Biomedical subjects

K Goossens

Publications and source records attributed to K Goossens.

9 recordsLinked to original sources

PubMed2001

Effects and characterization of paracrine factors produced by human prostate stromal cells in bioassays using rat Sertoli cells, LNCaP tumor cells, and cultured prostate epithelial cells.

BACKGROUND: Prostatic stroma affects both proliferation and differentiation of epithelial cells but the factors involved remain poorly understood. In order to identify and characterize potential paracrine mediators, we studied the effects of human prostate fibroblast-conditioned media (PFCM) in three bioassay systems. METHODS: The first bioassay uses transferrin secretion by cultured rat Sertoli cells as an endpoint for differentiating activity. Factors active in this (heterologous) assay were compared to PModS, a mediator of mesenchymal-epithelial interactions in the testis, also produced by rat prostate stromal cells. The two other (homologous) bioassays use LNCaP tumor cells or subcultured human prostate epithelial cells (PEC) as targets. Differentiation is evaluated by prostate-specific antigen (PSA) secretion and reverse transcriptase-polymerase chain reaction (RT-PCR) for a number of markers of epithelial function. Proliferation is assayed by measurements of DNA and thymidine incorporation. RESULTS: PFCM markedly stimulates transferrin production by Sertoli cells. The main factor(s) involved are acid stable and bind to heparin. However, both their size (approximately 37 kDa) and their behavior on reversed-phase chromatography differ from that of PModS. Although PFCM increases total RNA content of LNCaP, it does not increase or restore differentiated function of LNCaP or PEC. Proliferative effects are observed in LNCaP and these effects cannot be neutralized by an antiserum directed against basic fibroblast growth factor (bFGF). Antiproliferative effects are observed in PEC and these effects are largely due to transforming growth factor-beta (TGF-beta). CONCLUSIONS: PFCM provokes differentiating effects in a Sertoli cell bioassay, but unlike with rat stromal cells, the factor(s) involved differ from PModS. In the two homologous systems studied, differentiating effects could not be demonstrated and discordant effects were noted on proliferation. Various bioassay systems will be required to identify the spectrum of mediators present in PFCM.

Animals↗

PubMed2000

Selective activation of the fatty acid synthesis pathway in human prostate cancer.

A substantial subset of breast, colorectal, ovarian, endometrial and prostatic cancers displays markedly elevated expression of immunohistochemically detectable fatty acid synthase, a feature that has been associated with poor prognosis and that may be exploited in anti-neoplastic therapy. Here, using an RNA array hybridisation technique complemented by in situ hybridisation, we report that in prostate cancer fatty acid synthase expression is up-regulated at the mRNA level together with other enzymes of the same metabolic pathway. Contrary to the observations that in many cell systems (including androgen-stimulated LNCaP prostate cancer cells) fatty acid and cholesterol metabolism are co-ordinately regulated so as to supply balanced amounts of lipids for membrane biosynthesis, storage or secretion, no changes in the expression of genes involved in cholesterol synthesis were found. These findings point to selective activation of the fatty acid synthesis pathway and suggest a shift in the balance of lipogenic gene expression in a subgroup of prostate cancers.

Acetyl-CoA Carboxylase↗

PubMed1999

Androgens decrease and retinoids increase the expression of insulin-like growth factor-binding protein-3 in LNcaP prostatic adenocarcinoma cells.

Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all-trans- and 9-cis-retinoic acid: atRA and 9cRA), deltanoids (1alpha,25-dihydroxycholecalciferol: VD3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10(-10) M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10(-6)-10(-5) M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10(-10) M) as well as at growth inhibitory (10(-8) M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.

Adenocarcinoma↗

PubMed1997

Androgens stimulate fatty acid synthase in the human prostate cancer cell line LNCaP.

In addition to modulation of cell proliferation and stimulation of prostate-specific antigen secretion, one of the most striking effects of androgens on the human prostate cancer cell line LNCaP is the accumulation of neutral lipids. These lipids are synthesized de novo, suggesting that LNCaP cells express all enzymes required for endogenous lipogenesis and that the expression and/or activity of some of these enzymes is affected by androgens. One of the key enzymes involved in lipogenesis is fatty acid synthase (FAS), a potential prognostic enzyme and therapeutic target that is found to be frequently overexpressed in a variety of cancers including prostate cancer. Here, using Northern blot analysis, the gene encoding FAS is shown to be abundantly expressed in LNCaP cells and in two other prostate cancer cell lines tested (PC-3 and DU-145). In LNCaP cells, androgen treatment (10(-8) M R1881) causes a 3-4-fold increase in FAS mRNA levels. Concomitantly with the increase in FAS gene expression, androgens induce a 10-12-fold stimulation of FAS activity. Effects are dose- and time-dependent and follow courses similar to those of the androgen induction of lipid accumulation. In support of the involvement of the androgen receptor, steroid specificity of regulation of FAS activity is in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells. Stimulation of FAS activity is inhibited by the antiandrogen Casodex (bicalutamide) and is absent in the androgen receptor-negative cell lines PC-3 and DU-145. Taken together, these data demonstrate that androgens, mediated by the androgen receptor, stimulate the expression and activity of FAS and suggest that stimulation of FAS activity represents at least part of the mechanism by which androgens induce the accumulation of neutral lipids in LNCaP cells.

Adenocarcinoma↗

PubMed1997

Extreme heat- and pressure-resistant 7-kDa protein P2 from the archaeon Sulfolobus solfataricus is dramatically destabilized by a single-point amino acid substitution.

This study reports the characterization of the recombinant 7-kDa protein P2 from Sulfolobus solfataricus and the mutants F31A and F31Y with respect to temperature and pressure stability. As observed in the NMR, FTIR, and CD spectra, wild-type protein and mutants showed substantially similar structures under ambient conditions. However, midpoint transition temperatures of the denaturation process were 361, 334, and 347 K for wild type, F31A, and F31Y mutants, respectively: thus, alanine substitution of phenylalanine destabilized the protein by as much as 27 K. Midpoint transition pressures for wild type and F31Y mutant could not be accurately determined because they lay either beyond (wild type) or close to (F31Y) 14 kbar, a pressure at which water undergoes a phase transition. However, a midpoint transition pressure of 4 kbar could be determined for the F31A mutant, implying a shift in transition of at least 10 kbar. The pressure-induced denaturation was fully reversible; in contrast, thermal denaturation of wild type and mutants was only partially reversible. To our knowledge, both the pressure resistance of protein P2 and the dramatic pressure and temperature destabilization of the F31A mutant are unprecedented. These properties may be largely accounted for by the role of an aromatic cluster where Phe31 is found at the core, because interactions among aromatics are believed to be almost pressure insensitive; furthermore, the alanine substitution of phenylalanine should create a cavity with increased compressibility and flexibility, which also involves an impaired pressure and temperature resistance.

Alanine↗

PubMed1996

Identification of local carboxy-terminal hydrophobic interactions essential for folding or stability of chloramphenicol acetyltransferase.

The role of the carboxy terminal in folding and stabilization of type I chloramphenicol acetyltransferase (CAT1) has been studied by mutagenesis and Fourier transform infrared analysis. We have shown that a CAT mutant truncated by seven amino acid residues folds into active protein. In this study, the last three residues of this truncated CAT mutant were randomized to detect structural information required for achieving a native enzyme conformation. Statistical analysis of sequencing data from randomly chosen mutants revealed that the amino-terminal CAT fragment of 212 amino acid residues is the shortest deletion mutant able to adopt a soluble, enzymatically active structure. This minimal length corresponds to a protein with full-length alpha5-helix in the three-dimensional crystal structure of CAT type III. The amino acid preferences at the carboxy terminal in the randomization experiments suggest that this helix also forms completely in the shortened CAT mutants. In addition correct folding and/or stabilization requires the formation of a hydrophobic + microdomain at the end of the alpha5-helix. The role of this hydrophobic interaction in CAT folding and structure stabilization is discussed.

Amino Acid Sequence↗

PubMed1996

Pressure-tuning the conformation of bovine pancreatic trypsin inhibitor studied by Fourier-transform infrared spectroscopy.

A hydrostatic pressure of 1.5 GPa induces changes in the secondary structure of bovine pancreatic trypsin inhibitor (BPTI) as revealed by the analysis of the amide I' band with Fourier-transform infrared (FTIR) spectroscopy in the diamond anvil cell. The features of the secondary structure remain distinct at high pressure suggesting that the protein does not unfold. The fitted percentages of the secondary structure elements during compression and decompression strongly suggest that the pressure-induced changes are reversible. The pressure-induced changes in the tyrosine side chain band are also reversible. The results demonstrate that the infrared technique explores different aspects of the behaviour of proteins in comparison with two published molecular dynamics studies performed up to 1 GPa [Kitchen, D.B., Reed, L.H. & Levy, R.M.(1992) Biochemistry 31, 10083-10093] and 500 MPa [Brunne, R.M. & van Gunsteren, W.F.(1993) FEBS Lett. 323, 215-217]. A possible explanation for the difference is the time scale of the experiments.

Amides↗

PubMed1995

Pressure effects on the stability of lipoxygenase: Fourier transform-infrared spectroscopy (FT-IR) and enzyme activity studies.

Fourier transform infrared spectroscopy (FT-IR) studies of lipoxygenase at pressures of up to 1.2 GPa have shown changes in the amide I' band which correlate to structural changes of the enzyme. The shift of the frequency maximum of the amide I' band at about 600 MPa suggests a cooperative change in the secondary structure of the protein. Studies of the changes in band width have shown the structural changes at 600 MPa to be irreversible. This has been confirmed by studies of enzyme activity after pressure treatment: exposure to 600 MPa for 30 min (40 degrees C) clearly reduced the activity of lipoxygenase. Anodic gel electrophoresis under non-denaturating conditions revealed a decrease in native protein parallel to the activity loss. A pressure-temperature-phase diagram for soybean lipoxygenase was established.

Enzyme Stability↗

PubMed1995

The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from an mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.

Amino Acid Sequence↗