ATM-dependent expression of the insulin-like growth factor-I receptor in a pathway regulating radiation response

Abstract

The ATM gene is mutated in the syndrome of ataxia telangiectasia (AT), associated with neurologic dysfunction, growth abnormalities, and extreme radiosensitivity. Insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor with tyrosine kinase activity that can mediate mitogenesis, cell transformation, and inhibition of apoptosis. We report here that AT cells express low levels of IGF-IR and show decreased IGF-IR promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-IR promoter activity and elevated IGF-IR levels, whereas expression in wild-type cells of a dominant negative fragment of ATM specifically reduces IGF-IR expression, results consistent with a role for ATM in regulating IGF-IR expression at the level of transcription. When expression of IGF-IR cDNA is forced in AT cells via a heterologous viral promoter, near normal radioresistance is conferred on the cells. Conversely, in ATM cells complemented with the ATM cDNA, specific inhibition of the IGF-IR pathway prevents correction of the radiosensitivity. Taken together, these results establish a fundamental link between ATM function and IGF-IR expression and suggest that reduced expression of IGF-IR contributes to the radiosensitivity of AT cells. In addition, because IGF-I plays a major role in human growth and metabolism and serves as a survival and differentiation factor for developing neuronal tissue, these results may provide a basis for understanding other aspects of the AT syndrome, including the growth abnormalities, insulin resistance, and neurodegeneration.

Figure 1

Influence of ATM on IGF-IR expression. (A) Western blot analysis of IGF-IR expression levels in AT mutant, heterozygous, and wild-type human cells. All cells are SV40-transformed fibroblasts, except 3487 and 3489, which are primary fibroblasts from an affected individual and his heterozygous parent. (B) Analysis of ATM and IGF-IR expression in GM5849 cells, an SV40-transformed human fibroblast cell line from an AT individual, and a series of subclones transfected with wild-type ATM cDNA. ATM expression was determined by immune precipitation with anti-ATM antibody, followed by Western blot analysis with the same antibody. IGF-IR expression was determined directly by Western blot. (C) Confirmation of functional complementation of GM5849 cells by ATM cDNA expression based on radiation response. Clonogenic survival was determined following IR in the ATM-deficient GM5489 cells and in two subclones (5849cA2 and 5849cA4), transfected with a vector expressing the wild-type ATM cDNA. Also included for comparison is an unrelated wild-type cell line, GM637.

Figure 2

ATM-dependent activation of the IGF-IR promoter. To examine activation of the IGF-IR promoter as a function of ATM status, the indicated cells were transfected with a plasmid containing the luciferase gene driven by base pairs −2350 to +640 of the rat IGF-IR promoter region, along with a β-galactosidase expression vector as a control for transfection efficiency. The luciferase activity, as a measure of IGF-IR activation, was normalized to β-gal expression in each case. Experiments were performed in triplicate, and the results shown are the mean of three separate experiments. (A) Comparison of IGF-IR promoter activity in ATM-deficient GM5849 cells (SV40-transformed fibroblasts) and a subclone transfected with a vector expressing wild-type ATM cDNA (5849cA2), along with an unrelated wild-type SV40 transformed fibroblast cell line, GM637. (B) Comparison of IGF-IR promoter activity in primary AT fibroblasts obtained from an affected individual (GM3487) and from his heterozygous parent (GM3489).

Figure 3

Effect of expression in wild-type cells of a dominant negative ATM fragment on (A) IGF-IR and EGFR levels and (B) clonogenic survival following IR. An SV40-transformed fibroblast cell line from an apparently normal individual (GM637) was transfected with a vector expressing a fragment of ATM containing the leucine zipper domain, which has been shown to function as a dominant negative protein (14). IGF-IR and EGFR levels were determined by Western blot in the 637 cells and in the cells transfected with the dominant negative leucine zipper fragment. Subclones 1 and 4 (LucZip1 and LucZip4) were chosen for analysis of radiation survival in comparison with the parental 637 cells.

Figure 4

Increased radiation survival in AT cells upon forced expression of IGF-IR. (A) Western blot analysis of IGF-IR expression in GM5849 cells, which lack ATM, and in GM5849 subclones transfected with a plasmid-expressing IGF-IR driven by a viral promoter. (B) Clonogenic survival after exposure to IR of subclones 3 (5849cI3) and 7 (5849cI7), both of which were judged by Western blot in A to express increased levels of IGF-IR, in comparison with the parental 5849 AT mutant cells and to an unrelated wild-type cell line, 637.

Figure 5

Specific inhibition of the IGF-IR pathway blocks correction of the radiosensitivity in AT cells by the ATM cDNA. GM5849 cells, deficient in ATM, were transfected with an ATM expression plasmid, yielding subclones expressing wild-type ATM (see Fig. 1). (A) The radiation survival of one complemented subclone, 5849cA4, was determined in the presence and absence of a neutralizing anti-IGF-IR antibody added to the growth medium. (B) The ATM cDNA-complemented subclone 5849cA4 was further transfected with a vector expressing a dominant negative variant of IGF-IR (486stop). The clonogenic survival of one transfectant subclone (c1) and of a cell population consisting of a number of pooled transfected clones (pooled) was determined in comparison with 5849cA4 and with the original parent line, 5849.