Loss of progesterone receptor may lead to an invasive phenotype in human endometrial cancer.
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Endometrial Neoplasms↗
Biomedical subjects
P E de Ruiter
Publications and source records attributed to P E de Ruiter.
CAG repeat contraction in the androgen receptor gene in three brothers with mental retardation.
We report on three brothers with mental retardation and a contracted CAG repeat in the androgen receptor (AR) gene. It is known that expansion of the CAG repeat in this gene leads to spinal and bulbar muscular atrophy (SBMA or Kennedy disease); however, contracted repeats have not yet been implicated in disease. As the range of the length of CAG repeats in the AR gene, like those of other genes associated with dynamic mutations, follows a normal distribution, the theoretical possibility of disease at both ends of the distribution should be considered.
Adolescent↗
17Beta-hydroxysteroid dehydrogenase-3 deficiency: diagnosis, phenotypic variability, population genetics, and worldwide distribution of ancient and de novo mutations.
17Beta-hydroxysteroid dehydrogenase-3 (17betaHSD3) deficiency is an autosomal recessive form of male pseudohermaphroditism caused by mutations in the HSD17B3 gene. In a nationwide study on male pseudohermaphroditism among all pediatric endocrinologists and clinical geneticists in The Netherlands, 18 17betaHSD3-deficient index cases were identified, 12 of whom initially had received the tentative diagnosis androgen insensitivity syndrome (AIS). The phenotypes and genotypes of these patients were studied. Endocrine diagnostic methods were evaluated in comparison to mutation analysis of the HSD17B3 gene. RT-PCR studies were performed on testicular ribonucleic acid of patients homozygous for two different splice site mutations. The minimal incidence of 17betaHSD3 deficiency in The Netherlands and the corresponding carrier frequency were calculated. Haplotype analysis of the chromosomal region of the HSD17B3 gene in Europeans, North Americans, Latin Americans, Australians, and Arabs was used to establish whether recurrent identical mutations were ancient or had repeatedly occurred de novo. In genotypically identical cases, phenotypic variation for external sexual development was observed. Gonadotropin-stimulated serum testosterone/androstenedione ratios in 17betaHSD3-deficient patients were discriminative in all cases and did not overlap with ratios in normal controls or with ratios in AIS patients. In all investigated patients both HSD17B3 alleles were mutated. The intronic mutations 325 + 4;A-->T and 655-1;G-->A disrupted normal splicing, but a small amount of wild-type messenger ribonucleic acid was still made in patients homozygous for 655-1;G-->A. The minimal incidence of 17betaHSD3 deficiency in The Netherlands was shown to be 1: 147,000, with a heterozygote frequency of 1:135. At least 4 mutations, 325 + 4;A-->T, N74T, 655-1;G-->A, and R80Q, found worldwide, appeared to be ancient and originating from genetic founders. Their dispersion could be reconstructed through historical analysis. The HSD17B3 gene mutations 326-1;G-->C and P282L were de novo mutations. 17betaHSD3 deficiency can be reliably diagnosed by endocrine evaluation and mutation analysis. Phenotypic variation can occur between families with the same homozygous mutations. The incidence of 17betaHSD3 deficiency is 0.65 times the incidence of AIS, which is thought to be the most frequent known cause of male pseudohermaphroditism without dysgenic gonads. A global inventory of affected cases demonstrated the ancient origin of at least four mutations. The mutational history of this genetic locus offers views into human diversity and disease, provided by national and international collaboration.
17-Hydroxysteroid Dehydrogenases↗
Forskolin-induced dephosphorylation of the androgen receptor impairs ligand binding.
When androgen receptor containing cells are cultured in the presence of the PKA stimulator forskolin, a rapid dephosphorylation of the androgen receptor occurs resulting in a decrease in the amount of 112 kDa androgen receptor isoform and an increase in 110 kDa androgen receptor isoform on SDS-PAGE. To establish which amino acid residues in the androgen receptor were phosphorylated in control and forskolin-treated cells, trypsin-digested androgen receptors were subjected to RP-HPLC analysis and subsequently to Edman degradation. It was observed that serine residues 506, 641, and 653 were potentially phosphorylated in control cells, while after forskolin treatment strong evidence was obtained that phosphorylation of serines 641 and 653 was significantly reduced. When the dephosphorylated androgen receptor was analyzed for its transcription activation capacity, it was observed that androgen-induced transcriptional regulation of two endogenous genes (PSA) and beta 1-subunit of Na,K-ATPase), in cells cultured in the presence of forskolin, was inhibited as compared to the control situation. The observation that the dephosphorylated androgen receptor was transcriptionally less active was further strengthened by the finding that the dephosphorylated androgen receptor was markedly impaired in ligand binding (Bmax was found to be reduced by approximately 40%). The current investigations show for the first time a clear function for the rapid phosphorylation which occurs directly after synthesis of the androgen receptor, namely, effective ligand binding.
Amino Acid Sequence↗
Crystal structure of DNA photolyase from Anacystis nidulans.
The crystal structure at 1.8 A resolution of 8-HDF type photolyase from A. nidulans shows a backbone structure similar to that of MTHF type E. coli photolyase but reveals a completely different binding site for the light-harvesting cofactor.
Amino Acid Sequence↗
Androgen receptor phosphorylation.
Phosphorylation of transcription factors plays an important role in regulation of gene expression. DNA-binding, transactivation activity, and subcellular trafficking of specific transcription factors have been shown to be regulated by phosphorylation/dephosphorylation. Steroid hormone receptors are phospho-proteins, and mutations in phosphorylation sites significantly affect the transactivation capacity of these ligand-dependent transcription factors. At present, it is unknown which amino acid residues of the human androgen receptor are phosphorylated and whether phosphorylation of particular sites is a prerequisite for proper androgen receptor function. The aim of our future research is to map all phosphorylation sites in the human androgen receptor, and to analyze their importance by mutational analysis in vitro and in vivo using a number of functional assays.
Androgens↗
Synergism between androgens and protein kinase-C on androgen-regulated gene expression.
Androgen (R1881) induced transcriptional activity of the human androgen receptor, stably expressed in CHO cells, can be stimulated an extra 2-fold by the addition of the protein kinase C activator, 4 beta-phorbol 12-myristate 13-acetate (PMA). This extra stimulation is not observed when the protein kinase A activator bromoadenosine 3':5'-cyclic monophosphate (8-BrcAMP) is used. The transcriptional activity was measured using a reporter plasmid containing the MMTV-promoter, coupled to the luciferase gene. The effect of PMA on R1881-induced transcription was not due to a higher expression level of the androgen receptor. Also, no extra phosphorylation of the androgen receptor could be measured after incubation with PMA. When GRE-tk-LUC and PSA-LUC reporters were used, the synergistic effect of PMA could not be observed. The findings on the composite MMTV-LTR promoter can be explained by either a direct synergistic interaction between occupied AP-1 like responsive elements and the androgen receptor or via an unknown transcription factor activated by the PKC pathway and interacting with the androgen receptor.
Animals↗
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↗
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↗
Crystallization and preliminary X-ray diffraction studies of photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans.
Photolyase (photoreactivating enzyme) from the cyanobacterium Anacystis nidulans was crystallized by the hanging drop vapor diffusion procedure using ammonium sulfate as a precipitant. The pale-yellow crystals were grown to a size of 0.4 mm in length and 0.1 mm in diameter. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 90.7 A and c = 135 A. Assuming that the asymmetric unit contains one molecule, the Vm value is calculated as 2.6 A3/dalton. The crystals are stable towards X-ray exposure and diffract beyond 2.5 A resolution.
Crystallization↗
Localization and hormonal stimulation of phosphorylation sites in the LNCaP-cell androgen receptor.
Phosphorylation of the androgen receptor in human prostate tumour cells (LNCaP) is increased by addition of androgens to intact cells. Double-label studies, using [35S]methionine incorporation into receptor protein, and [32P]P(i) to label metabolically receptor phosphorylation sites, have enabled us to determine the phosphate content, relative to receptor protein, of both nontransformed and transformed and androgen receptors generated in intact LNCaP cells. No net change in the phosphorylation of the intact 110 kDa steroid-binding component of the androgen-receptor complex was found upon transformation to the tight nuclear binding form in the intact cell. Partial proteolysis of androgen receptor protein metabolically labelled with [32P]P(i) and photolabelled with [3H]R1881 (methyltrienolone) revealed that phosphorylation occurs mainly in the N-terminal trans-activation domain, whereas no phosphorylation was detected in the steroid- and DNA-binding domains. The location of most (> 90%) of the hormonally regulated phosphorylation sites in the N-terminal trans-activation domain suggests a role of phosphorylation of the androgen receptor in transcription regulation.
Androgens↗
Androgen receptor heterogeneity in LNCaP cells is caused by a hormone independent phosphorylation step.
Androgen receptor synthesis and modification were studied in the human LNCaP cell line. Immunoblotting showed that the androgen receptor migrated as a closely spaced 110-112 kDa doublet on SDS-PAGE gels. Most of the receptor protein is present in the higher molecular mass form. Labelling experiments with [35S]methionine showed that the androgen receptor is synthesized as a single 110 kDa protein which is rapidly converted to a 112 kDa protein. Upon alkaline phosphatase treatment a gradual elimination of the 112 kDa isoform with a concomitant increase of the 110 kDa isoform was seen, indicating that the observed 110 to 112 kDa upshift reflects androgen receptor phosphorylation. Furthermore, it is shown that both isoforms can bind hormone and undergo a hormone dependent transformation to a tight nuclear binding form, indicating that the 110 to 112 kDa conversion is not an obligatory step for hormone binding or receptor transformation.
Autoradiography↗
Detection of residual disease in translocation (14;18) positive non-Hodgkin's lymphoma, using the polymerase chain reaction: a comparison with conventional staging methods.
This paper reports the detection of residual lymphoma cells in blood and bone marrow samples from patients with translocation (t) (14;18) positive non-Hodgkin's lymphoma by the polymerase chain reaction (PCR) compared with conventional staging techniques. In 15 of 22 samples, in which no lymphoma cells could be detected by morphological examination, t(14;18) positive cells were detected by PCR. In 13 of 21 samples, in which a monoclonal B-cell population was not detectable by immunological marker analysis, PCR was positive. The clinical status (physical examination, imaging techniques, leucocyte count, and occasionally morphology and immunological marker analysis) was documented in 30 patients at the time of PCR analysis. In three of 19 patients with clinical evidence of disease, circulating t(14;18) positive cells were not detectable by PCR. Five of 11 patients in clinical remission from 7 to 47 months, showed t(14;18) positive cells in the blood. Our data show that PCR analysis in t(14;18) positive non-Hodgkin's lymphoma offers a powerful tool in the study of residual disease.
Biomarkers, Tumor↗
Synthesis and post-translational modification of the androgen receptor in LNCaP cells.
Androgen receptor synthesis and modification were studied in the human LNCaP cell line. Immunoblotting with a specific polyclonal antibody showed that the androgen receptor migrated as a closely spaced 110-112 kDa doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Most of the receptor protein is present in the higher molecular mass form. Pulse labelling experiments with [35S]methionine showed that the androgen receptor is synthesized as a single 110 kDa protein which is rapidly converted to a 112 kDa protein. Alkaline phosphatase treatment of cytosols from [35S]methionine pulse labelled cells caused a gradual elimination of the 112 kDa isoform with a concomitant increase of the 110 kDa isoform. This indicates that the observed 110 to 112 kDa upshift of the newly synthesized androgen receptor reflects receptor phosphorylation. Both isoforms can bind hormone and can undergo a hormone dependent transformation to a tight nuclear binding form, indicating that the 110 to 112 kDa conversion is not an obligatory step for hormone binding or receptor transformation.
Humans↗
Mechanisms of androgen receptor activation and function.
Androgens play a crucial role in several stages of male development and in the maintenance of the male phenotype. Androgens act in their target cells via an interaction with the androgen receptor, resulting in direct regulation of gene expression. The androgen receptor is a phosphoprotein and modulation of the phosphorylation status of the receptor influences ligand-binding and consequently transcription activation of androgen responsive genes. Androgen binding induces a conformational change in the ligand-binding domain, accompanied by additional receptor phosphorylation. Subsequently the liganded androgen receptor interacts with specific androgen response elements in the regulatory regions of androgen target genes, resulting in stimulation of gene expression. Anti-androgens induce a different conformational change of the ligand-binding domain, which does not or only partially result in stimulation of transactivation. Interestingly, different anti-androgens can induce different inactive conformations of the androgen receptor ligand-binding domain. Recent evidence strongly supports a ligand dependent functional interaction between the ligand-binding domain and the NH2-terminal transactivating domain of the androgen receptor. Two regions in the NH2-terminal domain are involved in this interaction, whereas in the ligand-binding domain the AF-2 AD core region is involved.
Androgens↗