We have raised specific antibodies to the second immunoglobulin-like domain of fibroblast growth factor receptors (FGFRs) and used these to investigate the expression and subcellular localization of FGFR-1, -2, -3, and -4 in breast epithelial cells. All four receptors classes could be detected in breast cell lines; however, FGFR-4 and FGFR-2 appeared to be expressed at a higher level in breast cancer cell lines than in normal epithelial cells. Surprisingly, FGFR-3 localized in the cell nucleus by immunofluorescence. A second antibody to a separate epitope confirmed this finding and showed that the form of FGFR-3 present must contain an intact kinase domain as well as the growth factor binding domain. Western analysis of fractionated cells revealed the presence of two forms of FGFR-3 of 135 and 110 kDa. The 110-kDa form was predominantly found in the nucleus, whereas the 135 kDa form was sometimes found in the nucleus. RT-PCR analysis of FGFR-3 mRNA showed the presence of a splice variant in which exons 7 and 8 are deleted. This results in the translation of FGFR-3 missing the transmembrane domain but with an intact kinase domain, which could be a soluble, intracellular receptor. Transfection experiments showed that FGFR-3 containing this deletion and no signal peptide gave an identical nuclear staining pattern to that seen in breast epithelial cells. We conclude that two forms of FGFR-3 are present in breast epithelial cells; a fulllength 135-kDa receptor, which has a conventional membrane localization, and a novel soluble form of 110 kDa.

Summary 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-lllb, which is a highaffinity receptor for KGF and find that it is present on malignant and non-malignant epithelial cells. The level of FGFR-2-lllb 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.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are growth factors which may have a physiological role in the normal breast and in breast cancer. A study of the effects of aFGF and bFGF on a variety of breast cell lines and epithelial cells purified from normal breast organoids showed that whereas normal breast cells did not exhibit membrane ruffling in response to either of these growth factors, some breast cancer cell lines did. This difference was not due to lack of receptor since all the cell lines tested were mitogenically stimulated by bFGF. Dominant negative mutations of FGF receptor 3 (FGFR-3) and the small GTP-binding protein p21rac inhibited membrane ruffling, showing that receptor dimerization and phosphorylation and p21rac activation are prerequisites for membrane ruffling in response to aFGF and bFGF. Transient transfection of individual FGFRs into cos-7 cells showed that FGFR-1, FGFR-2 and FGFR-3 could not mediate a membrane ruffling response whereas FGFR-4 could. These studies elucidate one signalling mechanism of FGF and point to differences in the response of normal and cancer breast epithelial cells which may be important in cell motility.

Summary Monoclonal antibodies against two epitopes of FGFR-1 have been used to investigate FGFR-1 expression in the normal and neoplastic human breast. Different forms are detected in the different cell types constituting the normal breast. Moreover, breast cancer cells lack one form of FGFR-1. Western blot analysis showed 115-kDa and 106-kDa forms of FGFR-1 within the human breast. The 115-kDa band corresponds to the beta form of FGFR-1, whereas the 106-kDa band is truncated at the carboxyl terminus. The 106-kDa form of FGFR-1 is the major form present in breast fibroblasts and myoepithelial cells, whereas epithelial cells contain equal amounts of the 11 5-kDa and 106-kDa forms. Breast cancer cells, however, appear to contain only the 115-kDa form of FGFR-1. This expression pattern is reflected in malignant and non-malignant tissue samples. Using reverse transcription polymerase chain reaction (RT-PCR) analysis, we have shown that the 1 06-kDa FGFR-1 isoform is not the previously described alpha 2 receptor that arises from a 25-base pair insertion in the second kinase domain. It is probable that the 106-kDa FGFR-1 has different signalling properties to the full-length receptor, having lost at least one tyrosine at amino acid 766, which is required for phospholipase C activation. This form of FGFR-1 appears to be lost in all breast cancer cells analysed and its absence may have a bearing on malignancy.Keywords: fibroblast growth factor receptor 1; splice variant; human breast cancer; epithelial/myoepithelial cell; monoclonal antibodyThe family of fibroblast growth factors comprises nine structurally related polypeptides that are mitogenic for a variety of cells and that are involved in embryogenesis, angiogenesis and differentiation (Burgess and Maciag, 1989;Miyamoto et al, 1993). Their cellular response is thought to be mediated through cell surface receptors that belong to the superfamily of tyrosine kinase receptors (Johnson et al, 1991;Keegan et al, 1991;Partanen et al, 1991;Jaye et al, 1992). Extracellular matrix and cell surface heparan sulphate proteoglycans are involved in the interaction of these growth factors with their receptors (Klagsbrun and Baird, 1991;Kan et al, 1993).The four fibroblast growth factor receptors are type four tyrosine kinase receptors and are encoded by four distinct genes. They consist of an extracellular ligand binding domain, a transmembrane part and an intracellular split kinase domain that is involved in signal transduction (Figure lA). They share a high amino acid sequence homology and are characterized by a large number of variant forms generated by alternative mRNA splicing (Hou et al, 1991;Jaye et al, 1992). Variants include: the full-length alpha form; the beta form lacking the first immunoglobulin domain; the intracellular gamma form lacking a signal peptide, the first immunoglobulin domain and the acidic box; and a truncated form, alpha 2, which results from a 25-bp insertion in the second kinase domain leading to a frame shift ( Figure IB). The precise biological func...