Biomedical subjects

J Trapman

Publications and source records attributed to J Trapman.

At least 91 records · Page 5Linked to original sources

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

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↗

PubMed1991

Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization.

A series of human androgen receptor (AR) deletion mutants was constructed to study the relationship between the structural domains and their different functions in the AR protein. Human AR mutants were expressed in COS-1 and HeLa cells to investigate hormone binding, transcriptional activation, and subcellular localization. The wild-type human AR (AR 1-910) was expressed as a 110- to 112-kDa doublet, as revealed on immunoblots. All mutant AR proteins also migrated as doublets, except for one. This AR has a deletion from amino acid residues 51-211 and migrated as a single protein band, possibly due to altered posttranslational modification. The AR steroid-binding domain is encoded by approximately 250 amino acid residues in the C-terminal end. Deletions in this domain as well as truncation of the last 12 C-terminal amino acid residues abolished hormone binding. Cotransfection studies in HeLa cells showed that transcriptional activation of an androgen-regulated reporter gene construct was induced by the wild-type human AR. Mutational analysis revealed two regions in the N-terminal part, encoded by amino acid residues 51-211 and 244-360, to be essential for this transcriptional activation. Deletion of the hormone-binding domain yielded a constitutively active AR protein, indicating that in the absence of hormone this domain displays an inhibitory function. In the presence of its ligand, the wild-type AR was located in the cell nucleus. In the absence of androgens the receptor was mainly nuclear, but cytoplasmic localization was observed as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

The promoter of the prostate-specific antigen gene contains a functional androgen responsive element.

Expression of prostate-specific antigen (PA) mRNA was tested at various time periods after incubation of the human prostate tumor cell line LNCaP with the synthetic androgen R1881. Androgen-stimulated expression was observed within 6 h after addition of R1881 to the cells. Run-on experiments with nuclei isolated from LNCaP cells showed that expression of the PA gene could be regulated by R1881 on the level of transcription. DNase I footprints of the promoter region of the PA gene (-320 to +12) with nuclear protein extracts from LNCaP cells showed at least four protected regions. The protected areas include the TATA-box, a GC-box sequence, and a sequence AGAACAgcaAGTGCT at position -170 to -156, which closely resembles the reverse complement of the consensus sequence GGTACAnnnTGTTCT for binding of the glucocorticoid receptor and the progesterone receptor. Fragments of the PA promoter region were cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression plasmid into COS cells in a transient expression assay. CAT activity of COS cells grown in the presence of 1 nM R1881 was compared to untreated controls. A 110-fold induction of CAT activity was found if a -1600 to +12 PA promoter fragment was used in the construct. By further deletion mapping of the PA promoter a minimal region (-320 to -155) was identified as being essential for androgen-regulated gene expression. Mutation of the sequence AGAACAgcaAGTGCT (at -170 to -156) to AAAAAAgcaAGTGCT almost completely abolished androgen inducibility of the reporter gene constructs. One or more copies of the sequence AGAACAgcaAGTGCT cloned in front of a thymidine kinase promoter-CAT reporter gene confers androgen regulation to the reporter gene. These findings provide strong evidence for transcription regulation of the PA gene by androgens via the sequence AGAACAgcaAGTGCT. Interestingly, in addition to the AGAACAgcaAGTGCT element, an upstream region (-539 to -320) is needed for optimal androgen inducibility of the PA promoter.

Androgens↗

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

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

The rat androgen receptor gene promoter.

The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was cloned from a genomic library and the promoter was identified. S1-nuclease protection analysis showed two major transcription start sites, located between 1010 and 1023 bp upstream from the translation initiation codon. The area surrounding these start sites was cloned in both orientations in a CAT reporter plasmid. Upon transfection of the constructs into COS cells, part of the promoter stimulated transcription in an orientation-independent manner, but the full promoter showed a higher and unidirectional activity. In the promoter/reporter gene constructs, transcription initiated from the same positions as in the native gene. Sequence analysis showed that the promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but one SP1 site is located at about 60 bp upstream from the major start site of transcription. Other possible promoter elements are TGTYCT sequences at positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505, resembling half-sites of the glucocorticoid-responsive element (GRE). Furthermore, a homopurine stretch containing a total of 8 GGGGA elements and similar to sequences that are present in several other GC-rich promoters, is located between -89 and -146 bp upstream from the major start site of transcription.

Animals↗

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 and phosphorylation in human LNCaP cells.

Androgen receptor heterogeneity and phosphorylation were studied in the human LNCaP cell line. Fluorography after photoaffinity labeling as well as immunoblotting with a specific polyclonal antibody revealed that the human androgen receptor migrated as a closely spaced 110 kD doublet on SDS-polyacrylamide gels. A time-dependent change in the ratio between the two isoforms was not observed after R1881 treatment of intact cells. In nuclear extracts of LNCaP cells that were incubated with [32P]orthophosphate in the presence of 10 nM R1881, a 110 kD phosphorylated protein was demonstrated after immunopurification using a monoclonal antibody against the human androgen receptor. Only a very small amount of this phosphoprotein was detected in the nuclear fraction from cells not treated with R1881. These results indicate that the human androgen receptor in LNCaP cells can be phosphorylated.

Autoradiography↗

PubMed1990

An MspI RFLP detected by the human glandular kallikrein gene (hGK) on chromosome 19q.

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Chromosomes, Human, Pair 19↗

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

Variation of prostate-specific antigen expression in different tumour growth patterns present in prostatectomy specimens.

A series of 55 randomly chosen radical prostatectomy specimens was analyzed for expression of prostate-specific antigen (PSA) by immunohistochemical techniques. Tissue sections were selected in such a manner that in addition to glandular benign prostatic hyperplasia (BPH), one or more different prostatic tumour growth patterns were present. Four monoclonal antibodies, directed against three different PSA epitopes, and one polyclonal anti-PSA antiserum were used. Expression of PSA was compared with that of prostate-specific acid phosphatase (PAP), recognized by two different polyclonal antisera. A critical dilution aimed at a maximum of staining intensity on BPH tissue sections was chosen for all antibodies. Anti-PSA and anti-PAP antisera stained essentially all BPH samples (over 90%). Irrespective of the nature of the antibodies used, PSA expression was found to be decreased in prostatic carcinoma. A clear cut relationship was found between immunoreactivity for PSA and the degree of differentiation of the tumour area. Under the experimental conditions used the PSA monoclonal antibodies stained only 1 out of 10 undifferentiated carcinomas, whereas 50% to 70% of the well- and moderately-differentiated carcinomas showed immunoreactivity. This correlation was less pronounced with the PAP staining pattern. If the PSA antibody titer was raised the percentage of clearly staining undifferentiated carcinomas could be considerably increased (up to 60%-100%), indicating that PSA expression is not absent, but lowered in most (if not all) undifferentiated carcinomas.

Acid Phosphatase↗

PubMed1990

Aberrant splicing of androgen receptor mRNA results in synthesis of a nonfunctional receptor protein in a patient with androgen insensitivity.

Androgen insensitivity is a disorder in which the correct androgen response in an androgen target cell is impaired. The clinical symptoms of this X chromosome-linked syndrome are presumed to be caused by mutations in the androgen receptor gene. We report a G----T mutation in the splice donor site of intron 4 of the androgen receptor gene of a 46,XY subject lacking detectable androgen binding to the receptor and with the complete form of androgen insensitivity. This point mutation completely abolishes normal RNA splicing at the exon 4/intron 4 boundary and results in the activation of a cryptic splice donor site in exon 4, which leads to the deletion of 123 nucleotides from the mRNA. Translation of the mutant mRNA results in an androgen receptor protein approximately 5 kDa smaller than the wild type. This mutated androgen receptor protein was unable to bind androgens and unable to activate transcription of an androgen-regulated reporter gene construct. This mutation in the human androgen receptor gene demonstrates the importance of an intact steroid-binding domain for proper androgen receptor functioning in vivo.

Animals↗

PubMed1990

Assignment of seven genes to distinct intervals on the midportion of human chromosome 19q surrounding the myotonic dystrophy gene region.

Hybridization studies using a panel of somatic cell hybrids with subchromosomal segments of 19q have localized the genes encoding hormone-sensitive lipase (LIPE), carcinoembryonic antigen (CEA), and small nuclear ribonucleoprotein polypeptide A (SNRPA) to various regions of 19q13.1; the cellular receptor for poliovirus sensitivity (PVS) to 19q13.2; and the genes coding for prostate-specific antigen (APS), human pancreatic kallikrein (KLK1), and small nuclear ribonucleoprotein 70-kD polypeptide (SNRP70) to 19q13.3----qter. Our results exclude several of these genes from being seriously considered as a candidate for the myotonic dystrophy gene on 19q.

Animals↗

PubMed1990

An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity.

We have discovered in the X-linked androgen receptor gene a single exonic nucleotide substitution that causes complete androgen insensitivity (resistance) in a sibship with three affected individuals. The mutation, a guanine-to-adenine transition, occurs at nucleotide number 2682 and changes the sense of codon 717 from tryptophan to a translation stop signal. Codon 717 is in exon 4, so the mutation predicts the synthesis of a truncated receptor that lacks most of its androgen-binding domain. The substitution abolishes a recognition sequence for the restriction endonuclease HaeIII. Amplification of exon 4 by the polymerase chain reaction followed by double digestion with HinfI and HaeIII permits facile recognition of hemizygotes and heterozygous carriers of the mutation.

Amino Acid Sequence↗

PubMed1989

The biological activity of interferon alpha is influenced by two distinct regions in the protein.

With the aim to assign differences in activity between murine interferon-alpha 1 and -alpha 4 to specific amino acids, we have constructed hybrid genes and analysed the antiviral properties of the corresponding hybrid proteins. The hybrid genes were constructed by means of homologous recombination between the alpha 1 and alpha 4 genes in Escherichia coli. Hybrids in which the N-terminal part is derived from alpha 1 show that two regions have a major effect on the activity: amino acid 10-20 and 55-67. When comparing hybrids with N-terminal alpha 4 sequences, transitions in activity are found in the same regions. Interestingly, the curves for the two sets of hybrids are exactly each others mirror image.

Amino Acid Sequence↗

PubMed1989

The prostate-specific antigen gene and the human glandular kallikrein-1 gene are tandemly located on chromosome 19.

Using a prostate-specific antigen cDNA as a hybridization probe, clones containing the kallikrein genes encoding prostate-specific antigen, human glandular kallikrein-1 and pancreas/kidney kallikrein were isolated from a human genomic library. Clones containing the prostate-specific antigen gene and the human glandular kallikrein-1 gene overlap and span a region of about 36 kb. The two genes are aligned in a head to tail orientation at a mutual distance of 12 kb. Southern blot analysis of DNA from a panel of human-hamster hybrid cells with specific probes revealed the genes to be situated on chromosome 19. Assuming that the pancreas/kidney kallikrein gene is located in the same cluster, the distance to the prostate-specific antigen gene and the human glandular kallikrein gene must be at least 15 kb.

Animals↗

PubMed1989

Characterization of the prostate-specific antigen gene: a novel human kallikrein-like gene.

Using Prostate-specific Antigen cDNA fragments as hybridization probes a clone containing the information for the gene encoding Prostate-specific Antigen was isolated form a human genomic DNA library. The complete gene (about 6 kb) was sequenced and shown to be composed of four introns and five exons. Two major transcription initiation sites were found. The sequence of the promoter region revealed the presence of various well known transcription regulatory elements including a TATA box. A high percentage of homology was found between the Prostate-specific Antigen gene and the hGK-1 gene (82%). This homology extended into the promoter region. Two previously described variant Prostate-specific Antigen cDNAs can now be explained by intron retention and alternative splicing of the primary transcript.

Amino Acid Sequence↗