Abstract
The level of expression of keratinocyte growth factor (KGF) mRNA has been measured in human breast cell lines, purified populations of epithelial cells, myoepithelial cells and fibroblasts from reduction mammoplasty tissue and a panel of 42 breast cancers and 30 non-malignant human breast tissues using a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) procedure. We found similar levels of KGF mRNA in malignant and non-malignant breast tissues. The study of the amount of KGF mRNA in breast cell lines and purified populations of cells revealed that fibroblasts are the predominant source of KGF with malignant and non-malignant epithelial cells containing very low levels of KGF mRNA. We have examined the distribution of fibroblast growth factor receptor (FGFR)-2-IIIb, which is a high-affinity receptor for KGF and find that it is present on malignant and non-malignant epithelial cells. The level of FGFR-2-IIIb present on breast cancer cell lines was sufficient for KGF stimulation of breast cancer cell proliferation. Other members of the fibroblast growth factor family have been either not expressed in the human breast (FGF3, FGF4) or have been found at much reduced levels in breast cancer (FGF1, FGF2) and this is the first member of the family to potentially influence the progression of breast cancer through stimulation of cell division.
Highlights
The breast cancer cell lines MCF7, ZR-75-1, MDA-MB-361, MDA-MB-453, MDA-MB-157, BT-20, SKBRIII, PMC42 and the non-malignant epithelial cell snes HBR-SV 161 and MCFlOa were tested by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the expression of keratinocyte growth factor (KGF) mRNA
Other non-breast cell lines were included in this assay as well as populations of epithelal cells, myoepithelial cells and fibroblasts purified from reduction mammoplasty tissue
The purity of the epitheral and myoepithelial cell populations was tested by RTPCR using primers for epithelial membrane antigen (EMA), which is a marker for epithelial cells, and common acute lymphoblastic leukaemia antigen (CALLA), which is a marker for myoepithelial cells
Summary
Reverse transcriptase was from Gibco-BRL (Paisley, UK), Taq polymerase from Peninsula Laboratories (UK), DNA polymerase klenow fragment and dNTPs from Pharmacia (Uppsala, Sweden). All but three of these cell lines were cultured in RPMI-1640 medium buffered with 25 mm Hepes and supplemented with 10% fetal calf serum (FCS), 100 units ml-' penicillin, 100 jig ml-1 streptomycin and 2 mM Lglutamine. The SKBR111 cells were grown in McCoy's SA medium with the same supplements as above and the MCF1Oa cells in a medium containing equal quantities of Dulbecco's modified eagle medium (DMEM) and Ham's nutrient mixture F-12 buffered with 15 mm Hepes with the following supplements: 10 gg ml-1 insulin, 1.4 nm hydrocortisone, 100 ng ml-l cholera enterotoxin, 20 ng ml-1 epidermal growth factor, 5% horse serum, 2 mm glutamine, 100 units ml-' penicillin and 100 jg mll streptomycin.
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