Michael T. Story

Amino-terminal sequence of a large form of basic fibroblast growth factor isolated from human benign prostatic hyperplastic tissue

Homogenization of human benign prostatic hyperplastic tissue in high ionic strength alkaline buffer containing protease inhibitors resulted in the isolation of a 17,400 molecular weight growth factor. When tissue was homogenized in ammonium sulfate at pH 4.5 without protease inhibitors a smaller, 16,600 dalton, growth factor was isolated. Both growth factors reacted with antisera against synthetic peptides whose sequences corresponded to the amino-terminal (1-12), Internal (33-43) and carboxyl-terminal (135-145) portions of basic fibroblast growth factor (bFGF). This suggested that the smaller growth factor was not a truncated form of (1-146) bFGF and that the larger growth factor may contain additional sequences. Amino-terminal sequencing showed the larger growth factor to have the sequence: Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu-Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly- Ala-Phe-Pro-. These results show that the larger growth factor is an 8 amino acid extended from of (1-146) bFGF and it is likely that the smaller growth factor is a proteolytic cleavage product of the larger growth factor produced during the extraction procedure.

Characterization of Growth Factors Derived from the Rat Ventral Prostate

Tissue homogenates of rat ventral prostate were examined for growth factor activity using a fibroblast mitogenesis assay. G-75 Sephadex gel filtration separated the growth factor activity into two peaks. A broad first peak contained 98% of the protein and several growth factor moieties. A smaller second peak (MW 6,000) contained epidermal growth factor (EGF) as determined by binding in both a competitive receptor binding assay and a radioimmunoassay using anti-mouse epidermal growth factor (anti-mEGF). The broad first peak also contained substantial amounts of EGF-like activity as higher MW forms of EGF. The broad first peak was further fractionated by heparin-Sepharose affinity chromatography. A major fraction with growth factor activity eluted at 1.5 M NaCl and this fraction was shown to contain bFGF by immunostaining with antisera prepared against synthetic peptides corresponding to amino acid sequences 1-12 (amino terminal), 33-43 (internal), and 136-145 (carboxy terminal) of basic fibroblast growth factor (bFGF). EGF-like and bFGF-like molecules account for the major growth factor activity in the rat ventral prostate.

Investigative Urology: Regional Concentration of Basic Fibroblast Growth Factor in Normal and Benign Hyperplastic Human Prostates

Basic fibroblast grown factor (bFGF) is a potent mitogen for mesenchymal cells, including fibroblasts cultured from prostate, and has been postulated to play a role in the development of benign prostatic hyperplasia (BPH). If this is the case, it might be expected that bFGF levels would be elevated in the adenomas of BPH and in the periurethral region of the prostate where BPH is believed to arise. This study was undertaken to test this hypothesis. The concentration of bFGF was evaluated in 31 prostates, 13 normal glands and 18 with BPH. A method for quantitating bFGF by radioimmunoassay was developed that enabled growth factor levels to be correlated to the geographic region of the prostate and the histopathology of the specimen. A 2- to 3-fold higher concentration of bFGF (ng./g. of tissue) was noted in the benign hyperplastic prostates when compared with the adult normal glands. Pubertal specimens demonstrated low growth factor levels comparable to those observed in the normal adult group. Two prepubertal prostates analyzed had high levels similar to those measured in the hyperplastic glands. While the levels of bFGF in the normal adult prostates were highest in the periurethral region, statistical analysis failed to demonstrate a significant difference. Similarly, quantitative morphometric evaluation failed to demonstrate any significant differences in bFGF concentration related to the proportion of stromal, epithelial, or lumenal elements in the tissue sections.

Characteristics of FGF-receptors Expressed by Stromal and Epithelial Cells Cultured from Normal and Hyperplastic Prostates

Three fibroblast growth factors (FGFs), acidic FGF (FGF1), basic FGF (FGF2), and keratinocyte growth factor (FGF7) have been identified in prostate. To understand how FGFs regulate growth of the prostate, and to determine if regulation is altered in benign prostatic hyperplasia (BPH), the mitogenic potential of FGFs, receptor binding, and FGF-receptor (FGFR) gene expression of stromal (PS) and epithelial cells (PE) cultured from normal human prostate and BPH where determined. FGF1 and FGF2, but not FGF7, were mitogens for PS. FGF1 and FGF7 were potent mitogens for PE, but FGF2 was a weak mitogen for these cells. Both PS and PE exhibited high affinity binding (pM K) of iodinated-FGF2. The K was 4-fold and 12-fold higher for PS than for PE cultured from normal prostate and BPH, respectively. Northern analysis indicated that PS, but not PE, expressed FGFR type 1 (FGFR1) mRNA. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to evaluate FGFR type 2 (FGFR2) expression. The size of amplified DNA fragments, and nucleotide sequences, indicated that PS also expressed transcripts for the exon IIIc RNA splice variant of FGFR2. A RT-PCR product with the FGFR2 exon IIIb nucleotide sequence joined with the exon IIIc sequence was amplified with poly A+ RNA from PE and primers spanning both exons. Thus, PE did not alternatively splice mRNA for FGFR2 exon IIIb and exon IIIc. No differences in the mitogenic potential of FGFs, receptor binding (K or number of sites), or FGFR gene expression were found in cells cultured from normal prostate and BPH.

Regulation of basic fibroblast growth factor expression by transforming growth factor beta in cultured human prostate stromal cells

Basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGFβ1) are potential autocrine growth regulators of the prostatic stroma, and therefore may play a role in the development of benign prostatic hyperplasia (BPH). We reported [Story et al.: Prostate 22:183–197, 1993] that TGFβ1 increased bFGF and bFGF mRNA expression in cultured human prostate stromal cells (PS). The current study extends those studies and investigates the mechanism by which TGFβ1 upregulates the level of bFGF mRNA. A solution hybridization assay was used to quantitate bFGF mRNA. Treatment of PS for 6 hr with TGFβ1 (1 ng/ml) maximally stimulated bFGF mRNA expression. TGFβ2 and TGFβ3 were similarly active in upregulating bFGF mRNA. TGFβ1 or cycloheximide each increased bFGF mRNA about 3‐fold. The effect of these agents was not additive. This suggested that a labile protein was involved in processing bFGF mRNA. Determination of message stability indicated that the half‐life of bFGF mRNA in TGFβ1‐treated PS was 6.8 hr, as compared to 4.3 hr in untreated cells. The data indicated that posttranscriptional mechanisms, that increased message stability were, at least in part, responsible for upregulation of bFGF mRNA by TGFβ1 in PS. Our studies suggest that growth of the prostatic stroma is regulated by the interaction of members of two families of growth modulators, bFGF and TGFβ. It remains to be determined if an imbalance in this system in favor of stroma hyperplasia plays a role in the development of BPH.

Epidermal Growth Factor is not the Major Growth-Promoting Agent in Extracts of Prostatic Tissue

Extracts of benign prostatic hyperplasia (BPH) contain a factor which is mitogenic for human foreskin fibroblasts in culture. Because of the similarity of BPH extract and epidermal growth factor (EGF) in stimulating quiescent fibroblasts to divide, it was of interest to determine if the prostate-associated growth factor competes for EGF receptor binding. BPH extract was found to compete poorly for 125I-EGF-receptor binding, did not influence the dissociation of cell-bound 125I-EGF and caused only a slight down-regulation of the EGF receptor. These findings indicate that EGF and BPH extract do not recognize the same receptor and that the major growth stimulating activity of BPH extract is not due to EGF.

Expression of transforming growth factor beta 1 (TGFβ1), -β2, and -β3 by cultured human prostate cells

Transforming growth factor betas (TGFβs) are members of a superfamily of polypeptides that control cell cycle progression and a variety of other cellular activities. TGFβ family members, ‐β1, ‐β2, and ‐β3, have been identified in prostate. The levels of expression of these TGFβ isotypes have been reported to vary with the pathologic state of the prostate. While the significance of these observations remains to be elucidated there is little doubt that TGFβs play an important role in controlling growth of the prostate. The prostatic cells expressing TGFβs have not been identified. This information would provide insight into the physiologic role of TGFβs and suggest ways that growth control may be altered in prostate disease. We used stromal (PS) and epithelial (PE) cells, cultured from normal human prostate and benign prostatic hyperplasia (BPH), to study the effect of TGFβs on cell proliferation and TGFβ transcript and protein expression. The proliferation of PS and PE was inhibited by pM quantities of TGFβ1, ‐β2, and ‐β3. Both cell types expressed transcripts for all three TGFβ isotypes, but PS primarily secreted TGFβ1, whereas PE secreted more TGFβ2 than TGFβ1. These observations suggest that TGFβs are antiproliferative agents in vivo, and that the stroma is the source of TGFβ1 while the epithelium is the major source of TGFβ2 in prostate. There were no significant differences in the growth response to TGFβs, the TGFβ‐isotype expressed, or the amount of TGFβ secreted by cells cultured from normal prostate or BPH.

Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

SummaryTo determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Cells were studied in Passages 2 to 8. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue.

Partial purification of a prostatic growth factor.

A 5-step fractionation procedure was used to isolate a prostatic growth factor from a tissue homogenate of human benign prostatic hyperplasia. The procedure made use of the property of prostatic growth factor to occur predominately as a large molecule (greater than 67,000) in low ionic strength solution (Tris with 0.05 M NaCl) but as a smaller molecule (less than 33,000) in high salt buffer (Tris with 1.55 M NaCl). The fractionation scheme consisted of ammonium sulfate precipitation, gel filtration (low ionic strength) and DEAE chromatography. The growth factor activity was eluted from DEAE with high ionic strength buffer followed by 2 gel filtration steps in high ionic strength buffer. The fractionation scheme produced greater than a 200-fold increase in prostatic growth factor activity and 10 per cent recovery. Additional separation steps will be required to purify prostatic growth factor to homogeneity.

Immunodiagnosis in ovarian cancer: Blocking factor activity☆☆☆

Tumor immunology studies have been utilized for development of a blocking factor assay for therapy monitoring in ovarian cancer. The blocking factor index was defined as the arithmetic difference between assays conducted in the presence and absence of the patients serum compared to incubations with normal control lymphocytes. Eighteen advanced ovarian epithelial malignancies have shown blocking factor activity during treatment. Blocking factor has abated in eight patients whose clinical disease completely regressed. Chemotherapy was discontinued after 18 to 24 months. In 10 patients, blocking factor persisted and chemotherapy has been continued. Some of these patients showed decreasing blocking factor; others have shown increases, which led to death due to disseminated disease in four cases. Blocking factor activity was found to correlate with tumor growth.