Fumio Wada

Changes in gene expression of growth factors and their receptors during castration-induced involution and androgen-induced regrowth of rat prostates

To find candidates for the mediator of the growth‐promoting action of androgen in rat prostates, the changes in the steady‐state levels of mRNAs coding for several growth factors and their receptors were examined by Northern blot analysis during castration‐induced involution, and subsequent regrowth induced by androgen in the ventral and dorsolateral lobes. The changes in the growth factor systems and a typical secretory protein in the ventral lobe were similar to, but more prominent than, those in the dorsolateral lobe, showing the higher androgen dependency of the ventral lobe. Among the growth factors and their receptors investigated, only epidermal growth factor (EGF) showed apparent positive androgen dependency: EGF mRNA content in the ventral lobe decreased to about 30% of the normal level within 24 hr after castration, and increased, attaining about 200–300% of the normal level 3–5 days after androgen administration to castrated rats. mRNAs coding for all other factors examined, i.e., transforming growth factor‐α (TGF‐α), EGF receptor, basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1, TGF‐β1, TGF‐β type II receptor, hepatocyte growth factor (HGF), and c‐MET/HGF receptor, increased after castration in greater or lesser degree, and after a brief pause or a decrease some of them increased again attaining a second peak 3–5 days after androgen replacement. The second increase was evident in TGF‐α, EGF receptor, KGF, and c‐MET mRNAs. These results indicate the possibility that multiple growth factor‐receptor systems participate in the androgen‐dependent regrowth of castrated rat prostates.

Participation of the microsomal electron transport system involving cytochrome P-450 in ω-oxidation of fatty acids

Abstract The participation of the microsomal electron transport system involving cytochrome P-450 in ω-oxidation of fatty acids by a rat liver preparation was examined since ω-oxidation involves microsomal reactions requiring both NADPH and molecular oxygen. ω-Oxidation of fatty acids was inhibited by CO and by the antibody against NADPH-cytochrome c reductase. The addition to the reaction mixture of drugs which interact with cytochrome P-450 inhibited ω-oxidation. It is concluded that the microsomal electron transport system involving cytochrome P-450 functions in ω-oxidation of fatty acids.

Studies on fatty acid ω-oxidation antiketogenic effect and gluconeogenicity of dicarboxylic acids

1. Administration of dicarboxylic acids to starving rats decreased the concentration of ketone bodies in the blood. 2. Incorporation of 14C into blood glucose was greater from dicarboxylic acids than from monocarboxylic acids. 3. These results suggest that omega-oxidation may be important for production of succinyl-CoA from fatty acids. 4. In starving or diabetic rats about 15% of palmitic acid were subjected to omega-oxidation and then beta-oxidation.

Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen

SummaryPrimary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC 404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively, of prostate epithelial cells in vivo.

A human prostatic growth factor (hPGF) : partial purification and characterization

A growth factor capable of stimulating DNA synthesis of BALB/3T3 cells was purified about 1,000-fold from the cytosol of human benign hypertrophic prostates by heparin-Sepharose chromatography; the growth factor bound to the column in the presence of 0.5 M NaCl was eluted with 1.5-1.7 M NaCl. Its molecular weight and isoelectric point were estimated to be 11,000-13,000 and 10.5, respectively. It was sensitive to heat- and acid-treatments but resistant to disulfide-reducing agent. The final preparation was able to stimulate DNA synthesis at 10 ng/ml. The degree of stimulation was dependent on serum concentration in the assay system; the degree of maximum stimulation increased about 5 times as serum concentration increased from 0.2 to 2%.

Stabilization of fibroblast growth factors by a non-cytotoxic zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS).

SummaryThe potential usefulness of a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), in the stabilization of acidic and basic fibroblast growth factors (FGFs) was examined. Among several detergents, CHAPS was found to be not only non-cytotoxic but also most useful in handing the diluted preparations of FGFs. The advantages are as follows: 1) at lower concentrations than 0.01% CHAPS did not affect growth factor activity of calf serum (CS) and the growth rate of BLAB/c 3T3 cells. The primary culture of rat prostate epithelium and colony formation of NRK-49F cells were hardly influenced by CHAPS lower than 0.003%; 2) the loss of FGFs that usually occurs due to their adherence to the surface of storage containers was effectively prevented by inclusion of 0.1% CHAPS; 3) the recovery of FGFs after storage or dialysis was significantly enhanced by inclusion of 0.1% CHAPS; 4) CHAPS at lower concentrations than 0.1% does not interfere with amino acid analysis, except that Thr may be misled only when the ratio of protein/CHAPS is low; 5) amino acid sequence analysis was hardly disturbed by CHAPS up to 0.5%. These results indicate that CHAPS is useful as a stabilizing agent for various kinds of polypeptides capable of showing biological activity at a low concentration.

Immunochemical studies on malate dehydrogenase (decarboxylating) (NADP)

Abstract 1. 1. Malate dehydrogenase (decarboxylating) (NADP) ( l -malate:NADP oxidoreductase (decarboxylating), EC 1.1.1.40) was purified from the supernatant fraction of rat liver. 2. 2. Antibody against this purified enzyme was prepared. This antibody inhibited the activity of the enzyme and precipitated it. 3. 3. Malate dehydrogenase (decarboxylating) (NADP) in supernatant fractions from kidney, heart, brain and adipose tissue of rats reacted with the antibody in the same manner as that of the ra tliver enzyme in the inhibition tests. Supernantant malate dehydrogenases from all these tissues except adipose tissue formed single precipitin bands with the antibody on Ouchterlony double-diffusion analysis which fused with each other. 4. 4. The enzyme from the livers of different species differed immunologically from that of rat liver. 5. 5. Mitochondrial enzyme was immunologically quite distinct from supernatant enzyme. 6. 6. Increase and decrease in enzyme activities in the cytoplasm of liver under various dietary and hormonal conditions were accompanied by proportional changes in the quantity of immunologically reactive protein.

Isolation and characterization of androgen-dependent non-histone chromosomal protein from dorsolateral prostate of rats

Abstract Rat prostate contains a unique androgen-dependent non-histone protein (Matuo et al. (1)). The non-histone protein was isolated in homogeneous form by extraction of nuclei from the dorsolateral prostate with 0.35 M NaCl in the presence of 1 mM PMSF and chromatography on a CM-Sepharose column. The final fraction was greater than 98% pure as judged by electrophoreses in SDS- and acid/urea-gels. The purified protein had a molecular weight of approximately 20,000, and an isoelectric point of approximately 11.5. Its absorption peak was at 276 nm and A(1%, 276nm)=9.3. The protein is characterized by the absence of cysteine, histidine and tryptophan, and by the high content of methionine, tyrosine and phenylalanine.

Production of IGF-II-related peptide by an anaplastic cells line (AT-3) established from the dunning prostatic carcinoma of rats

SummaryAT-3 cells, one of anaplastic cell lines established from the Dunning prostatic carcinoma of rats, were able to grow under serum-free conditions in a state of suspension detached from a substratum. Radioimmunoassays using monoclonal antibody against rat insulin-like growth factor II (IGF-II) revealed the presence of IGF-II-related peptide in acid-ethanol extracts extracsts of lyophilized serum-free media conditioned by AT-3 cell. The peptide contents in the culture media increased with increase in cell number; 71 ng at 3.0 × 106 cells and 449 ng at 4.6 × 107 cells. IGF-II-related peptide was hardly detectable in acid-ethanol extracts of AT-3 cells harvested after 13-days culture. These results indicate that AT-3 cells produce IGF-II-related peptide ana may release it into the culture media.

Difference in androgen-dependent change of non-histone proteins between dorsolateral and ventral prostates of rats

Abstract A novel species of non-histone protein having a molecular weight of approximately 20,000 (20K), abundantly localized in the dorsolateral prostate, was found to be decreased in the content by castration and to be restored by replacement of androgen, in addition to non-histone proteins of molecular weights ≥ 34,000. The content of 20K DNA was more rapidly decreased by castration, but more slowly restored by replacement of androgen, with the dorsolateral prostate than the ventral prostate. Of other nuclear proteins, non-histone proteins of molecular weights ≥ 90,000 in the dorsolateral prostate were more susceptible to the decrease by castration, whereas those of all kinds of histones were hardly dependent on the androgen level.