Stroma and the heparin-binding fibroblast growth factor (FGF) family influence normal epithelial cell growth and differentiation in embryonic and adult tissues. The role of stromal cells and the expression of isoforms of the FGF ligand and receptor family were examined during malignant progression of epithelial cells from a differentiated, slowly growing, nonmalignant model rat prostate tumor. In syngeneic hosts, a mixture of stromal and epithelial cells resulted in nonmalignant tumors which were differentiated and slowly growing.In the absence of the stromal cells, epithelial cells progressed to malignant tumors which were independent of the stroma and undifferentiated. The independence of the malignant epithelial cells from stromal cells was accompanied by a switch from exclusive expression of exon Illb to exclusive expression of exon IlIc in the FGF receptor 2 (FGF-R2) gene. The FGF-R2(IIIb) isoform displays high affinity for stromal cell-derived FGF-7, whereas the FGF-R2(Ic) isoform does not recognize FGF-7 but has high affinity for the FGF-2 member of the The development of prostate tumors is age related and believed to progress by a series of genetic changes and selection of cells with increasing malignant character. Most marked is the shift from relatively slowly growing, nonmetastatic, androgen-sensitive tumors to a rapidly growing, androgen-independent, highly malignant stage (15). The androgen-sensitive tumors are subject to antiandrogen therapies which can result in regression of the tumor, but frequently appearance of a highly malignant tumor that is resistant to treatment follows such therapies. A lack of knowledge of mechanisms underlying the appearance of the malignant tumors has hampered design of strategies for prediction and prevention of their appearance as well as intervention after they appear. The epithelial cell compartment of slowly growing, androgen-sensitive tumors usually exhibits some degree of morphological differentiation that distinguishes it from the stromal compartment. In contrast, malignant tumors are undifferentiated and exhibit no apparent relationship between epithelial and stromal cells (15). Mesenchyme plays an active role in growth and differentiation of the epithelium during prostate development (4, 6), and the stroma has been implicated in maintenance of adult epithelium (5, 9). However, the role of the stroma in development and progression of tumors has received less attention. Advances in isolation and maintenance of specific prostate cell types in vitro and the identification of purified * Corresponding author. t Present address: Department of Urology, Gunma University, Maebashi, Gunma 371, Japan. polypeptide regulators that act directly on them has renewed optimism for understanding the cellular and molecular basis of the progression of prostate tumors (25,(27)(28)(29)(30)39). Isolated epithelial and stromal cells from both normal prostate tissue and slowly growing, androgen-sensitive tumors are insensitive to androgen, yet they are responsive to multiple polypeptid...

Apoptosis of mammalian cells is accompanied by various morphological changes including nuclear condensation, DNA fragmentation and cell surface changes. Methods developed over the past few years have focused on detection of DNA-associated changes that occur rather late in apoptosis. However, detection of apoptosis at early stages, before gross morphological changes, is critical for understanding the pathways of programmed cell death. In this report, we describe a rapid and reliable assay for detecting early stages of apoptosis. This assay is based on the observation that soon after initiating apoptosis, most mammalian cell types translocate phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be specifically detected by staining with fluorescein isothiocyanate (FITC)-labeled annexin V (annexin V-FITC), a protein with a strong, natural affinity for PS. Using this assay, we have detected apoptotic cells in culture, in real time, using fluorescence microscopy and flow cytometry. In combination with vital dye staining, the progressive stages of apoptosis were observed. PS redistribution occurs earlier than DNA-associated changes and membrane leakage. In addition, PS externalization occurs during apoptosis induced by a variety of stimuli. Therefore, the annexin V binding assay provides an excellent indicator for the early stages of apoptosis.

The growth of isolated epithelial and stromal cells from both androgen-dependent normal rat prostate and an androgen-responsive model rat prostate tumor is androgen-independent. When added to co-cultures of epithelial and stromal cells separated by a semipermeable membrane, androgen stimulated epithelial cell growth without an effect on stromal cell growth. Northern blot and nuclease protection analysis of mRNA revealed that stromal cells specifically expressed an androgen-sensitive secreted member of the heparin-binding fibroblast growth factor family [keratinocyte growth factor (KGF)/fibroblast growth factor-7]. KGF was mitogenic for epithelial cells, but not for stromal cells. Epithelial cells expressed specifically a splice variant of the bek receptor gene that specifically binds KGF. Expression of the bek receptor gene in stromal cells was undetectable by Northern blot and nuclease protection analyses. The results suggest that stromal cell-derived KGF has the properties of an andromedin, which mediates the indirect control of epithelial cell proliferation by androgen through a directional stromal-to-epithelial cell paracrine mechanism.

Two tandem immunoglobulin-like disulfide loops (Loops II and III) linked by a short connecting sequence in the ectodomain of the fibroblast growth factor receptor kinase compose the binding sites for glycosaminoglycan and fibroblast growth factor (FGF) ligands. Alternate splicing of exons IIIb and IIIc coding for the COOH-terminal half of Loop III confers high affinity for FGF-7 or FGF-2, respectively, on the fibroblast growth factor receptor ectodomain without effect on the binding of FGF-1. Here we show that a 139-amino acid fragment composed of Loop II, the inter-Loop II/III sequence, and a short segment of the NH2 terminus of Loop III is sufficient and near the minimal requirement for binding of FGF-1, FGF-2, and FGF-7. Extension of the fragment by five additional highly conserved residues (SD(P/A)QP) within a distinct constitutive structural domain (fl1) in Loop III restricts the binding of FGF-7 without effect on FGF-1 and FGF-2. Since the presence of exon IIIc in the full-length ectodomain does not change this ligand binding profile, we suggest that alternately spliced exon IIIc plays no active role in binding of the three ligands. In contrast, exon IIIb actively abrogates the restriction on the binding of FGF-7 and concurrently lowers the affinity for FGF-2.

Alternate splicing of a single exon encoding an NH2-terminal immunoglobulin (Ig) disulfide loop in the ectodomain of the fibroblast growth factor receptor (FGFR) types 1 and 2 results in alpha and beta isoforms that exhibit 3- and 2-Ig loops, respectively. Previously we demonstrated that alternately spliced Loop I has no independent ligand binding activity but is sufficiently interactive with the ligand- and heparin-binding site formed by Loops II and III to lower affinity for the same fibroblast growth factor (FGF) ligand. Here we show that a lower affinity of FGFR1 alpha for heparin parallels the lower affinity for FGF-1. A mutant of FGFR1 alpha in which the sequence between Loops I and II was deleted exhibits high affinity for both FGF-1 and heparin and other properties of the FGFR1 beta isoform, which include resistance to degradation by trypsin and display of specific antibody epitopes. This suggests that the interloop sequence facilitates the interaction of Loop I with Loops II and III. Lack of expression of both exons coding for Loop I and the sequence between Loops I and II in the FGFR2 gene characterizes rat prostate tumor cells, which exhibit a loss of the low affinity class of FGF receptors. Although the exon coding for the sequence between Loops I and II is alternately spliced in the FGFR2 beta isoform, coordinate expression with the exon coding for Loop I results in the functional differences between the FGFR alpha and FGFR beta variants.