title: Overexpression of mature insulin-like growth factor (IGF)-II leads to growth arrest in Caco-2 human colon cancer cells.
abstract: Caco-2 cells produce both mature 7500 M(r) and higher M(r) forms of IGF-II (pro IGF-II - pIGF-II) and pIGF-II production is much higher than that of mature IGF-II (mIGF-II). The present study was performed to determine whether overexpression of mIGF-II or pIGF-II stimulates Caco-2 cell growth. A pIGF-II cDNA construct that expresses IGF-II including the E-domain was prepared by cloning a 1250 bp BamH I-Apa I human prepro IGF-II cDNA fragment downstream of the CMV promoter in pcDNA3. To create a mIGF-II cDNA construct which does not express the E-domain, two stop codons were inserted right after the glutamine residue of the D-peptide by site directed mutagenesis utilizing the pIGF-II cDNA expression construct as the template. Caco-2 cells were stably transfected with the mIGF-II or pIGF-II construct. Secretion of the mature and higher M(r) forms of IGF-II into serum-free medium was higher in clones transfected with the mIGF-II or pIGF-II expression constructs compared to vector controls. Both IGF-II clonal cell lines grew faster than the control Caco-2 cells until six days of culture. However, at day 12 the final cell density of the pIGF-II expressing cells was higher than that of the mature IGF-II clones. Western blot analysis of cell lysates at day 8 through day 12 with anti-IGF-I receptor (IGF-IR) beta subunit antibody revealed that the mature IGF-IR levels were lower in both IGF-II overexpressing cell lines compared to the vector control clone. Furthermore, it was shown that at day 12 the IGF-IR levels were significantly lower in mIGF-II clones than in pIGF-II clones. These results indicate that mIGF-II is more effective in down-regulating the IGF-IR than pIGF-II. We propose that overexpression of mIGF-II causes down-regulation of the IGF-IR, leading to growth arrest of Caco-2 cells.
authors.affiliation: Division of Life Sciences, Hallym University, 1 Okchon Dong, Chunchon 200-702, Republic of Korea.
authors.forename: Eun Ji
authors.initials: EJ
authors.lastname: Kim
authors.forename: P Elly
authors.initials: PE
authors.lastname: Holthuizen
authors.forename: Jaebong
authors.initials: J
authors.lastname: Kim
authors.forename: Jung Han Yoon
authors.initials: JH
authors.lastname: Park
mesh_terms: D015153:Blotting, Western; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003062:Codon; D018388:Codon, Terminator; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D005786:Gene Expression Regulation; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis* / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection
chemical_list: D003062:Codon; D018388:Codon, Terminator; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D010455:Peptides; D005973:Glutamine; D007335:Insulin-Like Growth Factor II