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 th e observation that insulin-like growth factor I (IGF-I) is necessary f or the acquisition and maintenance of the transformed phenotype in hep atocarcinoma, 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 transfec ted 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 cl ones, me have quantified, by different approaches, the number of apopt otic 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 clo nes, by morphological and DNA fragmentation analyses, flow cytometry a nd comet assay. Results: We have demonstrated that IGF-I inhibits the development of apoptosis in parental cells, that the transfected clone s are able to restore the spontaneous apoptotic programme, and that ap optosis increases massively when overexpression of IGF-I antisense is caused by zinc stimulation of the metallothionein I promoter. Conclusi on: The present results allow us to conclude that the level of apoptot ic pathway in liver cell lines is directly related to the amount of IG F-I deficiency.