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Biomedical subjects

J Trapman

Publications and source records attributed to J Trapman.

At least 55 records · Page 3Linked to original sources
PubMed1995

Identification of two transcription activation units in the N-terminal domain of the human androgen receptor.

To locate in detail the regions in the human androgen receptor (AR) involved in transcription activation, a series of N-terminal deletions was introduced in the wild type AR and in a constitutively active AR. The different constructs were tested for their capacity to activate transcription. Almost the entire N-terminal domain (residues 1-485) was necessary for full wild type AR activity when cotransfected with the (GRE)2tkCAT reporter in HeLa cells. In contrast, a smaller part of the N-terminal domain (amino acids 360-528) was sufficient for the constitutively active AR to induce transcription of the same (GRE)2tkCAT reporter in HeLa cells. This demonstrates the capacity of the AR to use different regions in the N-terminal domain as transcription activation units (TAUs). To obtain additional information of AR N-terminal TAUs, the GAL4 DNA binding domain was linked to either the entire or parts of the AR N-terminal domain and cotransfected with the (UAS)2tkCAT reporter in HeLa cells. The results confirmed that the first 485 amino acid residues accommodate a transcription activation function. When the chimeric AR-GAL4 constructs were tested on a different reporter ((UAS)5E1bCAT), a small shift in position of the TAU, responsible for full transcription activation, was observed. The data presented show that the size and location of the active TAU in the human AR is variable, being dependent on the promoter context and the presence or absence of the ligand binding domain.

Animals↗
PubMed1995

Androgen receptor mutations.

Male sexual differentiation and development proceed under direct control of androgens. Androgen action is mediated by the intracellular androgen receptor, which belongs to the superfamily of ligand-dependent transcription factors. At least three pathological situations are associated with abnormal androgen receptor structure and function: androgen insensitivity syndrome (AIS), spinal and bulbar muscular atrophy (SBMA) and prostate cancer. In the X-linked androgen insensitivity syndrome, defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46,XY individuals. Complete or gross deletions of the androgen receptor gene have not been found frequently in persons with complete androgen insensitivity syndrome. Point mutations at several different sites in exons 2-8 encoding the DNA- and androgen-binding domain, have been reported for partial and complete forms of androgen insensitivity. A relatively high number of mutations were reported in two different clusters in exon 5 and in exon 7. The number of mutations in exon 1 is extremely low and no mutations have been reported in the hinge region, located between the DNA-binding domain and the ligand-binding domain and which is encoded by the first half of exon 4. Androgen receptor gene mutations in prostate cancer are very rare and are reported only in exons 4-8. The X-linked spinal and bulbar muscle atrophy (SBMA; Kennedy's disease) is associated with an expanded length (> 40 residues) of one of the polyglutamine stretches in the N-terminal domain of the androgen receptor.

Amino Acid Sequence↗
PubMed1994

Loss of heterozygosity of chromosome 8 microsatellite loci implicates a candidate tumor suppressor gene between the loci D8S87 and D8S133 in human prostate cancer.

To search for specific chromosome 8 aberrations in human prostate cancer, DNA was isolated from 44 human prostate tumor samples. Twenty six tumor samples were obtained from locally progressive tumors by transurethral resection, 12 were from radical prostatectomy specimens, and 6 were from lymph node metastases. Tumor DNAs were screened for allelic losses using 16 highly polymorphic microsatellite loci (14 covering the p arm, 2 on the q arm). In general, the detected deletions were large. In 59% of the tumor DNAs, allelic loss of 3 or more 8p loci was observed. Loss of 8p loci occurred in between 36 and 69% of the informative cases; for the two 8q markers, the percentages of loss were 11 and 25%, respectively, indicating preferential loss of (part of) 8p. In one tumor, two separate 8p deletions were found. The percentage of loss of heterozygosity was considerably higher in transurethral resection (65%) and lymph node metastases (83%) than in radical prostatectomy specimens (33%), suggesting that 8p deletion is a relatively late step in tumor progression. The maximal overlapping deleted region in all tumor DNAs is between the distal locus D8S133 and the proximal locus D8S87, indicating the localization of a candidate tumor suppressor gene within this region.

Chromosome Deletion↗
PubMed1994

Changes in the abundance of androgen receptor isotypes: effects of ligand treatment, glutamine-stretch variation, and mutation of putative phosphorylation sites.

The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) migration pattern of wild-type and mutated human androgen receptors (ARs) expressed in COS-1 cells was analyzed. In the absence of hormone, the wild-type AR migrated as a closely spaced 110-112 kDa doublet. Alkaline phosphatase treatment resulted in a single 110 kDa band showing that the 112 kDa upshift reflects receptors phosphorylation. Deletion of the N-terminal amino acids 46-101 or 100-142 resulted in mutant ARs migrating as single protein bands. Three consensus phosphorylation sites in this region were substituted, and the resulting mutated proteins were analyzed. Two Ser-Pro-directed kinase consensus sites at positions Ser-80 and Ser-93 were both necessary for the AR 112 kDa upshift. Substitution of the putative casein kinase II Ser-118 site had no effect on the AR migration pattern. Surprisingly, deletion of the glutamine repeat, located directly N-terminal of the Ser-Pro sites, resulted also in an AR single form. Lengthening of the glutamine repeat caused an increase in the spacing between the two isotypes of the doublet, showing that the number of glutamine residues determines the extent of the upshift. Hormone treatment induced an extra isotype with an apparent molecular mass of 114 kDa, resulting in a 110-112-114 kDa AR triplet. The hormone-induced upshift was dependent on the Ser-80 consensus phosphorylation site. Mutations in the DNA binding domain caused a different distribution of receptor protein over the three AR isotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Structure, chromosome localization, and regulation of expression of the interferon-regulated mouse Ifi54/Ifi56 gene family.

The interferon-alpha (IFN-alpha) regulated mouse Ifi54/Ifi56 gene family, which is composed of at least four members (Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2), was isolated and characterized. In addition, the chromosomal localization of the four genes was determined. The Ifi54 and Ifi56 genes show an identical organization. Both are composed of a very small first exon and a second exon, which contains the complete open reading frame, except for the ATG start codon and the first two nucleotides of the second codon. In both genes, the two exons are separated by a small intron (5 and 2.5 kb, respectively). Expression of both genes is rapidly induced by IFN-alpha (within 2 h). The Ifi54 promoter region contains two sequences, which are closely related to the interferon stimulated response element (ISRE) consensus sequence (ISRE 1, GGTTTCAATTTCT, and ISRE 2, AGTGTTACTTTCT). The two elements are located directly adjacent to each other. A similar organization was recently established for the hamster Ifi54 promoter (Bluyssen et al., 1994). However, the mouse promoter is 70% less active than the hamster promoter. It turned out that ISRE 2 is hardly active, due to the G at position 4, which is a T in the hamster Ifi54 ISRE 2 and in the ISRE consensus sequence. The Ifi56 promoter region contains at a similar position two functional ISREs of identical strength (ISRE 1, AGTTTCAGTTTCT, and ISRE2, AGTTTCACTTTCC). In the Ifi56 promoter, the two ISRE motifs are separated by 6 bp. In addition to the Ifi56 gene, parts of two closely related genes (Ifi56-ps1 and Ifi56-ps2) were isolated. Both fragments contain an Ifi56-related open reading frame. However, we were unable to isolate the presumed first exon of Ifi56-ps1 and Ifi56-ps2, nor could we show expression of the genes. The Ifi54, Ifi56, Ifi56-ps1, and Ifi56-ps2 genes could all be assigned to mouse chromosome 19D1, suggesting a tight clustering.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor.

Aberrant activation of the androgen receptor (AR) may be one of the mechanisms which contribute to progression of prostatic carcinoma to an androgen-independent stage. We investigated effects of growth factors on stimulation of the AR-mediated gene transcription in human prostatic tumor cell lines. DU-145 cells, which do not contain endogenous AR, were cotransfected with an androgen-inducible chloramphenicol acetyltransferase (CAT) reporter gene and an AR expression vector. The reporter gene (CAT) was driven either by artificial promoters consisting of one or two androgen-responsive elements in front of a TATA box or by the promoter of the prostate-specific antigen (PSA) gene, a naturally occurring androgen-inducible promoter. Insulin-like growth factor-I (IGF-I), at a concentration of 50 ng/ml, stimulated AR-mediated reporter gene transcription to the same extent as the synthetic androgen methyltrienolone. This growth factor was effective irrespective of the nature of the androgen-inducible promoter. Keratinocyte growth factor (KGF) and epidermal growth factor (EGF), at concentrations of 50 ng/ml, activated CAT reporter gene transcription only in experiments in which the artificial promoter with two androgen-responsive elements was used. Insulin-like growth factor-II and basic fibroblast growth factor displayed no effect on AR-mediated gene transcription. None of the growth factors stimulated reporter gene activity in control experiments when added to cells cotransfected with the CAT gene and an empty expression vector. AR activation by IGF-I, KGF, and EGF was completely inhibited by the pure AR antagonist casodex, showing that these effects are AR mediated. Activation of endogenous AR by growth factors was studied in the LNCaP cell line by determination of PSA secretion. IGF-I, at a concentration of 50 ng/ml, increased the PSA level in the supernatant of this cell line 5-fold. Again, the IGF-I effect on PSA secretion was blocked by casodex. Our results provide evidence that IGF-I, KGF, and EGF directly activate the AR in the absence of androgens, which means that the androgen-signaling chain may be activated by growth factors in an androgen-depleted environment. These findings may have implications for endocrine therapy for metastatic prostatic carcinoma.

Androgen Antagonists↗
PubMed1994

Frequency and characterization of p53 mutations in primary and metastatic human prostate cancer.

We have studied the frequency of mutations in the p53 gene in human prostate cancer. The investigated material consisted of 20 primary-tumor tissue specimens, obtained by transurethral resection and tissue specimens of 15 lymph-node metastases, obtained at total prostatectomy. The applied methods encompassed immunohistochemistry on frozen sections, using the monoclonal antibody PAb 1801, and single-strand conformation polymorphism (SSCP) analysis, after amplification of single exon sequences by PCR, on exons 5 to 8 of the p53 gene. The mutations, leading to aberrantly migrating bands in the PCR-SSCP analysis, were identified by direct sequencing of the PCR product. Immunohistochemical and PCR-SSCP analysis were completely confirmative. In the primary tumors, mutations were found in 10% of the specimens (codons 232 and 273), and in lymph-node metastases in 15% of the specimens (codons 248 and 273). In one case (codon 273), the same mutation was found both in the primary tumor and in the lymph-node metastasis. Our results show that p53 mutations are infrequent in both primary and metastatic prostate tumors. In addition, they indicate that there is no strict correlation between p53 mutation and tumor metastasis.

Amino Acid Sequence↗
PubMed1994

The interferon-stimulated gene 54 K promoter contains two adjacent functional interferon-stimulated response elements of different strength, which act synergistically for maximal interferon-alpha inducibility.

The interferon-alpha(IFN-alpha)-regulated hamster ISG-54 K gene, which is activated in hamster CHO-12 cells at least 40-fold, was isolated and the promoter region was characterized in detail. Sequence analysis revealed the presence of two elements, closely related to the interferon-stimulated-response-element (ISRE) consensus sequence [AGTTTCNNTTTC(CT)]. The putative ISRE-I sequence (GGTTTCAATTTCT) is located at position -97 to -85; ISRE-II (AGTTTTACTTTCT), which differs at three positions from ISRE-I, is found directly upstream of ISRE-I at position -110 to -98. In a transient transfection assay in CHO-12 cells the wild-type hamster ISG-54K-promoter-chloramphenicol-acetyl-transferase (CAT) reporter construct showed a 40-80-fold induction, offering an excellent model to study the functional properties of the two ISRE. To find out whether both elements were functional in interferon regulation of the promoter, selected point mutations were introduced in the -110 to -85 region and in flanking sequences. The (mutated) ISG-54 K promoter was linked to the CAT reporter gene and transiently expressed in CHO cells in the absence and presence of murine (Mu)IFN-alpha 6. Transfections showed that both the -97 to -85 (ISRE-I) and the -110 to -98 (ISRE-II) segment were needed for optimal interferon induction of the ISG-54 K promoter. However, ISRE-I has an approximately sevenfold stronger activity compared to ISRE-II. Sequential substitution of the three ISRE-I bases, which differ in ISRE-II showed that the T at position -105 causes the lower activity of ISRE-II. Transfection of ISG-54 K promoter constructs, in which ISRE-I was replaced by ISRE-II, which generates a promoter with two ISRE-II segments, and vice versa (two ISRE-I), provided further evidence for a role of both elements in IFN-alpha induction. Importantly, all data obtained in transfection studies show that the two ISRE cooperate synergistically. The mechanism of synergism is most probably an indirect interaction between transcription factors binding to the ISRE, because an increase in the spacial arrangement of the two ISRE with a complete helical turn or half a turn did not result in a substantial decrease in promoter activity.

Amino Acid Sequence↗
PubMed1994

A practical approach to the detection of androgen receptor gene mutations and pedigree analysis in families with x-linked androgen insensitivity.

Androgen insensitivity syndrome (AIS) is an X-linked disorder in which defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46,XY individuals. This survey reports the analysis of 11 AIS subjects. The androgen receptor gene of these subjects was analyzed using polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis and sequencing or sequencing of PCR-amplified androgen receptor gene fragments alone. In total, 10 single base changes and one partial gene deletion were detected. Seven single base changes resulted in an amino acid change, one resulted in the introduction of a premature stop codon, one event represented a single base insertion resulting in a frame-shift, and one single base change affected a donor splice site. The androgen receptor protein in genital skin fibroblasts from several patients was studied with respect to molecular mass after immunoprecipitation and SDS-PAGE. Two patients expressed a truncated receptor protein in agreement with the established genomic mutation. Pedigree analysis was performed to identify possible carriers for the syndrome in families of AIS patients using single-strand conformation polymorphism and restriction site analysis of PCR products. In one case, the polymorphic (CAG)n(CAA) repeat in exon 1 encoding a polyglutamine stretch was used to identify the mutant allele in a family with X-linked partial androgen insensitivity before the identification of the actual genomic mutation. PCR-single-strand conformation polymorphism analysis proved to be a fast and reliable technique to screen for androgen receptor gene mutations and to study the androgen receptor gene of family members of AIS-affected individuals.

Base Sequence↗
PubMed1994

Androgen receptor status in localized and locally progressive hormone refractory human prostate cancer.

Heterogeneity in human androgen receptor (hAR) expression in prostate cancer is considered to be implicated in tumor progression. hAR expression was therefore studied immunohistochemically in localized and locally progressive, hormone refractory (HR) prostate cancer. Because altered functional activity of the hAR may be due to changes in the structural integrity of the hAR gene, exons 2 to 8 of the hAR gene were assessed for mutations by single-strand conformation polymorphism (SSCP) analysis and exon 1 was analyzed for the size of the CAG repeat. The hormone binding capacity, a prerequisite for ligand-regulated receptor function, was determined by a ligand binding assay. Coexpression of the hAR and prostate-specific antigen (PSA) was studied by a sequential double immunoenzymatic staining to verify whether PSA expression is a parameter of hAR function. Almost all human prostatic carcinomas revealed heterogeneous hAR expression, regardless of tumor differentiation and progression. Putative predominance of hAR-negative tumor areas in HR prostate cancer was not observed. No hAR gene mutations or major changes in the CAG repeat were found in the 18 HR carcinomas or in the 9 control samples. Moreover, all selected hAR-expressing cancers were able to bind the synthetic androgen methyltrienolone (R1881). Immunoenzymatic double staining revealed even PSA expression in hAR-negative tumor areas. PSA immunohistochemistry in human prostatic carcinomas therefore is of no use in determining hAR functional activity. Thus, most prostatic carcinomas, even when progressed to a state of hormone insensitivity, contain a structurally intact hAR gene, heterogeneously expressed with retained androgen binding capacity.

Antibodies, Monoclonal↗
PubMed1994

Differential splicing of human androgen receptor pre-mRNA in X-linked Reifenstein syndrome, because of a deletion involving a putative branch site.

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of > 6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS.

Alternative Splicing↗
PubMed1993

In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein.

Translation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with high affinity for R1881 (Kd = 0.3 nM). All AR molecules synthesized specifically bound steroid. No evidence for AR phosphorylation during in vitro synthesis was found. When AR was labelled with [3H]R1881 and analysed on sucrose-density gradients, a complex of approx. 6 S was observed. The complex was shifted to a higher sedimentation coefficient after incubation with a monoclonal AR antibody directed against an epitope in the DNA-binding domain. In the presence as well as the absence of hormone, AR molecules were able to bind to DNA-cellulose without an activation step. Gel retardation assays revealed that the AR forms complexes with a DNA element containing glucocorticoid-responsive element/androgen-responsive element sequences. Receptor-DNA interactions were stabilized by different polyclonal antibodies directed against either the N- or C-terminal part of the AR and were abolished by an antibody directed against the DNA-binding domain of the receptor. In conclusion, translation of AR cRNA in vitro yields an activated AR protein which binds steroid with high affinity. It is proposed that AR antibodies enhance AR-DNA binding by stabilizing AR dimers when bound to DNA.

Adenosine Triphosphate↗
PubMed1993

Threonine on amino acid position 868 in the human androgen receptor is essential for androgen binding specificity and functional activity.

The human androgen receptor gene in the androgen sensitive prostate tumor cell line (LNCaP) contains a point mutation in codon 868 resulting in the substitution of threonine by alanine. This amino acid change is responsible for the increased affinity of the mutant receptor protein for progestagens and estrogens. To further elucidate the role of threonine 868 on androgen binding capacity, specificity and functional activity, threonine 868 was substituted by six different amino acid residues. Substitution by aspartic acid, lysine or tyrosine totally eliminated androgen binding and the mutated androgen receptors did not have any transcriptional activating potential with either R1881, R5020 or estradiol. Introduction of a serine or an alanine broadened the steroid specificity, as did the introduction of a cysteine to a lesser degree. It is concluded that threonine on position 868 of the human androgen receptor limits the ligand specificity of the receptor to androgens.

Amino Acid Sequence↗
PubMed1993

Nuclear import of the human androgen receptor.

Nuclear import of the human androgen receptor was investigated by immunocytochemical analysis of androgen receptor deletion and substitution mutants, which were transiently expressed in COS-1 cells. The signal responsible for nuclear import is encoded by amino-acid residues 608-625 and is functionally similar to the bipartite nucleoplasmin nuclear-localization signal. Although the subcellular distribution of androgen receptors mutated in the DNA-binding domain was unchanged compared with the wild-type androgen receptor, in the presence of ligand these mutations resulted in part of the receptor population forming clusters. Depending on the presence or absence of the bipartite nuclear localization signal, clusters were formed in the nucleus or in the cytoplasm, respectively. Expression of the wild-type androgen receptor in different cell lines revealed a cell-line-specific subcellular distribution of the unliganded receptor. The androgen receptor was predominantly nuclear when expressed in HeLa cells, whereas mainly cytoplasmic staining was observed when it was expressed in COS-1 cells. In the presence of hormone, the androgen receptor was located in the nucleus, independent of the cell line that was expressing the receptor. Anti-androgens and various steroid hormones induced the nuclear localization of the wild-type androgen receptor in a dose-dependent way, without activating transcription of an androgen-regulated reporter gene. This indicates that the inability of the tested compounds to activate transcription is not due to inhibited nuclear import.

Amino Acid Sequence↗
PubMed1993

Two different, overlapping pathways of transcription initiation are active on the TATA-less human androgen receptor promoter. The role of Sp1.

In this study, the minimal promoter requirements of the TATA-less human androgen receptor (hAR) gene promoter are described. The hAR promoter is characterized by a short GC-box (-59/-32) and a long homopurine stretch (-117/-60). Two major transcription initiation sites, AR transcription initiation site I (AR-TIS I, (+1/2/3)) and AR transcription initiation site II (AR-TIS II, (+12/13)) are located in a 13-base pair region (Faber, P. W., van Rooij, H. C. J., van der Korput, J. A. G. M., Baarends, W. M., Brinkmann, A. O., Grootegoed, J. A., and Trapman, J. (1991) J. Biol. Chem. 266, 10743-10749). Transient transfection of COS cells with hAR promoter deletion and mutant constructs, followed by RNA isolation and S1 nuclease protection analysis showed that the process of transcription initiation through AR-TIS I and AR-TIS II is regulated by different promoter sequences. The GC-box directed initiation from AR-TIS II but did not affect AR-TIS I utilization, which is dependent upon sequences between positions -5 and +57. Band shift analysis identified the transcription factor Sp1 as the protein interacting with the GC-box. A single Sp1 binding sequence was found to be present in the GC-box. Footprint analysis confirmed the interaction of Sp1 with this sequence. The differential initiation through AR-TIS I and AR-TIS II was substantiated by the introduction of point mutations in the Sp1 binding sequence: only mutations that specifically abolished Sp1 binding interfered with AR-TIS II utilization, but all mutations left AR-TIS I initiation intact.

Animals↗