Sequence analysis of the transcription control region upstream of the human FGF-3 gene.

With the purpose of studying the transcriptional regulation of the human FGF-3 gene, we have cloned and determined the nucleotide sequence of the 11-kbp region flanking its 5' end. Analysis of the sequence disclosed the presence of multiple repetitive elements. Remarkably, all of them were found to have inserted in the same orientation as the FGF-3 gene, suggesting that the whole upstream region could play a role in the control of its transcription. Unique regions within the sequence were scanned for the presence of transcriptional regulatory elements. A potential "Initiator" sequence preceded by several motifs homologous to binding sites for transcription factors pinpointed a putative promoter, 6 kbp upstream of the ATG codon for the FGF-3 protein. A 250-nt sequence stretch surrounding the "Initiator" was found to display punctate homology with the first (P1) of the three promoters (P1, P2 and P3) of the mouse Fgf-3/int-2 gene, specifically in the region of the transcriptional start sites. These data should be useful in studying the mechanisms of regulation of the FGF-3 transcription unit.

Induction of apoptosis in rat hepatocarcinoma cells by expression of IGF-I antisense c-DNA.

BACKGROUND/AIMS: We have developed a gene therapy strategy based on the observation that insulin-like growth factor I (IGF-I) is necessary for the acquisition and maintenance of the transformed phenotype in hepatocarcinoma. This strategy consists in transfecting the rat hepatoma cell line with an episomal vector expressing the antisense IGF-I c-DNA under the control of the metallothionein I promoter inducible by zinc, decreasing therefore the level of IGF-I in these cells. The transfected clones lost their tumorigenic properties, and were able to induce, in vivo, the regression of an established tumor in syngeneic rats. To understand the loss of tumorigenic properties of these transfected clones, we have quantified, by different approaches, the number of apoptotic cells according to the level of IGF-I expression. METHODS: IGF-I antisense synthesis in transfected cells was stimulated using zinc. We then characterized and quantified apoptosis, in these transfected clones, by morphological and DNA fragmentation analyses, flow cytometry and comet assay. RESULTS: We have demonstrated that IGF-I inhibits the development of apoptosis in parental cells, that the transfected clones are able to restore the spontaneous apoptotic programme, and that apoptosis increases massively when overexpression of IGF-I antisense is caused by zinc stimulation of the metallothionein I promoter. CONCLUSION: The present results allow us to conclude that the level of apoptotic pathway in liver cell lines is directly related to the amount of IGF-I deficiency.