Immunohistochemical staining of human breast tissues, using an antibody against fibroblast growth factor receptor 3 [FGFR-3], showed differences in cellular distribution. Both malignant and non-malignant epithelial cells contained FGFR-3 immunoreactivity, but myoepithelial cells and stroma were negative. The staining pattern in malignant epithelial cells was predominantly nuclear, whereas epithelial cells in normal breast tissue showed both cytoplasmic and nuclear elements. Reverse transcription-polymerase chain reaction (RT-PCR) revealed two isoforms of FGFR-3 corresponding to the FGFR-3-IIIb variant and a previously described exon-deleted nuclear form of FGFR-3, which were present in both malignant and non-malignant epithelial cells. The higher level of nuclear staining and loss of cytoplasmic staining seen in malignant epithelial cells did not correspond to an increase in expression of the exon-deleted form of FGFR-3, nor to any detectable activating point mutations. Since receptor activation can result in its movement to a perinuclear localization, an alternative explanation for the redistribution of FGFR-3-IIIb could be different degrees of activation by a ligand (FGF1 or FGF9). No FGF9 was detected by immunohistochemistry in breast tissues. FGF1, however, is present in the majority of breast cancers and a different tissue distribution of FGF1 was found in breast tissues, showing predominantly nuclear, or a mix of nuclear and cytoplasmic FGFR-3. The difference in FGFR-3 staining patterns may implicate this ligand-receptor pair in breast cancer.

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Summary Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 370C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast.Keywords: breast cancer; fibroblast growth factor 1; protease; immunohistochemistry Fibroblast growth factor 1 (FGF1) belongs to a family of multifunctional polypeptides that are involved in a wide array of biological processes, which include cellular proliferation and differentiation, angiogenesis, chemotaxis, embryonal development and tissue repair (Basilico and Moscatelli, 1992). To date, the FGFs consist of a family of nine homologous polypeptide growth factors that include FGF1 (acidic FGF), FGF2 (basic FGF), FGF3 (int-2), FGF4 (hst-l/Kaposi FGF), FGF5, FGF6 (hst-2), FGF7 (keratinocyte growth factor), FGF8 (androgen-induced growth factor) and FGF9 (glial-activating factor) (Basilico and Moscatelli, 1992;Tanaka et al, 1992;Miyamoto et al, 1993). These proteins share 35-50% overall homology of their amino acid sequences (Basilico and Moscatelli, 1992;Givol and Yayon, 1992).Unlike other members of the family, FGF1, FGF2 and FGF9 are synthesized without a signal peptide sequence and thus may remain sequestered in the cell (Basilico and Moscatelli, 1992;Cao and Pettersson, 1993). However, release of FGF may occur through leakage from damaged cells or from viable cells via a novel mechanism (Mignatti et al, 1992;Cao and Pettersson, 1993). Yeoman (1993) has postulated that proteoglycan-bound FGF may be released from the cell surface or extracellular matrix by the action of proteases, and Briozzo et al (1991) have shown that Received 10 June 1996 Revised 26 November 1996 Accepted 3 December 1996 Correspondence to: J Gomm MCF7 breast cancer cells secrete cathepsin D, which is able to digest the extracellular matrix and releas...

In order to investigate the specific factors controlling the growth of normal breast cell types, purified populations of human breast epithelial and myoepithelial cells from reduction mammoplasties were grown in primary culture in three defined media and their response to foetal calf serum (FCS), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) measured using MTT growth assays. Epithelial and myoepithelial cells differed markedly in their growth requirements. Whereas epithelial cell survival was dependent on the presence of FCS, myoepithelial cell growth was dramatically inhibited by serum. EGF and FGF2 were mitogenic for epithelial cells but not myoepithelial cells, the addition of insulin being the only essential supplement required for myoepithelial cell growth. Heparin inhibited FGF2-stimulated epithelial cell growth but also basal myoepithelial cell proliferation and this inhibition could be overcome by the addition of EGF. Neutralizing antibodies to EGF also inhibited basal myoepithelial cell growth. This suggests the possibility of an autocrine role for a heparin-binding member of the EGF family in the growth of myoepithelial cells. Purified cells combined to form lobuloalveolar structures when incubated in a reconstituted basement membrane matrix (Matrigel) in the presence of EGF and FGF2.

Summary Limonene, a monocyclic monoterpene, occurs naturally in orange peel oil. It has been shown to exhibit both chemopreventive and chemotherapeutic activity without toxicity in rodent models. In this study we examined the effect of limonene both at maximally optimal and suboptimal doses and in combination with suboptimal doses of 4-hydroxyandrostrenedione on nitrosmethylurea-induced rat mammary tumours. A 10% limonene dose mixed in the diet caused tumour regression in all animals. A 5% limonene dose was only able to cause regression in 50% of the rats (P <0.05). A suboptimal dose of 4-hydroxyandrostrenedione (12.5mgkg 1) resulted in tumour re ression in 75% of rats. A combination of 5% limonene with 4-hydroxyandrostrenedione (12.5mgkg-) resulted in a greater tumour regression (83.3%) than either agent given individually (P <0.001 and 0.006 for limonene/4-hydroxyandrostrenedione vs limonene alone and 4-hydroxyandrostrenedione alone respectively).