OVEREXPRESSION OF BASIC FIBROBLAST-GROWTH-FACTOR IN MCF-7 HUMAN BREAST-CANCER CELLS - LACK OF CORRELATION BETWEEN INHIBITION OF CELL-GROWTHAND MAP KINASE ACTIVATION

Basic fibroblast growth factor (bFGF, FGF-2) is progressively lost fro m mammary epithelial cells as they become malignant. To investigate th e effects of restoring the expression of bFGF in breast cancer cells, we constructed MCF-7 cells that permanently overexpress 18-kD cytoplas m-localizing bFGF (MCF-7/Delta A(FCF(18)) cells) and cells that expres s both the 18-kD along with the 22- and 24-kD nucleus-localizing bFGF peptides (MCF-7/NCFFGF(18,22,24) cells), using retroviral transduction . These stable cell constructs grew more slowly and had a larger fract ion of their populations in the G(0)/G(1) phase of the cell cycle than control cells. All forms of bFGF were eluted from MCF-7/NCFFGF(18,22. 24) cell monolayers with 2 M NaCl, in contrast to fibroblasts that wer e demonstrated to secrete only the 18-kD bFGF isoform. High-affinity b inding of 18-kD I-125-bFGF to these cells was significantly decreased, probably because of competitive binding by the autocrine-secreted bFG F. Recombinant 18-kD bFGF that was previously demonstrated in our labo ratory to inhibit proliferation, activate MAP kinase, and induce the c yclin-dependent kinase inhibitor p21(WAF1/CIP1) in MCF-7 cells, furthe r inhibited MCF-7/Delta A(FGF(18)) cells but had no effect on MCF-7/NC FFGF(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 M CF-7/NCFFGF(18,22,24) cells. Both cell types overexpressing bFGF isofo rms had elevated levels of the cyclin-dependent kinase inhibitor p27(K ip1) but not that of p21(WAF1/CIP1). In MCF-7/Delta A(FGF(18)) cells, FGFR1 and MAP kinase were constitutively phosphorylated. Exogenous rec ombinant 18-kD bFGF did not accentuate these effects but did induce an increase in the levels oi p21(WAF1/CIP1) corresponding to the further inhibition induced by exogenous bFGF in these cells. In MCF-7/NCFFGF( 18,22,24) cells, FGFR1 and MAP kinase were not phosphorylated at basel ine nor upon stimulation with recombinant bFGF, and exogenous bFGF onl y had a minimal effect on low steady-state p21(WAF1/CIP1) levels. Howe ver, stimulation of these cells with phorbol ester or insulin did resu lt in MAP kinase phosphorylation. While growth-inhibited in the G(1) p hase of the cell cycle, MCF-7/NCFFGF(18,22,24) cells retained active i soforms of cdk2 and the hyperphosphorylated form oi Rb. These data sug gest that high molecular weight forms of bFGF overexpressed in MCF-7 c ells do not activate the receptor-mediated MAP kinase pathway, and do not induce p21(WAF1/CIP1) in an autocrine manner, but inhibit prolifer ation through other, possibly direct nuclear signalling mechanisms. (C ) 1998 Wiley Liss Inc.