Proinsulin-like growth factor-II overexpression does not alter monoallelic H19 gene expression in transfected human...

Insulin-like growth factor-II (IGF-II) is a potent mitogen for cells in culture. The H19 gene is a developmentally regulated gene with putative tumor suppressor activity, and loss of H19 expression may be involved in tumorigenesis. The H19 gene is closely linked to the human IGF-II gene (IGF2) on chromosome 11p15.5 and these genes are reciprocally imprinted in most fetal tissues. H19 is expressed only from the maternal and IGF2 from the paternal chromosome. We have asked whether overexpression of proIGF-II alters H19 imprinting status and/or expression. Human embryonal kidney fibroblasts (293 cells) were stably transfected with a PCMV5 vector containing the full length human IGF-II cDNA or a control cDNA. Transfectant clones expressed large quantities of IGF-II mRNA and secrete 1-5 ug/ml and 150-230 ng/ml proIGF-II within 24 hours of serum-free culture (transfectant 293-9 and -11 respectively) (1). Cells were genotyped at the exon 5, RsaI restriction fragment length polymorphism (RFLP) and found to be informative (+/-). H19 expression was monoallelic (+) indicating preservation of H19 imprinting in all cell lines. Using quantitative RT-PCR with internal competitors for H19 and for IGF-II cDNA, overexpression of IGF2 in 293-11 and 293-9 cells was confirmed. In contrast, no significant difference with respect to H19 expression was detected between the overexpressing cells and control lines. In conclusion, (1) human embryonal fibroblasts express the H19 gene. (2) H19 imprinting is preserved in these cells. (3) proIGF-II overexpression does not alter H19 expression.