Abstract

While numerous researchers have used rat models to investigate the in vivo actions of IGF-I, interpretation of the results in terms of true concentrations of rat IGF-I (rIGF-I) in plasma has been hampered by the absence of homologous reference standards. In order to overcome this we have produced recombinant rIGF-I (rrIGF-I) from Escherichia coli using procedures similar to those we have previously described for the production of other recombinant IGFs. The rrIGF-I is indistinguishable from serum-derived rIGF-I when characterized in a number of in vitro assays including ability to stimulate protein synthesis and inhibit protein degradation in cultured rat cells, as well as in interactions with the rat type-1 IGF receptor and with rat IGF-binding proteins. Moreover, both the serum-derived and the recombinant rat proteins are similar to recombinant human IGF-I (rhIGF-I) in these assays. However, differences between the human and rat IGFs are apparent when tested in immunoassays using some antibodies raised against rhIGF-I. Furthermore, the differences between rhIGF-I and rrIGF-I are even greater when rhIGF-I is used as the competing radiolabel in these assays, a situation that can lead to a two- to threefold underestimation of the actual concentration of IGF-I in rat plasma. These results indicate that, while immunoassays employing antibodies raised against rhIGF-I and rhIGF-I reference standards reliably indicate trends in IGF-I concentrations in rat plasma, the true amounts of rIGF-I present can only be assured in an assay using homologous tracer and reference peptides.

Journal of Endocrinology (1996) 149, 379–387

To explore the clinical significance of p53 in the pathogenesis of adrenal neoplasms, we investigated the incidence of p53 gene mutations in functioning human adrenal tumours using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to screen p53 exons 4 to 9. We examined 29 adrenocortical adenomas (primary aldosteronism, n=17; Cushing's syndrome, n=12, all benign), and 33 phaeochromocytomas (benign solitary, n=18; benign multiple, n=5; malignant, n=10) in Japanese and Chinese patients. PCR-SSCP did not show any abnormal band-shifts in any of the adrenocortical adenoma and benign solitary phaeochromocytoma tissues. In contrast, six phaeochromocytoma tissues (two cases benign multiple, four cases malignant) showed PCR-SSCP band-shifts. Subsequent DNA sequencing analysis of the shifted bands revealed six cases with nine mutations or intronic sequence alterations: three cases contained sequence alterations within intronic regions, three cases with silent mutation (sequence alteration in codon without amino acid alteration), and three cases contained missense mutations (one case each in exons 5, 6 and 9). Immunohistochemical staining demonstrated that two of three cases with missense mutations and one case with an intronic sequence alteration over-expressed p53 protein in tumour cell nuclei. We observed no association between p53 gene mutation and p21/WAF1/Cip-1 expression. The relatively high incidence of p53 gene mutations or intronic sequence alteration in multiple and malignant phaeochromocytomas, but not in benign solitary cases, suggests that p53 mutation could play some role in the pathogenesis of multiple and/or malignant phaeochromocytomas.