low molecular weight bFGF isoforms exhibit a distinct capacity to modify cellular radiosensitivity. However, in the HeLa cell sytem used for analysis of bFGF radiation resistance after transfection with different bFGF isoforms, only those cells that were engineered to produce the 24-kDa isoform exhibited a significant increase in clonogenic survival after irradiation (81). The mechanism through which endogenous expression ofthe 24-kDa bFGF isoform mediates radiation resistance is not known, but recent work has demonstrated that in neurons, an

Radiation Resistance

Fig. 17. bFGF protein expression from nonirradiated HeLa wild-type, HeLa PINA, HeLa 3A, and HeLa 5A cell lines. 40 p,g of extracted proteins from each cell line were loaded on 12.5% SDS-PAGE and Western blotting was performed. The migration of the different bFGF isoforms is indicated by molecular weight.

Fig. 17. bFGF protein expression from nonirradiated HeLa wild-type, HeLa PINA, HeLa 3A, and HeLa 5A cell lines. 40 p,g of extracted proteins from each cell line were loaded on 12.5% SDS-PAGE and Western blotting was performed. The migration of the different bFGF isoforms is indicated by molecular weight.

Clonogenic Survival Curves

Fig. 18. Radiation survival curves of HeLa, HeLa PINA, HeLa 3A, HeLa5A. Cells were exposed to various ionizing radiation doses and the radiosensitivity ofthe different cell lines calculated using the clonogenic survival assay. A semi-logarithmic plot of data from the different cell lines is shown. Radiation survival curves were obtained using the quadratic linear model. (A) (•), HeLa and (O), HeLa PINA cells; (B) (O), HeLa PINA and (■), HeLa 3A; (C) (O), HeLa PINA and (▲), HeLa 5A. Each bar represents the mean ± SDofthree different experiments for one selected clone of each transfected cell line. (SD representing less than 1% of variation do not appear on the graph).

Fig. 18. Radiation survival curves of HeLa, HeLa PINA, HeLa 3A, HeLa5A. Cells were exposed to various ionizing radiation doses and the radiosensitivity ofthe different cell lines calculated using the clonogenic survival assay. A semi-logarithmic plot of data from the different cell lines is shown. Radiation survival curves were obtained using the quadratic linear model. (A) (•), HeLa and (O), HeLa PINA cells; (B) (O), HeLa PINA and (■), HeLa 3A; (C) (O), HeLa PINA and (▲), HeLa 5A. Each bar represents the mean ± SDofthree different experiments for one selected clone of each transfected cell line. (SD representing less than 1% of variation do not appear on the graph).

intracellular bFGF receptor is present in the nuclear membrane and that this FGFR binds acidic FGF and with a lower affinity than bFGF (reviewed in ref. 82). The 18-kDa form is secreted and binds to membrane tyrosine kinase receptors leading to the activation of serial intracellular messengers including Ras and Raf, but no interaction between the 24-kDa endogenous pathway and these oncogenes has as yet been described. It cannot yet be excluded that Ras or a small G protein in the Ras family transduces a signal originating with the 24-kDa bFGF isoform. Alternatively, the pathway of radiation resistance induced by the 24-kDa bFGF isoform may be altogether distinct from that observed in Ras transformed cells, and define a new mechanism of radiation resistance.

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