Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor.

The androgen receptor (AR) is involved in the development, growth and progression of prostate cancer (CaP). CaP often progresses from an androgen-dependent to an androgen-independent tumor, making androgen ablation therapy ineffective. However, the mechanisms for the development of androgen-independent CaP are unclear. More than 80% of clinically androgen-independent prostate tumors show high levels of AR expression. In some CaPs, AR levels are increased because of gene amplification and/or overexpression, whereas in others, the AR is mutated. Nonetheless, the involvement of the AR in the transition of CaP to androgen-independent growth and the subsequent failure of endocrine therapy are not fully understood. Here we show that in CaP cells from a patient who failed androgen ablation therapy, a doubly mutated AR functioned as a high-affinity cortisol/cortisone receptor (ARccr). Cortisol, the main circulating glucocorticoid, and its metabolite, cortisone, both equally stimulate the growth of these CaP cells and increase the secretion of prostate-specific antigen in the absence of androgens. The physiological concentrations of free cortisol and total cortisone in men greatly exceed the binding affinity of the ARccr and would activate the receptor, promoting CaP cell proliferation. Our data demonstrate a previously unknown mechanism for the androgen-independent growth of advanced CaP. Understanding this mechanism and recognizing the presence of glucocorticoid-responsive AR mutants are important for the development of new forms of therapy for the treatment of this subset of CaP.

Immunochemical localization of NADP-specific isocitrate dehydrogenase in Escherichia coli.

The intracellular localization of isocitrate dehydrogenase was determined by immunochemical techniques with ultrathin sections of Escherichia coli. The thin sections, which were obtained by ultracryomicrotomy, were incubated first with antiserum specific for the enzyme and then with a protein A-gold complex. Transmission electron microscopy showed that the gold label was dispersed mainly in the cytoplasm.

Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets.

Hypocalcemic vitamin D-resistant rickets is a human genetic disease resulting from target organ resistance to the action of 1,25-dihydroxyvitamin D3. Two families with affected children homozygous for this autosomal recessive disorder were studied for abnormalities in the intracellular vitamin D receptor (VDR) and its gene. Although the receptor displays normal binding of 1,25-dihydroxyvitamin D3 hormone, VDR from affected family members has a decreased affinity for DNA. Genomic DNA isolated from these families was subjected to oligonucleotide-primed DNA amplification, and each of the nine exons encoding the receptor protein was sequenced for a genetic mutation. In each family, a different single nucleotide mutation was found in the DNA binding domain of the protein; one family near the tip of the first zinc finger (Gly----Asp) and one at the tip of the second zinc finger (Arg----Gly). The mutant residues were created in vitro by oligonucleotide directed point mutagenesis of wild-type VDR complementary DNA and this cDNA was transfected into COS-1 cells. The produced protein is biochemically indistinguishable from the receptor isolated from patients.

Hereditary vitamin D resistant rickets caused by a novel mutation in the vitamin D receptor that results in decreased affinity for hormone and cellular hyporesponsiveness.

Mutations in the vitamin D receptor (VDR) result in target organ resistance to 1alpha,25-dihydroxyvitamin D [1,25(OH)2D3], the active form of vitamin D, and cause hereditary 1,25-dihydroxyvitamin D resistant rickets (HVDRR). We analyzed the VDR of a patient who exhibited three genetic diseases: HVDRR, congenital total lipodystrophy, and persistent mullerian duct syndrome. The patient was treated with extremely high dose calcitriol (12.5 microg/d) which normalized serum calcium and improved his rickets. Analysis of [3H]1,25(OH)2D3 binding in the patient's cultured fibroblasts showed normal abundance of VDR with only a slight decrease in binding affinity compared to normal fibroblasts when measured at 0 degrees C. The patient's fibroblasts demonstrated 1,25(OH)2D3-induction of 24-hydroxylase mRNA, but the effective dose was approximately fivefold higher than in control cells. Sequence analysis of the patient's VDR gene uncovered a single point mutation, H305Q. The recreated mutant VDR was transfected into COS-7 cells where it was 5 to 10-fold less responsive to 1,25(OH)2D3 in gene transactivation. The mutant VDR had an eightfold lower affinity for [3H]1,25(OH)2D3 than the normal VDR when measured at 24 degrees C. RFLP demonstrated that the patient was homozygous for the mutation while the parents were heterozygous. In conclusion, we describe a new ligand binding domain mutation in the VDR that causes HVDRR due to decreased affinity for 1,25(OH)2D3 which can be effectively treated with extremely high doses of hormone.

The molecular basis of hereditary 1,25-dihydroxyvitamin D3 resistant rickets in seven related families.

Hereditary 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant rickets (HVDRR) is an autosomal recessive disease caused by target organ resistance to the action of 1,25(OH)2D3, the active form of the hormone. The defect in target cells is heterogenous and commonly appears to be a mutation in the gene encoding the vitamin D receptor (VDR). We have studied cultured skin fibroblasts and Epstein-Barr virus transformed lymphoblasts of seven family branches of an extended kindred having eight children affected with HVDRR. We have previously shown that cells from three affected children in this group contain an "ochre" nonsense mutation coding for a premature stop codon in exon 7 within the steroid-binding domain of the VDR gene. In the current studies, we found that cells from affected children failed to bind [3H]1,25(OH)2D3 and had undetectable levels of VDR as determined by immunoblots using an anti-VDR monoclonal antibody. Measurement of VDR mRNA by hybridization to a human VDR cDNA probe showed undetectable or decreased abundance of steady-state VDR mRNA. Parents, expected to be obligate heterozygotes, showed approximately half the normal levels of [3H]1,25(OH)2D3 binding, VDR protein, and mRNA. The mutation at nucleotide 970 (counting from the mRNA CAP site) results in the conversion of GTAC to GTAA, which eliminates an Rsa I restriction enzyme site and facilitates identification of the mutation. We found that polymerase chain reaction (PCR) amplification of exons 7 and 8 from family members and subsequent Rsa I digestion allows detection of the specific genotype of the individuals. When Rsa I digests of PCR-amplified DNA are subjected to polyacrylamide gel electrophoresis, children with HVDRR exhibit a homozygous banding pattern with loss of an Rsa I site. Parents exhibit a heterozygotic DNA pattern with detection of both normal and mutant alleles. In summary, our data show that the genetic abnormality is a point mutation within the steroid-binding domain of the VDR in all seven related families with HVDRR. Analysis of restriction fragment length polymorphism at the 970 locus of PCR-amplified DNA fragments can be used to diagnose this mutation in both affected children and parents carrying the disease.

Hereditary 1,25-dihydroxyvitamin D-resistant rickets due to an opal mutation causing premature termination of the vitamin D receptor.

Mutations in the vitamin D receptor (VDR) gene have been shown to cause hereditary vitamin D-resistant rickets (HVDRR). The patient in this study is a young French-Canadian boy with no known consanguinity in his family. The child exhibited the clinical characteristics of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated 1,25-dihydroxyvitamin D (1,25(OH)2D) levels as well as total alopecia. Fibroblasts were cultured from a skin biopsy of the patient and used to assess the VDR. Northern blot analysis showed that a normal size VDR transcript was expressed; however, [3H]1,25(OH)2D3-binding levels were very low and Western blot analysis failed to detect any VDR protein. Total resistance to 1,25(OH)2D3 was demonstrated by the failure of the cultured fibroblasts to induce the transcription of the 25-hydroxyvitamin D-24-hydroxylase gene when treated with high concentrations of 1,25(OH)2D3. Analysis of the DNA sequence revealed a unique C to T base change corresponding to nucleotide 218 of the VDR cDNA. This single base substitution changes the codon for arginine (CGA) to an opal stop codon (TGA), resulting in the truncation of the VDR at amino acid 30. The Arg30stop mutation prematurely terminates translation and deletes 398 amino acids including most of the zinc fingers as well as the entire ligand-binding domain. Restriction fragment length polymorphism analysis of a DdeI restriction site created by the mutation showed that the parents were heterozygous for the mutant allele. In conclusion, the Arg30stop mutation truncates the VDR and leads to a hormone-resistant condition which is the molecular basis of HVDRR in this patient.

The presence of a polymorphism at the translation initiation site of the vitamin D receptor gene is associated with low bone mineral density in postmenopausal Mexican-American women.

We examined the association of bone mineral density (BMD) with a polymorphism in the gene encoding the vitamin D receptor (VDR) that causes a change in the predicted protein sequence. The polymorphism results from a C-to-T transition and creates an initiation codon (ATG) three codons proximal to a downstream start site. The polymorphism can be defined by a restriction fragment length polymorphism (RFLP) using the restriction endonuclease FokI. The presence of a FokI site, designated f, allows protein translation to initiate from the first ATG. The allele lacking the site (designated F), initiates from a second ATG site. Thus, translation products from these alleles are predicted to differ by three amino acids with the f variant elongated. In a group of 100 postmenopausal Mexican-American Caucasian women, subjects with the ff genotype (15% of the study population) had a 12.8% lower BMD at the lumbar spine than FF subjects (37% of the population) (p = 0.01). Heterozygote (Ff) subjects (48% of the population) had an intermediate BMD. This association between BMD and genotype was not apparent at the femoral neck or forearm. Over a 2-year follow-up period, a decrease in BMD at the femoral neck was greater in ff compared with FF subjects (-4.7% vs. -0.5%, p = 0.005). This trend was not apparent at the lumbar spine or forearm. There were no differences between genotype groups in measurements of 25-hydroxyvitamin D (25(OH)D), calcitriol, parathyroid hormone (PTH), osteocalcin, or urinary pyridinolines. We conclude that the FokI polymorphism of the VDR gene correlates significantly with decreased BMD at the lumbar spine and with an increased rate of bone loss at the hip in ff subjects. We emphasize that these initial data should be interpreted with caution but that the utility of this polymorphism as a genetic marker to determine BMD and osteoporosis risk warrants further study in larger populations with subjects of diverse ethnic backgrounds.

The vitamin D receptor gene start codon polymorphism: a functional analysis of FokI variants.

The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where "f" indicates the presence of the restriction site and the first ATG, while "F" indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F-VDR) being three amino acids shorter than the f variant (f-VDR). To determine whether there are functional differences between the f-VDR and the F-VDR, we studied the two VDR forms expressed in COS-7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f-VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]-1,25-dihydroxyvitamin D3 ([3H]-1,25(OH)2D3) (Kd = 131+/-78 pM, f-VDR; Kd = 237+/-190 pM, F-VDR; p = 0.24); however, a 2-fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24-hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose-response (0.1-10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF, Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24-hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f-VDR and F-VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.

Biological activity of 19-nor, 10-keto, 25-hydroxyvitamin D3.

19 nor, 10 keto, 25-hydroxyvitamin D3 (19/10-25OHD3) is a metabolite of 25-OHD3 produced in vitro by various phagocytes including normal human blood monocytes and transformed cell lines, U937 and HL-60. We recently reported that 19/10-25OHD3, induced differentiation of U937 cells. In these studies, 19/10-25OHD3 alone produced no detectable effect on the growth rates, surface adherence, and oxidative metabolism of U937 and HL-60 cells. When combined with lymphocyte-conditioned medium (LCM), 19/10-25OHD3 reduced proliferation, increased surface adherence and stimulated luminol-dependent luminescence (LDL) of the U937 cells. In contrast, the combination of 19/10-25OHD3 and LCM had no effect on the growth of HL-60 cells but did increase the surface adherence and the expression of a complement receptor component. 19/10-25OHD3 competed for tritium-labeled 1,25(OH)2D3 binding to receptors extracted from cultured human skin fibroblasts. This displacement capacity was 600 times weaker than that of unlabeled 1,25(OH)2D3. Incubation of human skin fibroblasts for 24 hr with 19/10-25OHD3 induced 25OHD3-24-hydroxylase activity in the fibroblasts. The inductive potency of 19/10-25OHD3 was 1/50 that of 1,25(OH)2D3. These results demonstrate bioactivity of 19/10-25OHD3 in several systems. At least one of these responses, the induction of 25OHD3-24-hydroxylase, is a receptor-mediated event. Some of the other responses may be independent of the cellular receptor for 1,25(OH)2D3. Interestingly, the potency of 19/10-25OHD3 was highest in the receptor-mediated response (1:50) and lower in the other parameters, ranging from 1:100 to 1:600 compared to 1,25(OH)2D3. This range of bioactivity in phagocytes and fibroblasts is presently explained.

Hereditary 1 alpha,25-dihydroxyvitamin D-resistant rickets resulting from a mutation in the vitamin D receptor deoxyribonucleic acid-binding domain.

Hereditary 1 alpha,25-dihydroxyvitamin D-resistant rickets (HVDRR) is a genetic disease that results from mutations in the gene encoding the vitamin D receptor (VDR). In this study of two siblings showing classical features of HVDRR, cultured dermal fibroblasts were used to characterize their VDR and assess responsiveness to 1,25-dihydroxyvitamin D3 treatment. The VDR displayed normal affinity and binding capacity for [3H]1,25-dihydroxyvitamin D3; however, the cells failed to exhibit induction of 25-hydroxyvitamin D 24-hydroxylase activity when treated with hormone. A decreased affinity of liganded VDR for DNA cellulose suggested that the defect was localized to the DNA-binding domain. Exons 2 and 3 of the VDR gene, which encode the two zinc fingers in the DNA-binding domain, were amplified and sequenced by polymerase chain reaction. Both siblings exhibited a G to A missense mutation (CGG to CAG) in exon 3, which results in the replacement of Arg77 by Gln at the base of the second zinc finger. This mutation has been described previously in two unrelated cases of HVDRR by Sone et al. It is unclear at this time whether these kindreds might be distantly related and, therefore, harbor the same mutation, or whether this represents a mutational hot spot in the VDR gene.

Abnormal binding of vitamin D receptors to deoxyribonucleic acid in a kindred with vitamin D-dependent rickets, type II.

Vitamin D-dependent rickets, type II, is a hereditary disease that results from target organ resistance to the action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We describe here a family (designated G) with a defect in the DNA-binding domain of the 1,25-(OH)2D receptor (VDR) manifested by normal steroid binding and decreased VDR affinity for DNA. The phenotypically normal parents are heterozygous, expressing both normal and defective forms of VDR. The affected children in this family had early-onset rickets, alopecia, hypocalcemia, and elevated serum 1,25-(OH)2D3 levels. The VDR of cultured skin fibroblasts of the affected children (G1 and G2) as well as the parents (G3 and G4) bound [3H]1,25-(OH)2D3 normally (Kd = 2-3 X 10(-11) mol/L; maximal number of binding sites = 20-40 fmol/mg protein). The cells from G1 and G2 were resistant to 1,25-(OH)2D3 action, as measured by induction of 24-hydroxylase activity, while the cells from G3 and G4 responded normally. Western blot analysis using the anti-VDR monoclonal antibody 9A7 showed that hypertonic extracts of fibroblasts from both affected children (G1 and G2) and their parents (G3 and G4) had immunoreactive bands of 48K, identical to the size of the VDR in normal cells. The VDR from G1 and G2 eluted as a single peak from DNA-cellulose columns at a lower salt concentration (0.1 mol/L) than that from normal subjects (0.2 mol/L), indicating an apparent decreased affinity for DNA. Fibroblast VDR from G3 and G4 each eluted from DNA-cellulose columns as two peaks, the normal peak (0.2 mol/L) and the abnormal peak (0.1 mol/L), which was found in G1 and G2. Western blot analysis of the 0.1 and 0.2 mol/L KCl peak fractions also confirmed that VDR was present in only the 0.1 mol/L fraction and not the 0.2 mol/L fraction from G1. However, VDR was present in both fractions from G3. In summary, this vitamin D-dependent rickets, type II, kindred has a defect in the DNA-binding domain of VDR. The parents are phenotypically normal and express both the normal and defective VDR alleles, as demonstrated by both DNA-cellulose chromatography and Western blot analysis. The affected children are resistant to 1,25-(OH)2D3 action and are homozygous for the defective VDR.

A novel mutation in the deoxyribonucleic acid-binding domain of the vitamin D receptor causes hereditary 1,25-dihydroxyvitamin D-resistant rickets.

Mutations in the vitamin D receptor (VDR) result in hereditary 1,25-dihydroxyvitamin D3-resistant rickets (HVDRR), an autosomal recessive disease caused by target organ resistance to the action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. In this study, we investigated the molecular basis of HVDRR in a child from Saudi Arabia who was previously shown to be resistant to 1,25-(OH)2D3 action, but whose cultured skin fibroblasts exhibited normal [3H]1,25-(OH)2D3 binding. Using the PCR, exons 2 and 3 of the VDR gene that encode the DNA-binding region of the receptor were amplified and sequenced. A novel point mutation at nucleotide 252 in exon 2 of the VDR was identified. This missense mutation (GGC to GAC) resulted in the conversion of glycine to aspartic acid at amino acid position 46 (G46D), located at the base of the first zinc finger. This single base change was introduced into wild-type VDR complementary DNA by site-directed mutagenesis, and the mutant VDR was then expressed in COS-1 cells. The expressed mutant VDR displayed a normal binding affinity (Kd = 1.2 x 10(-10) mol/L) for [3H]1,25-(OH)2D3 as determined by Scatchard analysis. However, the mutant VDR was shown to have reduced binding affinity for DNA by DNA-cellulose chromatography. In COS-7 cells cotransfected with a vitamin D response element-chloramphenicol acetyltransferase reporter construct and the mutant VDR complementary DNA expression vector, the mutant VDR was unable to activate gene transcription in cells treated with up to 100 nmol/L 1,25-(OH)2D3. Restriction fragment length polymorphism analysis using MwoI restriction digests of exon 2 demonstrated that the affected child is homozygous for the mutation, whereas the child's father is heterozygous and a carrier of the defective allele. In conclusion, a new mutation was identified in exon 2 of the VDR gene. This mutation, which occurs in the first zinc finger of the DNA-binding domain of the receptor, blocks 1,25-(OH)2D3 action and leads to the syndrome of HVDRR.

Cloning and characterization of the gene encoding the ADP-ribosylation factor in Candida albicans.

We have cloned and sequenced the gene (ARF) encoding the ADP-ribosylation factor (ARF) of Candida albicans. The gene contains an open reading frame of 537 nucleotides (nt) that codes for a protein with an Mr of 20,259. The C. albicans ARF gene is 67-70% identical at the nt level to other ARF sequences including those of humans; the deduced amino acid sequence of C. albicans ARF shows a 78-83% identity and 89-92% similarity to the other ARFs. Southern analysis of C. albicans genomic DNA suggested the presence of a second ARF gene. The presence of multiple ARF genes is a consistent finding among the other organisms previously shown to have ARFs.

Cyclic AMP-independent phosphorylation of Escherichia coli isocitrate dehydrogenase.

The phosphorylation of NADP-specific isocitrate dehydrogenase in a wild-type and in an adenylate cyclase deletion mutant of Escherichia coli has been investigated. The results obtained clearly indicate that cyclic AMP is not required for the phosphorylation reaction per se, not is it for the synthesis or possible activation of the phosphoprotein kinase in this organism. This data are in contrast to results observed in Salmonella typhimurium, and indicate that important differences exist in the phosphorylation of the isocitrate dehydrogenase in these two organisms.

Phosphorylation of isocitrate dehydrogenase in Escherichia coli mutants with a non-functional glyoxylate cycle.

The phosphorylation of NADP-specific isocitrate dehydrogenase in an isocitrate lyase and in a malate synthase mutant of Escherichia coli has been investigated. The results clearly demonstrate that isocitrate dehydrogenase may undergo an acetate-induced phosphorylation in organisms which do not have a functional glyoxylate cycle. This observation, together with those reported in Salmonella typhimurium, suggest that the current notion concerning the interrelationship between the glyoxylate cycle and the reversible phosphorylation of NADP-isocitrate dehydrogenase in microbial physiology should be reevaluated, and that phosphoenolpyruvate may be a key factor in the regulation of the reversible covalent modification of this enzyme in vivo.

Detection of femoral head avascular necrosis in adults by SPECT.

Twenty-one adult patients with the clinical diagnosis of avascular necrosis (AVN) of the femoral head were examined with radionuclide angiography, planar bone scintigraphy, and single photon emission computed tomography (SPECT). A final diagnosis of AVN was established for 15 symptomatic patients with a total of 20 involved hips. SPECT and planar bone scintigraphy were considered positive for AVN only if a photopenic bony defect could be identified. Using SPECT bone scintigraphy, 12 of 15 symptomatic patients and 17 of 20 involved hips (sensitivity of 0.85) were correctly identified, whereas with planar imaging only eight of 15 patients and 11 of 20 involved hips were detected. There were no false-positive diagnoses on SPECT or planar bone scintigraphy. In addition, hyperemia in the region of the proximal femoral metaphysis was demonstrated in six of 20 involved hips. It is concluded that by identifying a photopenic defect that is not evident on planar views, SPECT can contribute to the diagnosis of AVN of the femoral head. In addition, metaphyseal hyperemia appears to be a promising new scintigraphic sign of AVN worthy of further investigation.