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N Boulle, H Schneid, A Listrat, P Holthuizen, M Binoux, and A Groyer


Initial observations have indicated similarities between bovine and human IGF-II production during development. The aim of the present study was to investigate whether cattle could provide an experimental model that would mimic the complex pattern of human IGF-II gene expression. Expression of bovine IGF-II gene during development was studied by RNA hybridization using various human IGF-II probes. In fetal tissues and in adult muscle, the bovine IGF-II gene was expressed as a family of eight transcripts ranging in size from 5·2 to 1·1 kb. In adult bovine liver, a major IGF-II transcript of 4·4 kb was expressed that could not be detected in any fetal or adult extra-hepatic tissue. During fetal life, quantitative IGF-II mRNA expression differed in liver and muscle, and the relative amounts of the different transcripts varied with the tissue of origin. These observations suggest that the regulation of bovine IGF-II gene expression is specific to the stage of development and the tissue concerned. Moreover its pattern is very similar to that in its human counterpart.

In order to identify a putative homology between human and bovine gene structures, bovine mRNAs were examined for cross-hybridization with various non-coding exons of the human gene. Cross-hybridization was detected with human untranslated exons 5 and 6, suggesting the presence of two distinct promoters similar to the human promoters P3 and P4. The 4·4 kb mRNA species expressed in adult bovine liver failed to hybridize to a probe for human exons 1 and 2, suggesting that the leader sequences of this transcript were different from those present in the human gene. Finally, results obtained with a probe containing the 3′ untranslated end of exon 9 suggested the presence of at least two polyadenylation sites in the bovine gene.

Although differences in IGF-II gene structures were found between cattle and man, the similarities in the pattern of gene expression between the two species suggest that cattle may be a useful model to investigate some developmental aspects of the expression of the human IGF-II gene.

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A Logie, N Boulle, V Gaston, L Perin, P Boudou, Y Le Bouc, and C Gicquel

In adrenocortical tumors, the malignant phenotype is associated with rearrangements (paternal isodisomy) at the 11p15 locus and IGF-II gene overexpression, strongly suggesting that the IGF system is a major determinant of adrenocortical tumor progression. The aim of this study was to validate an in vitro model for investigating the involvement of the IGF system in adrenocortical tumorigenesis. We analyzed the production of IGF mRNA and proteins, IGF-binding proteins (IGFBPs) and IGF receptors by the NCI H295R cell line, which is derived from a human adult adrenocortical carcinoma. H295R cells were shown to proliferate for a long period (26 days) in the absence of serum or any added growth factor. Northern blot analyses showed high IGF-II mRNA contents in H295R cells. The cells secreted large amounts of IGF-II protein (14 ng/10(6) cells per 48 h) although no IGF-I protein was detected. Western ligand blot analyses of conditioned media detected the presence of large amounts of a 34 kDa protein, which was identified as IGFBP-2 by immunoblotting. The presence of high-affinity binding sites for IGF-I and IGF-II on H295R cells was shown by binding experiments using radiolabeled IGFs and confirmed by reverse transcription PCR analyses showing type 1 and type 2 IGF receptors. Proliferation of H295R cells was inhibited by anti-IGF-II antibody (45%) and by anti-type 1 IGF receptor antibody (53%) indicating that IGF-II is an autocrine growth factor for these cells and that its effects are, at least in part, mediated by the type 1 IGF receptor. These findings confirm the involvement of the IGF system in adrenocortical tumors and suggest that the H295R cell line is a suitable in vitro model for studying the molecular mechanisms of adrenocortical tumor proliferation.