Search PubMed⌕ Search

Biomedical subjects

W Rombauts

Publications and source records attributed to W Rombauts.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of a functional androgen-response element in the exon 1-coding sequence of the cystatin-related protein gene crp2.

Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2, +40/+63). It displays an androgen response element-like sequence motif 5'-AGAAGAaaaTGTACA-3' and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5' upstream of the transcription start point and are located at an identical position (-142/ -120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (-273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5'-TGTACA-3' sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (-273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.

Androgens↗
PubMed1996

Androgenic induction of cystatin-related protein and the C3 component of prostatic binding protein in primary cultures from the rat lacrimal gland.

Cystatin-related protein (CRP) and the C3 component of prostatic binding protein (C3) are synthesized in vivo under androgen control in the lacrimal gland and ventral prostate of adult male rats [1,2]. Androgen administration to female or 7-day castrated male rats, which do not express CRP, can induce its synthesis [3]. In this study, we show androgen-dependent expression of CRP1 and C3 in primary cultures of acinar cells of the lacrimal gland of female rats. Addition of androgens to the culture medium results in the synthesis and secretion of both proteins in a time- and dose-dependent way. Estradiol or progesterone are unable to induce their expression. Dexamethasone in low concentrations and present as a basal component of the serum free defined medium, is needed to sensitize the culture system for androgens. In high concentrations, this synthetic glucocorticoid seems to play a similar role as androgens in CRP1 and C3 regulation.

Androgen-Binding Protein↗
PubMed1996

The androgen-specific probasin response element 2 interacts differentially with androgen and glucocorticoid receptors.

The nuclear receptors constitute a large family of transcription factors characterized by a well conserved DNA-binding domain. The receptors for glucocorticoids, progestins, mineralocorticoids, and androgens constitute a subgroup because they bind in vitro with high affinity to DNA elements containing a partial palindrome of the core sequence 5'-TGTTCT-3'. In vivo, however, the corresponding steroids differentially regulate the expression of their target genes, even when more than one receptor type is present in a particular cell. The DNA-binding domains of the androgen and of the glucocorticoid receptors bind most androgen response elements with similar relative affinities. In contrast, one element (5'-GGTTCTTGGAGTACT-3') which was recently described in the promoter region of the probasin gene selectively interacts with the DNA-binding domain of the androgen receptor and not with that of the glucocorticoid receptor. From studies with chimeric elements, it can be deduced that it is the left subsequence 5'-GGTTCT-3' which excludes the glucocorticoid receptor domain from binding. In co-transfection experiments where the ARE of the C3(1) gene is responsive to both androgens and glucocorticoids, the probasin element is induced only by androgens and not by glucocorticoids. The existence of response elements which are recognized preferentially by the androgen receptor provides yet another possible mechanism to explain the differences of the in vivo effects between androgens and other steroids of the subgroup.

Androgen-Binding Protein↗
PubMed1996

A human gene encoding diazepam-binding inhibitor/acy1-CoA-binding protein: transcription and hormonal regulation in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP.

Diazepam-binding inhibitor (DBI)/acyl-CoA-binding protein (ACBP) is a highly conserved 10-kD polypeptide expressed in various organs and implicated in the regulation of multiple biological processes such as GABAA/benzodiazepine receptor modulation, acyl-CoA metabolism, steroidogenesis, and insulin secretion. To extend our knowledge about the biology of DBI/ACBP and to elucidate the molecular mechanisms responsible for regulating DBI/ACBP gene expression, we have studied the androgen-regulated expression of DBI/ACBP transcripts in the human prostatic adenocarcinoma cell line LNCaP and have cloned and characterized a human gene encoding DBI/ACBP. Northern blotting, reverse transcription-assisted polymerase chain reaction (RT-PCR), ribonuclease protection, and 5' RACE analysis (rapid amplification of cDNA ends) of DBI/ACBP transcripts in LNCaP cells revealed androgen-regulated expression of multiple transcripts originating from multiple transcription start sites and alternative processing. The most abundant type of transcripts (referred to as type 1 transcripts) encodes genuine DBI/ACBP of 86 amino acids, while the minor type (type 2 transcripts) harbors an insertion of 86 bases and might encode an unrelated protein of 67 amino acids. Examination of a cloned DBI/ACBP gene revealed a structural organization of four exons present in all transcripts and one alternatively used exon present only in type 2 transcripts. The promoter region is located in a CpG island and lacks a canonical TATA box. Transient transfection of DBI/ACBP promoter fragments into LNCaP cells demonstrated that a region of 1.1 kb upstream of the translation start site is able to drive high-level expression of luciferase in LNCaP cells in an androgen-regulated fashion. Taken together these data indicate that the isolated human gene encoding DBI/ACBP is functional, has a high degree of structural similarity with the corresponding rat gene, exhibits hallmarks of a typical housekeeping gene, and harbors cis-acting elements that are at least partially responsible for androgen-regulated transcription in LNCaP cells.

Adenocarcinoma↗
PubMed1995

The genes coding for rat cystatin-related prostate protein (Cstrp) map to chromosome 3q41.

Two genes encoding rat cystatin-related prostate protein (Cstrp), previously called CRP (Devos et al., 1993), were mapped to chromosome 3q41 by fluorescent in situ hybridization. The results were confirmed using a panel of mouse-rat hybrids that segregate rat chromosomes. Analysis of genomic DNA indicates that the Cstrp locus comprises probably more than three very similar genes.

Animals↗
PubMed1994

Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the human prostatic adenocarcinoma cell line LNCaP.

To study the mechanisms by which androgens intervene in the regulation of growth and differentiation of human prostatic epithelial cells, cDNA clones encoding putative prostate-secreted proteins were characterized and tested as potential markers for androgen action. One of the isolated cDNAs expressed diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP), suggesting that this polypeptide, that has been implicated in a large number of biochemical processes, is expressed and secreted by prostate cells. As demonstrated by Northern blot analysis, the mRNA encoding DBI/ACBP was expressed in prostate tissue and in the three human prostatic adenocarcinoma cell lines tested: LNCaP, PC-3 and DU-145. In androgen-sensitive LNCaP cells, the synthetic androgen R1881 stimulated the DBI/ACBP steady state mRNA levels with half maximal effects at a concentration of 0.2 nM. Increases were a maximal 12 h after addition of the synthetic hormone. DBI/ACBP mRNA levels could also be stimulated by the synthetic androgen mibolerone and by the natural androgens testosterone and dihydrotestosterone. In agreement with the altered steroid specificity of the androgen receptor in LNCaP cells, estradiol and progesterone also exerted a stimulatory effect. Cortisol and the synthetic glucocorticoid dexamethasone were without effect. Androgen stimulation of DBI/ACBP mRNA levels was abolished in the presence of the protein synthesis inhibitor cycloheximide, implying a role for labile or androgen-induced proteins in this androgen stimulation. This is in contrast to the androgen stimulation of the mRNA encoding prostate-specific antigen (PSA), suggesting that different mechanisms are involved in the androgen regulation of these two genes. Although further experiments are required to confirm that DBI/ACBP is secreted by prostatic epithelial cells, these data demonstrate that the mRNA encoding DBI/ACBP is expressed in prostate cells and is affected by androgens in androgen-responsive LNCaP cells.

Adenocarcinoma↗
PubMed1994

Nuclear extracts enhance the interaction of fusion proteins containing the DNA-binding domain of the androgen and glucocorticoid receptor with androgen and glucocorticoid response elements.

Comparable fragments of the androgen receptor (AR) (amino acids 540-607) and of the glucocorticoid receptor (GR) (amino acids 412-515) were expressed in E. coli as fusion proteins with protein A. Both fusion proteins, denoted ARF1 and GRF1, contain the DNA-binding domain and some flanking amino acids. In vitro binding assays have shown that both fusion proteins interact with androgen/glucocorticoid response elements (ARE/GREs) in an intron fragment of the C3(1) gene of the androgen-regulated rat prostatic binding protein and in the typically glucocorticoid-responsive long terminal repeat (LTR) promoter of mouse mammary tumour virus. Present results indicate that the interaction of both ARF1 and GRF1 with the C3(1) as well as the LTR fragments is enhanced in the presence of nuclear extract. The factor that gives rise to this enhancement appears to be ubiquitous and sensitive to trypsin and temperature treatment. In the C3(1) fragment, the enhancing effect requires the presence of an intact functional ARE/GRE (Core II) as well as a region spanning the ARE/GRE half-site Core I.

Androgens↗
PubMed1993

Structure of rat genes encoding androgen-regulated cystatin-related proteins (CRPs): a new member of the cystatin superfamily.

Cystatin-related proteins (CRPs) are abundant androgen-regulated secretory glycoproteins that are specifically synthesized in the ventral prostate and lachrymal gland of the rat. Two complete 6-kb genes, Crp1 and Crp2, have been cloned and characterized. They are differentially expressed and encode slightly different proteins. The genes each contain four exons which are interrupted by large introns. An alignment of their sequences demonstrates an overall homology of 90%. The 3' end of a third gene, Crp3, from which only a 1.5-kb fragment was isolated, displays a sequence identity of 84%. These data indicate the existence of a Crp multigene family. The 5' flanking regions of Crp1 and Crp2 are highly homologous and contain a GATAAA sequence 29 nt upstream from the transcription start point. This TATA-box-like element is also found in the promoters of the genes encoding cystatin type-2 proteins. No other recognizable transcription control elements can be detected. Potential binding sites (ARE) for the androgen receptor are scattered throughout the entire genes. The exon/intron organization of the genes encoding CRPs, the size of the exons and their encoding amino acid sequences exhibiting a characteristic spacing of the Cys residues are structural elements displaying a remarkable similarity with the corresponding elements in the genes encoding cystatin type-2 proteins. CRPs must therefore belong to the cystatin superfamily. However, due to their additional domain encoded in an extra exon 2, CRPs must be classified as a new family, type 5.

Amino Acid Sequence↗
PubMed1993

Intronic androgen response elements of prostatic binding protein genes.

Transcription of the genes that code for the different components of Prostatic Binding Protein is dependent on androgens. DNA-cellulose competition assays have demonstrated the presence of regions with affinity for the androgen receptor located immediately upstream and in the first intron of these genes. DNase I footprinting in vitro now reveals the exact binding site of a receptor dimer in the first intron of the C3(1), C3(2) and C1 genes. Gene transfer experiments resulted in the description of functional androgen response elements in the C3 genes. The C1 intronic binding site, however, has a much weaker affinity for the DNA-binding domain of the androgen receptor, a characteristic that might explain its inability to function as an androgen response element in chimeric gene constructs.

Androgen-Binding Protein↗
PubMed1993

Interaction of androgen and glucocorticoid receptor DNA-binding domains with their response elements.

Fusion proteins containing the glucocorticoid and the androgen receptor DNA-binding domain (ARF1 and GRF1) were produced in Escherichia coli. DNAse I footprinting was used to compare the interaction of these proteins with responsive elements (REs) in a typically glucocorticoid-responsive gene (mouse mammary tumour virus (MMTV)) and in an androgen-responsive gene (the C3(1) gene of rat prostatic binding protein). It is demonstrated that response elements which most closely resemble the consensus sequence show identical footprinting patterns for ARF1 and GRF1. The protected regions suggest that these sequences are occupied by two DNA-binding domains (DBDs) forming a dimer. Regions that constitute imperfect RE sequences, however, are apparently recognized by only one DBD, which mainly protects the TGTTCT motif. At these REs, the protection patterns produced by ARF1 and GRF1 are not identical. In the long terminal repeat (LTR) of MMTV but not in C3(1), a mechanism other than classical dimer formation seems to increase the affinity of ARF1 and GFR1 for these imperfect REs.

Androgen-Binding Protein↗
PubMed1993

Proteins interacting with an androgen-responsive unit in the C3(1) gene intron.

The expression of the three genes encoding the components C1, C2 and C3 of prostatic binding protein (PBP) is under androgen control and restricted to the rat ventral prostate. The SstI-PvuII fragment of the first intron of the C3(1) gene displays two binding sites for ubiquitous transcription factors and one for a tissue-specific factor in a 80-bp region upstream of its androgen response element (ARE). The octamer transcription factor 1 (OTF-1) binds to the most distal element (site 1) while a member of the nuclear factor I (NF-I) family recognizes site 2. A third unidentified prostate-specific factor, which also occurs in castrated rats, interacts with the proximal element (site 3). In T-47D cells, both the OTF-1 and the NF-I-like factor can modulate the androgen response of the promoter in a reporter gene construct containing the C3(1) intronic fragment.

Androgen-Binding Protein↗
PubMed1992

The 4.4-kilodalton proline-rich polypeptides of the rat ventral prostate are the proteolytic products of a 637-kilodalton protein displaying highly repetitive sequences and encoded in a single exon.

We have determined the complete sequence of the 637-kilodalton precursor for the proline-rich polypeptides (PRPs). This protein is encoded in one large exon of a single copy gene. The acidic precursor of 5761 residues comprises a signal peptide and three large domains displaying a high proline content (11-15%). The sequence of domain A (928 residues) is unique and contains several small clusters of acidic amino acids. Domain B (830 residues) exhibits seven tandem repeats, four of them displaying a strongly diverged sequence. In domain C (3914 residues) 39 units, of which only 8 are degenerate, occur in a tandem repeat. Their sequence of 100 amino acids shows a high structural similarity (76-92%) and contains all the PRP variants which are produced by specific proteolytic processing. The COOH-terminal part (35 residues) is basic. Two variant PRP-precursor alleles occur which slightly differ in the number of repeats in domain C. The high degree of sequence conservation within the repeat regions suggests that the gene presumably evolved by multiple amplification and dispersion of two internal segments. In the 5097-base pair genomic region 5' upstream from the translation start, several control elements for transcription are recognized. A potential binding site for the Sp1 factor (GGGCGG) separated by 47 nucleotides from an initiator motif, most probably elements of the promoter, is detected in the vicinity of the ATG codon. Several putative androgen response elements (TGTYCT) are found in the 5' adjacent region and far upstream two Alu type III repeats and two (CA)n repeats are located. These results provide the basis for a detailed study of the androgen-regulated and tissue-specific expression of the PRP-precursor gene.

Amino Acid Sequence↗
PubMed1991

Interaction of androgen response elements with the DNA-binding domain of the rat androgen receptor expressed in Escherichia coli.

A fragment of the rat androgen receptor (amino acids 533-637) containing the DNA-binding domain was produced in Escherichia coli as a fusion product with protein A of Staphylococcus aureus. The fusion protein was purified on IgG-Sepharose, a method that does not involve the use of denaturing agents. Approximately 4 mg of fusion protein was obtained from 500 ml of bacterial culture. In gel shift assays, the recombinant DNA-binding domain displays an affinity for a fragment of the long terminal repeat of mouse mammary tumor virus and for an intronic fragment of the gene coding for the C3 component of the androgen-regulated rat prostatic binding protein. In a DNase I footprinting assay, the fusion protein protects a sequence in the C3 fragment that has previously been shown to act as a functional androgen response element. Interestingly, a single base pair mutation in the response element, which abolishes androgen inducibility, also destroys the ability to interact with the recombinant androgen receptor DNA-binding domain.

Androgens↗