Abstract
Insulin-like growth factor-II (IGF-II) is a potent mitogen for cells in culture. TheH19gene is a developmentally regulated gene with putative tumor suppressor activity, and loss ofH19expression may be involved in tumorigenesis. TheH19gene is closely linked to the human IGF-II gene (IGF2) on chromosome 11p15.5 and these genes are reciprocally imprinted in most fetal tissues.H19is expressed only from the maternal andIGF2from the paternal chromosome. We have asked whether overexpression of proIGF-II altersH19imprinting 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,RsaIrestriction fragment length polymorphism (RFLP) and found to be informative (+/−).H19expression was monoallelic (+) indicating preservation ofH19imprinting in all cell lines. Using quantitative RT-PCR with internal competitors for H19 and for IGF-II cDNA, overexpression ofIGF2in 293-11 and 293-9 cells was confirmed. In contrast, no significant difference with respect toH19expression was detected between the overexpressing cells and control lines. In conclusion, (1) human embryonal fibroblasts express theH19gene. (2)H19imprinting is preserved in these cells. (3) proIGF-II overexpression does not alterH19expression.
Published Version
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