Although insulin-like growth factor I (IGF-I) is a mitogenic growth factor, its role in tumorigenesis is unclear. We therefore transfected wild-type and truncated (-subunit mutant ('52STOP) human IGF-I receptor cDNAs into Rat-1 fibroblasts. Rat-1 transfectants expressed 2.5-to 7-fold increased IGF-I receptor mass, while the Kd for IGF-I binding was unchanged. The Rat-i cells transfected with wild-type receptor cDNA responded to in vitro IGF-I treatment by increased proliferation and DNA synthesis. Cells overexpressing wild-type receptors were also transformed as emdenced by ligand-dependent colony proliferation in soft agar. After injection into athymic nude mice, all wild-type transfectants formed solid sarcomas within 3 weeks, and ex vivo tumor cell assays confirmed continued overexpression of human IGF-I receptors. In contrast, both DNA synthesis and proliferation of 952STOP-transfected cells were attenuated below that of untransfected cells. 952STOP cells were nonresponsive to IGF-I in vitro and were unable to sustain anchorageindependent growth. No tumors were induced for up to 8 weeks after h jection of 952STOP transfectants into athymic mice, despite the presence of demonstrable endogenous IGF-I receptors on the 952STOP-transfected cells. Therefore, "52STOP behaves as a dominant negative inhibitor of endogenous IGF-I receptor function, probably by assembling nonfunctional hybrid rat/mutant human receptor tetramers.

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In vitro transcription experiments using a Xenopus laevis cell-free extract have demonstrated that a DNA fragment containing a glucocorticoid response element (GRE) significantly enhances the expression of a methionine tRNA gene. This stimulation can be observed when the element is located in cis or trans. In the cis configuration, the element can be located 2900 basepairs from the gene and still display transcriptional enhancement. In trans, the enhancement can occur at a low element to template molecular ratio. The nucleosome positioning and chromatin structure near the tRNA gene appear to be unaffected by the presence of the long terminal repeat of the mouse mammary tumor virus which contains the GRE. Taken together, these data suggest that GREs can stimulate transcription of hormonally unresponsive genes, perhaps by providing a "molecular sink" to which inhibitors may bind. Further experimentation may reveal a correlation between these in vitro studies and in vivo gene regulation.