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Biomedical subjects

E Mulder

Publications and source records attributed to E Mulder.

At least 37 records · Page 2Linked to original sources
PubMed1991

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones.

The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGF alpha-like activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mutated amino acid in the steroid binding domain of the androgen receptor.

Cell Division↗
PubMed1991

Androgen receptor abnormalities.

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2-8, we identified a G----T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the activation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G----C) results in an aspartic acid----histidine substitution (with negligible androgen binding), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A----G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors in the human androgen receptor have an enormous impact on phenotype and fertility.

Amino Acid Sequence↗
PubMed1991

Autoradiographic analysis of diaminopimelic acid incorporation in filamentous cells of Escherichia coli: repression of peptidoglycan synthesis around the nucleoid.

Peptidoglycan synthesis rate in nonconstricting filaments of Escherichia coli dnaX(Ts) has been studied by autoradiography of incorporated [3H]diaminopimelic acid. Analysis of autoradiograms of whole cells and sacculi showed that peptidoglycan is synthesized at a reduced rate in the nucleoid-containing parts of these filaments. The lower rate of peptidoglycan synthesis in the cell center coincides with a higher local rate of protein synthesis. DNA-less cell formation in dnaX(Ts), dnaX(Ts) sfiA, and the minB minicell-forming mutant is accompanied by a local increase in peptidoglycan synthesis at the constriction site.

Autoradiography↗
PubMed1991

Androgen receptor expression in human tissues: an immunohistochemical study.

The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.

Antibodies, Monoclonal↗
PubMed1990

The androgen receptor: functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines.

The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT codon encoding a threonine residue to GCT, encoding alanine.

Amino Acid Sequence↗
PubMed1990

Stimulatory effects of antiandrogens on LNCaP human prostate tumor cell growth, EGF-receptor level and acid phosphatase secretion.

LNCaP cells (derived from a lymph node carcinoma of the human prostate) show androgen responsive growth. Progestagens, estradiol and antiandrogens competed with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen-sensitive systems. Optimal growth (3-4 fold increase in DNA content of 6 day cell cultures vs controls) was observed after addition of the synthetic androgen R1881 (0.1 nM). Both steroidal and non-steroidal antiandrogens did not suppress the androgen responsive growth. At a concentration of 10 nM cyproterone acetate or 100 nM RU 23908, growth was even stimulated to an extent comparable to that observed after addition of androgen. Cyproterone acetate and RU 23908 also increased the number of epidermal growth factor receptors expressed at the cell surface to a comparable level as did the androgen. Like androgens, cyproterone acetate, RU 23908 or estradiol stimulated the secretion per cell of prostate specific acid phosphatase in the culture fluid. In conclusion, antiandrogens can exert striking stimulatory effects on the proliferation of LNCaP cells probably due to a defective androgen receptor system. It is discussed that comparable changes in the specificity of the androgen receptor in prostate cancer cells may give these cells an advantage in growth rate and may contribute to development of tumors characterized as hormone independent.

Acid Phosphatase↗
PubMed1990

A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens.

LNCaP prostate tumor cells contain an abnormal androgen receptor system. Progestagens, estradiol and anti-androgens can compete with androgens for binding to the androgen receptor and can stimulate both cell growth and excretion of prostate specific acid phosphatase. We have discovered in the LNCaP androgen receptor a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or Hela cells. Androgens, progestagens, estrogens and anti-androgens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene construct (GRE-tk-CAT). The mutation therefore influences both binding and the induction of gene expression by different steroids and antisteroids.

Binding Sites↗
PubMed1990

Unusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: high affinity for progestagenic and estrogenic steroids.

UNLABELLED: LNCaP tumor cells, derived from a metastatic lesion of a human prostatic carcinoma, are androgen-sensitive in cell culture. Although increase in growth rate is observed with low doses of progestagens or estradiol, these cells contain exclusively androgen receptors. In the present study the binding affinity of different ligands for both non-DNA- and DNA-binding (transformed) forms of the androgen receptor were analyzed. The cytosolic (non-transformed) form of the receptor displayed an abnormal high affinity for progestagens and estradiol when compared with the cytosolic androgen receptor from other sources. Subsequently the non-transformed forms of the androgen receptor obtained from LNCaP cell nuclei was studied. A high binding affinity was found not only for dihydrotestosterone, but also for progesterone and the synthetic progestagen R5020 (relative binding affinity 42% and 10% of dihydrotestosterone). The binding characteristics of the transformed androgen receptor were examined in intact cells at 37 degrees C. LNCaP cells were compared in this respect with COS cells containing the cloned human androgen receptor, normal human skin fibroblasts and PC3 (prostate) and NHIK (cervix) human tumor cell lines. The affinity of the transformed androgen receptors for the progestagen R5020 in LNCaP cells was significantly higher than in the other cell systems, although the differences were less pronounced than for the non-transformed receptor form. IN CONCLUSION: the LNCaP tumor cells contain an androgen receptor with an abnormal binding site. This might be due to a mutation and/or a post-transcriptional effect.

Androgens↗
PubMed1990

Androgen receptor heterogeneity in human prostatic carcinomas visualized by immunohistochemistry.

Expression of the human androgen receptor was examined in 26 primary prostatic carcinomas by immunohistochemical staining with a polyclonal antibody reactive with the N-terminal domain of the human androgen receptor. Eighteen carcinomas showed homogeneous staining for the androgen receptor, whereas in seven cases a considerable heterogeneity in expression of the receptor was found. In one case, only a very limited number of immunoreactive tumour cells were detected. Comparison of androgen receptor expression with the tumour grading score, according to the MD Anderson grading system, revealed that the proportion of immunostained tumour cells and--to a lesser extent--the intensity of immunostaining were decreased in the more aggressive (grade III) tumours. The use of immunohistochemistry for detection of expression of androgen receptor in prostatic carcinomas may become a new and sensitive method for predicting prostatic tumour behaviour under hormonal therapy and prognosis.

Aged↗
PubMed1990

The Escherichia coli minB mutation resembles gyrB in defective nucleoid segregation and decreased negative supercoiling of plasmids.

Nucleoid segregation in the Escherichia coli minB mutant and in cells that over-produce minB gene products appeared defective as measured from fluorescence micrographs. Electrophoretic resolution of topoisomers of plasmid isolates from the minB strain revealed a decreased level of negative supercoiling; in addition, multimerization was observed. Over-production of the minB gene product also resulted in a decreased level of negative supercoiling. This phenotype is typical of the gyrB(ts) mutant, which is known to be affected in chromosome decatenation and supercoiling. We propose that the minB mutation and over-production of the minB gene products cause a defect in nucleoid segregation, which may be related to the decrease in negative supercoiling. As in the gyrB(ts) mutant, retardation of nucleoid segregation is proposed to inhibit constriction initiation in the cell centre and to give rise to nucleoid-free cell poles. As a consequence, these cells divide between nucleoid and cell pole, resulting in minicell and (sometimes) in anucleate cell formation.

Bacterial Proteins↗
PubMed1990

Antiproliferative effects of suramin on androgen responsive tumour cells.

UNLABELLED: The effect of the polyanionic drug suramin on two androgen responsive tumour cell lines was studied. Human prostate tumour (LNCaP) cell growth is stimulated two- to three-fold by the synthetic androgen R1881 (0.1 nM) or EGF (1 ng/ml). Suramin (0.01-1.0 mM) inhibited the growth of LNCaP cells in a dose dependent way, both in the presence and absence of androgen or EGF. Growth was arrested in the G0/G1 phase of the cell cycle, but was resumed after removal of suramin. DDT-1 hamster ductus deferens tumour cells are stimulated by PDGF (25 ng/ml), b-FGF (10 ng/ml) and testosterone (10 nM). Suramin inhibited PDGF and b-FGF stimulated cell growth. However in the presence of testosterone, suramin showed a biphasic effect: stimulatory at low dose (0.01 mM) and inhibitory above 0.01 mM. Suramin decreased the apparent affinity of EGF binding sites on LNCaP cells with a two- to eight-fold increase in Kd at 0.1 and 1.0 mM suramin, respectively. IN CONCLUSION: suramin counteracts the growth stimulatory effects of both androgens and growth factors on androgen sensitive tumour cells. The effects are reversible after withdrawal of suramin.

Androgen Antagonists↗
PubMed1989

Glucocorticoid receptors in alveolar macrophages: methodological aspects of the determination of the number of glucocorticoid receptors per cell.

Among patients with asthma or chronic obstructive pulmonary diseases the response to glucocorticoids varies considerably. To investigate the possible relationship between clinical response and number of glucocorticoid receptors in alveolar macrophages or their KD value a micro receptor assay was developed. Assay conditions were adjusted because of receptor occupancy by endogenous or therapeutically used glucocorticoids and high aspecific binding.

Cell Line↗
PubMed1989

The human androgen receptor: domain structure, genomic organization and regulation of expression.

The domain structure and the genomic organization of the human androgen receptor (hAR) has been studied after molecular cloning and characterization of cDNA and genomic DNA encoding the hAR. The cDNA sequence reveals an open reading frame of 2751 nucleotides encoding a protein of 917 amino acids with a calculated molecular mass of 98,845 D. The N-terminal region of the hAR is characterized by a high content of acidic amino acid residues and by several homopolymeric amino acid stretches. The DNA-binding domain showed a high homology with the DNA-binding domain of the human glucocorticoid receptor (hGR) and the human progesterone receptor (hPR). The predominantly hydrophobic steroid binding domain of the hAR is 50-55% homologous with the ligand binding domains of the hGR and hPR. Transient expression of recombinant AR cDNA in COS-cells resulted in the production of a 110 kDa protein with the expected binding specificity of androgen receptors. Co-transfection with a reporter-gene construct [CAT(chloramphenicol acetyl transferase) under direction of the androgen regulated MMTV-promoter] showed that the protein is functionally active with respect to transcription regulation. In the LNCaP prostate carcinoma cell line two major (11 and 8 kb) and one minor (4.7 kb) mRNA species can be found which can be down-regulated by androgens. The hAR protein coding region was shown to be divided over eight exons with an organization similar to that of the progesterone and oestrogen receptor. The sequence encoding the N-terminal domain was found in one large exon. The two DNA-binding fingers were encoded by two small exons; the information for the androgen-binding domain was found to be distributed over five exons. Southern blot analysis of genomic DNA revealed that the hAR is encoded by one single gene, which is situated on the X-chromosome.

Animals↗
PubMed1989

Mechanism of androgen action: recent observations on the domain structure of androgen receptors and the induction of EGF-receptors by androgens in prostate tumor cells.

In this paper two different aspects of androgen action are reviewed. Polyacrylamide gel electrophoresis of androgen receptors, photoaffinity labeled with R1881 showed that receptors isolated from both human prostate cells and calf uterine cytosol cells are proteins with a molecular mass of approx 110 kD. Purification to homogeneity of this form of the receptor from calf uterus also yielded a 110 kD protein. A molecular model for the DNA-binding form of the receptor is presented in which one polypeptide comprises three active domains: one for ligand binding, one for interaction with nuclear acceptor sites, and a third domain which modulates nuclear interaction. Mild digestion with chymotrypsin or a protease from rat prostates removes the modulating domain and leaves the ligand binding and nuclear interaction domain intact. Trypsin treatment yields a fragment of lower molecular mass containing the ligand binding domain with some affinity for RNA, but not DNA. In vitro studies with a human prostate tumor cell line (LNCaP), suggest that androgens not only directly effect cell growth, but also act indirectly. Both epidermal growth factor (EGF) and androgens stimulate cell growth. In addition androgens stimulate synthesis of receptors for EGF. Thus androgens effect tumor cell growth by autocrine or paracrine mechanisms by making the cells more sensitive for growth factor mediated stimuli.

Androgens↗
PubMed1989

Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor.

Antibodies against the N-terminal domain of the human androgen receptor (hAR) were prepared by two different approaches. Firstly, rabbits were immunized with a beta-galactosidase-hAR (amino acids (aa) 174-353) fusion protein. Secondly, two synthetic peptides corresponding to potentially antigenic sites located within this fragment (aa 201-222 and 301-320) were used as immunogens. The obtained antisera contained high titer anti-hAR antibodies as was established with several independent methods (e.g. sucrose gradient centrifugation, immunoprecipitation, Western blotting). The two anti-peptide antisera specifically stained nuclei of glandular epithelial cells in frozen sections of human prostate tissue. Progesterone, estradiol and glucocorticoid receptors were not immunoprecipitated with these antisera. The specific hAR antibodies provide new tools for the characterization of this steroid receptor as well as for diagnostic purposes in pathology of the human prostate and androgen resistance.

Animals↗
PubMed1989

Actively replicating nucleoids influence positioning of division sites in Escherichia coli filaments forming cells lacking DNA.

The positioning of constrictions in Escherichia coli filaments pinching off anucleate cells was analyzed by fluorescence microscopy of dnaX(Ts), dnaX(Ts) sfiA, dnaA46(Ts), gyrA(Am) supF(Ts), and gyrB(Ts) mutants. In filaments with actively replicating nucleoids, constrictions were positioned close to the nucleoid, whereas in nonreplicating filaments, positioning of constrictions within the anucleate region was nearly random. We conclude that constriction positioning depends in an unknown way on nucleoid replication activity.

Cell Division↗