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


CONCLUSIONS
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.
Ellouk-Achard
S
S
Laboratoire de Génétique Moléculaire, CNRS UPR 42, Villejuif, France.
Djenabi
S
S
De Oliveira
G A
GA
Desauty
G
G
Duc
H T
HT
Zohair
M
M
Trojan
J
J
Claude
J R
JR
Sarasin
A
A
Lafarge-Frayssinet
C
C
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology*; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology*; D016376:Oligonucleotides, Antisense; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D007334:Insulin-Like Growth Factor I