title: Overexpression of basic fibroblast growth factor in MCF-7 human breast cancer cells: lack of correlation between inhibition of cell growth and MAP kinase activation.
abstract: Basic fibroblast growth factor (bFGF, FGF-2) is progressively lost from mammary epithelial cells as they become malignant. To investigate the effects of restoring the expression of bFGF in breast cancer cells, we constructed MCF-7 cells that permanently overexpress 18-kD cytoplasm-localizing bFGF (MCF-7/deltaA(FGF)(18) cells) and cells that express both the 18-kD along with the 22- and 24-kD nucleus-localizing bFGF peptides (MCF-7/NCF(FGF)(18,22,24) cells), using retroviral transduction. These stable cell constructs grew more slowly and had a larger fraction of their populations in the G0/G1 phase of the cell cycle than control cells. All forms of bFGF were eluted from MCF-7/NCF(FGF)(18,22,24) cell monolayers with 2 M NaCl, in contrast to fibroblasts that were demonstrated to secrete only the 18-kD bFGF isoform. High-affinity binding of 18-kD 125I-bFGF to these cells was significantly decreased, probably because of competitive binding by the autocrine-secreted bFGF. Recombinant 18-kD bFGF that was previously demonstrated in our laboratory to inhibit proliferation, activate MAP kinase, and induce the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in MCF-7 cells, further inhibited MCF-7/deltaA(FGF)(18) cells but had no effect on MCF-7/NCF(FGF)(18,22,24) cells. The total cellular content of the high-affinity FGF receptors 1-3 was unchanged, but FGF receptor 4 was decreased in MCF-7/NCF(FGF)(18,22,24) cells. Both cell types overexpressing bFGF isoforms had elevated levels of the cyclin-dependent kinase inhibitor p27Kip1 but not that of p21WAF1/CIP1. In MCF-7/deltaA(FGF)(18) cells, FGFR1 and MAP kinase were constitutively phosphorylated. Exogenous recombinant 18-kD bFGF did not accentuate these effects but did induce an increase in the levels of p21WAF1/CIP1 corresponding to the further inhibition induced by exogenous bFGF in these cells. In MCF-7/NCF(FGF)(18,22,24) cells, FGFR1 and MAP kinase were not phosphorylated at baseline nor upon stimulation with recombinant bFGF, and exogenous bFGF only had a minimal effect on low steady-state p21WAF1/CIP1 levels. However, stimulation of these cells with phorbol ester or insulin did result in MAP kinase phosphorylation. While growth-inhibited in the G1 phase of the cell cycle, MCF-7/NCF(FGF)(18,22,24) cells retained active isoforms of cdk2 and the hyperphosphorylated form of Rb. These data suggest that high molecular weight forms of bFGF overexpressed in MCF-7 cells do not activate the receptor-mediated MAP kinase pathway, and do not induce p21WAF1/CIP1 in an autocrine manner, but inhibit proliferation through other, possibly direct nuclear signalling mechanisms.
authors.affiliation: Department of Medicine, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103, USA.
authors.forename: R
authors.initials: R
authors.lastname: Wieder
authors.forename: E
authors.initials: E
authors.lastname: Fenig
authors.forename: H
authors.initials: H
authors.lastname: Wang
authors.forename: Q
authors.initials: Q
authors.lastname: Wang
authors.forename: S
authors.initials: S
authors.lastname: Paglin
authors.forename: T
authors.initials: T
authors.lastname: Menzel
authors.forename: J
authors.initials: J
authors.lastname: Gabrilove
authors.forename: Z
authors.initials: Z
authors.lastname: Fuks
authors.forename: J
authors.initials: J
authors.lastname: Yahalom
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D016213:Cyclins / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D016193:G1 Phase / Q000502:physiology; D006801:Humans; D010766:Phosphorylation; D010770:Phosphotransferases / Q000037:antagonists & inhibitors; D011506:Proteins / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D016213:Cyclins; D011506:Proteins; D017468:Receptors, Fibroblast Growth Factor; D016222:Fibroblast Growth Factor 2; D010770:Phosphotransferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases