Insulin-like growth factor (IGF)-binding protein-3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in human prostate cancer cells.
Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can stimulate apoptosis and inhibit cell proliferation directly and independently of binding IGFs or indirectly by forming complexes with IGF-I and IGF-II that prevent them from activating the IGF-I receptor to stimulate cell survival and proliferation. To date, IGF-independent actions only have been demonstrated in a limited number of cells that do not synthesize or respond to IGFs. To assess the general importance of IGF-independent mechanisms, we have generated human IGFBP-3 mutants that cannot bind IGF-I or IGF-II by substituting alanine for six residues in the proposed IGF binding site, Ile(56)/Tyr(57)/Arg(75)/Leu(77)/Leu(80)/Leu(81), and expressing the 6m-hIGFBP-3 mutant construct in Chinese hamster ovary cells. Binding of both IGF-I and IGF-II to 6m-hIGFBP-3 was reduced >80-fold. The nonbinding 6m-hIGFBP-3 mutant still was able to inhibit DNA synthesis in a mink lung epithelial cell line in which inhibition by wild-type hIGFBP-3 previously had been shown to be exclusively IGF-independent. 6m-hIGFBP-3 only can act by IGF-independent mechanisms since it is unable to form complexes with the IGFs that inhibit their action. We next compared the ability of wild-type and 6m-hIGFBP-3 to stimulate apoptosis in serum-deprived PC-3 human prostate cancer cells. PC-3 cells are known to synthesize and respond to IGF-II, so that IGFBP-3 could potentially act by either IGF-dependent or IGF-independent mechanisms. In fact, 6m-hIGFBP-3 stimulated PC-3 cell death and stimulated apoptosis-induced DNA fragmentation to the same extent and with the same concentration dependence as wild-type hIGFBP-3. These results indicate that IGF-independent mechanisms are major contributors to IGFBP-3-induced apoptosis in PC-3 cells and may play a wider role in the antiproliferative and antitumorigenic actions of IGFBP-3.
Hong
Jiang
J
Clinical Endocrinology Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Zhang
George
G
Dong
Feng
F
Rechler
Matthew M
MM
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D016466:CHO Cells; D016923:Cell Death; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics* / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008297:Male; D008907:Mink; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D018972:Insulin-Like Growth Factor Binding Protein 3; D008024:Ligands; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D007930:Leucine