Overexpression of insulin-like growth facloi-II gene (IGF2) in Wilms' tumor occurs through a process independent on the loss of heterogosity or loss of imprinting (W.U. Wang el al. : J. Biol. Them. 271,27X63, 1996) In the human chondrosarcoma-denved HCS-2/8 and -2/A cell lines, cloned by M Takigawa et al. (Cancel Res. 49 39%, 1989;Int J. Cancel 48. 717, 1991), total expression from 4 promoters (PI, P2, P3, P4) of 1GF2 and expression of H19 were 4-ft times and 1-0.5 times, respectively, compared to human normal chondrocytcs. In order to probe the mechanism of IGF2 Overexpression and repressive H19 expression in HCS-2/8 and -2/A cells, the imprinting and hetcro/ygosity ot 1GF2 and H19 were examined by restriction fragment length poly moi phi suis (RFLP) and sequence analyses with their RT-PCR products. RT-PCR products from all promoters and genomic PCR product of 1GF2 in HCS-2/8 were perfectly digested at Apal site on Exon9 (Ex9), suggesting the paternal allelc expression. However, the RT-PCR product from PI was partially cut at Alu\ site on the 5'-region of Ex3, of which the sequence data indicated both alleles expression. In RT-PCR product from P4, lacking of 4 bases in Ace I site at joint portion between Ex6-E.x7 occured bv the alternative splicing. While, RT-PCR product of the 5′-rcgion of E.x5 on H19 in HCS-2/K was not digested by Rsa] at all, suggesting the maternal allelc expression. Sequence data of the RT-PCR product suggested the existence of other RF''LP sites (MscI, DdeI, BsgI,) besides Alu\ and Rsal sites. These results imply that IGF2 Overexpression and repressive H19 expression may not relate directly with the imprinting status of IGF2 and HI9 in the chodrosarccma cells.
|Publication status||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology