AbstractWe investigated the effect of overexpression of the gene for gamma‐glutamyltranspeptidase (GGTP) on the gamma irradiation sensitivity of human bronchoepithelial cell line IB3–1 or A549 lung carcinoma cells. The enzyme has been considered a putative radioprotector because it is the only enzyme capable of degrading extracellular glutathione to ultimately provide cysteine for resynthesis of intracellular glutathione. Clonal sublines of each cell line were isolated after transfection with a plasmid containing both the rat GGTP cDNA and neor gene, and selected in G418. Each transfected clone demonstrated a different degree of GGTP enzymatic expression by biochemical analysis. The D0 of parent IB3–1 cells was 1.434 ± 0.138 Gy, n 2.331 ± 0.685, and was unchanged by GGTP overexpression, D0 1.932 ± 0.690 Gy (P = 0.133), n 2.916 ± 2.7 (P = 0.628). In contrast, overexpression of the MnSOD transgene in IB3–1 cells increase the shoulder on the irradiation survival curve for IB3–1 cells, showing significant increase in the shoulder, D0 1.239 ± 0.1 Gy (P = 0.079), n 7.276 ± 0.1 (P < 0.001). Lung cancer cell line A549 was subcloned after expression of the GGTP transgene, and clonal subline 22 demonstrated stably increased GGTP activity and protein production by Western analysis. The parental A549 line was also redoned by limiting dilution. A series of clonal sublines of clonal line 22 expressed up to a 97‐fold increase in GGTP expression compared to control clones. In clonogenic cell survival assays in the presence or absence of added glutathione, no difference in irradiation sensitivity was detected between parent or GGTP transfected and overexpressing subclones despite an increase in GGTP activity. The D0 of the subclone 10 of parent A549 cells was 1.6 ± 0.40 Gy and n was 4.8 ± 3.0, α 0.13 ± 0.10, β 0.052 ± 0.02; while the D0 for GGTP overexpressing clone 17, a subclone of line 22, was 1.3 ± 0.05 Gy, n 2.6 ± 0.18, a 0.279 ± 0.01, p 0.059 ± 0.01. The cell lines were not significantly different with respect to D0 (P = 0.55), indicating no significant effect of GGTP overexpression on radiosensitivity. Thus, GGTP overexpression in either normal lung bronchoalveolar cell line IB3–1 or lung cancer cell line A549 does not induce radioresistance in vitro. © 1995 Wiley‐Liss, Inc.