No abstract

Scatchard analysis of binding of 125I-basic fibroblast growth factor (FGF) to baby hamster kidney (BHK) cells revealed the presence of two binding sites: a high affinity site with KD of 20 pM and 80,000 sites per cell and a low affinity site with KD of about 2 nM and 600,000 sites per cell. The binding to the two sites could be separated by first washing the cells with 2 M NaCl at pH 7.5 which released the low affinity binding and then extracting the cells with 0.5% Triton X-100 to recover the 125I-basic FGF bound to high affinity sites. The binding to the high affinity site was acid sensitive, suggesting that it represented binding to the receptor. Binding to the low affinity site could be competed strongly by heparin and less strongly by heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. Treatment of BHK cells with heparinase abolished 62% of the low affinity binding, suggesting that the low affinity binding represented binding to cell-associated, heparin-like molecules. A variety of other cell types, including bovine capillary endothelial (BCE) cells, also demonstrated both low and high affinity binding sites. To test whether the low affinity binding might play a role in the basic FGF stimulation of plasminogen activator (PA) production by BCE cells, heparin was added to BCE cultures at concentrations which totally blocked binding of 125I-basic FGF to the low affinity sites. Addition of the heparin did not diminish the increased PA production induced by basic FGF. This suggests that the low affinity binding has no direct role in the stimulation of PA production in BCE cells.

Abstract. Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity

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Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propose a model of prostatic homeostasis in which mouse prostatic epithelial stem cells are concentrated in the proximal region of prostatic ducts while the transit-amplifying cells occupy the distal region of the ducts. This model can account for many biological differences between cells of the proximal and distal regions, and has implications for prostatic disease formation.

We previously showed that prostatic stem cells are concentrated in the proximal regions of prostatic ducts. We now report that these stem cells can be purified from isolated proximal duct regions by virtue of their high expression of the cell surface protein stem cell antigen 1 (Sca-1). In an in vivo prostate reconstitution assay, the purified Sca-1-expressing cell population isolated from the proximal region of ducts was more effective in generating prostatic tissue than a comparable population of Sca-1-depleted cells (203.0 ؎ 83.1 mg vs. 11.9 ؎ 9.2 mg) or a population of Sca-1-expressing cells isolated from the remaining regions of ducts (transit-amplifying cells) (31.9 ؎ 24.1 mg). Almost all of the proliferative capacity of the proximal duct Sca-1-expressing cell population resides within the fraction of cells that express high levels of Sca-1 (top one-third), with the proximal region of prostatic ducts containing 7.2-fold more Sca-1 high cells than the remaining regions. More than 60% of the high-expressing cells coexpress ␣6 integrin and the antiapoptotic factor Bcl-2, markers that are also characteristic of stem cells of other origins. Further stratification of the phenotype of the stem cells may enable the development of rational therapies for treating prostate cancer and benign prostatic hyperplasia.prostate ͉ ␣6 integrin ͉ Bcl-2

No abstract

A protein that stimulates the production of plasminogen activator and latent collagenase in cultured bovine capillary endothelial cells has been purified 106-fold from term human placenta by using a combination of heparin affinity chromatography, ion-exchange chromatography, and gel chromatography. The purified molecule has a molecular weight of 18,700 as determined by NaDodSO4/PAGE under both reducing and nonreducing conditions. The purified molecule stimulates the production of plasminogen activator and latent collagenase in a dose-dependent manner between 0.1 and 10 ng of protein/ml. The purified protein also stimulates DNA synthesis and chemotaxis in capillary endothelial cells in the same concentration range. Thus, this molecule has all of the properties predicted for an angiogenic factor.During neovascularization of tumors, new capillaries arise as sprouts from existing microvessels in response to local release of angiogenic factors by the tumor cells (1). It has been proposed that the response of microvascular endothelial cells to angiogenic factors has three major components: an increase in endothelial cell protease production that allows the endothelial cells to penetrate the surrounding tissues, a stimulation of endothelial cell migration toward the source of the angiogenic factor, and an increase in the rate of endothelial cell proliferation (2). Possible correlates of each of these components have been identified in the response of cultured microvascular endothelial cells to angiogenic preparations-increased collagenase and plasminogen activator (PA) production (2), increased endothelial cell motility and chemotaxis (3, 4), and increased rate of multiplication (5, 6). However, it has not been demonstrated whether the stimulation of these different processes is due to a single molecule in the preparations or to several different molecules. Several groups have used heparin-Sepharose affinity chromatography to purify endothelial cell mitogens from various sources (7-12). We have investigated whether heparin-Sepharose affinity chromatography could also be used to purify the PA and collagenase-inducing activity from a preparation with known angiogenic activity and whether the purified proteaseinducing molecule was also able to induce the other activities associated with angiogenesis-increased endothelial cell mitosis and motility. As a source of the protease-inducing activity, we have chosen term human placenta, which has been shown to contain an angiogenic activity (13) and to stimulate PA and collagenase production in cultured capillary endothelial cells (21). and the substance to be tested. After incubation at 370C for 24 hr, the medium was collected from the cultures and was assayed for collagenase as described (16). All collagenase was in a latent form and was activated with trypsin to detect activity. The cell layers from these same cultures were washed twice with cold phosphate-buffered saline, pH 7.5, and were extracted with 0.5% Triton X-100 in 0.1 M sodium phosphate, pH 8.1, and the cell...