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Purpose: Prostate cancer metastasizes to the skeleton to form osteoblastic lesions. Androgen ablation is the current treatment for metastatic prostate cancer. This therapy is palliative, and the disease will return in an androgen-independent form that is preceded by a rising titer of prostate-specific antigen (PSA). Here, we investigated the possibility that human osteoblasts might secrete factors that contribute to the emergence of androgen-independent prostate cancer. Experimental Design: Primary cultures of human osteoblasts were used as a source of conditioned medium (OCM). Proliferation, expression of androgen-regulated genes, and transactivation of the androgen receptor (AR) were monitored in LNCaP human prostate cancer cells in response to OCM using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Northern blot analysis, and reporter gene constructs. Levels of interleukin-6 (IL-6) present in OCM were measured, and its contribution to proliferation and expression of PSA were investigated by neutralization studies with anti IL-6 antibodies. Results: OCM increased the proliferation and expression of PSA at both the protein and RNA levels in LNCaP cells. Synergistic increases in the activities of PSA (6.1 kb)- and pARR3-tk-luciferase reporters were measured in cells cotreated with both OCM and androgen. OCM targeted the NH2-terminal domain of the AR. The effect of OCM on transcriptional activity of the AR was inhibited by an antiandrogen. Neutralizing antibodies to IL-6 blocked proliferation and expression of PSA by OCM. Conclusion: Osteoblasts secrete factors, such as IL-6, that cause androgen-independent induction of PSA gene expression and proliferation of prostate cancer cells by a mechanism that partially relies on the AR. Identifying such molecular mechanisms may lead to improved clinical management of metastatic prostate cancer.

In previous studies we established that human bone and human osteoblast-like cells (hOB cells) cultured from bone express 5alpha-reductase (5alpha-R) activity, as demonstrated by the conversion of testosterone and androstenedione to their corresponding 5alpha-reduced metabolites, 5alpha-dihydrotestosterone (DHT) and 5alpha-androstanedione. Two 5alpha-R isozymes (types 1 and 2) have been identified in various tissues. As their nature in bone is unknown, we investigated which isozymes were expressed in first passage hOB cells cultured from bone specimens obtained from six donors (five women and one man). For comparison, 5alpha-reductase isozyme expression in genital skin fibroblasts cultured from foreskin of three males was determined. Pharmacological and biochemical studies using selective inhibitors of the 5alpha-R isozymes were performed, and gene expression was assessed by RT-PCR. In hOB cells, LY191704, a potent nonsteroidal selective inhibitor of 5alpha-R type 1, and the 4-azasteroid 17beta-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5alpha-androstan-3-one (a dual inhibitor of 5alpha-R types 1 and 2) inhibited 5alpha-R activity with a 50% inhibitory concentration (IC(50)) of approximately 4 nM. Finasteride, a selective inhibitor of 5alpha-R type 2, blocked 5alpha-R activity with an IC(50) of approximately 60 nM. The IC(50) of progesterone, a physiological substrate for 5alpha-R, was approximately 200 nM. In genital skin fibroblasts, LY191704 inhibited 5alpha-R with an IC(50) of more than 5000 nM, whereas finasteride and 17beta-(N,N,-diethyl-carbamoyl)-4-methyl-4-aza-5alpha-androstan-3-one effectively inhibited 5alpha-R with IC(50) of approximately 4 nM. Experiments to determine 5alpha-reductase activity in homogenates of hOB cells as a function of pH showed very low activity at pH 5.5, but a broad shoulder of activity from pH 6.0-9.0, which was not inhibited by finasteride, but was nearly completely blocked by LY191704. RT-PCR revealed that 5alpha-R type 1 and 2 mRNAs were expressed in both bone and genital skin fibroblasts. Based on our pharmacological and biochemical studies, it appears that 5alpha-R activity in hOB cells is catalyzed predominantly by the type 1 rather than the type 2 isozyme. This expression pattern is in contrast to that in genital skin fibroblasts, where the activity of the type 2 isozyme prevails. As in most androgen target tissues DHT is biologically more active as an androgen than testosterone, DHT is formed in bone by 5alpha-R type 1 action from circulating testosterone, and bone cells also express the androgen receptor, local DHT production may play a physiological role in human bone homeostasis.

Expression of CD44 is involved in the maturation as well as the homing of haemopoietic progenitor cells. Whether these processes are mediated by CD44 standard (CD44s) or variant (CD44v) isoforms is unknown. To assign functional activities of CD44 in haemopoiesis of the rat to distinct isoforms, ligand binding of haemopoietic progenitor cells was inhibited by monoclonal antibodies recognizing an epitope on CD44s (Ox50) or CD44 exon v6, (1.1ASML). The vast majority of rat bone marrow cells (BMC) as well as stromal cells and non-adherent cells in long-term bone marrown culture (LTBMC) expressed CD44s. Bone marrow cells and non-adherent cells in LTBMC, but not the stromal cells, also contained a population of large and granulated cells, which stained with anti-CD44v6. In vivo and in vitro reconstitution experiments revealed that homing of BMC as well as settlement on stromal elements was influenced exclusively by anti-CD44s, which also inhibited proliferation of progenitor cells. Anti-CD44v6 had no influence on homing and seeding, but interfered with stroma formation and progenitor maturation. Finally, restoration of functional activity of T-lineage cells was impaired in the presence of anti-CD44v6. The data indicate that CD44s and CD44v6 fulfilled distinct functions in haemopoiesis of the rat. Although CD44s facilitated homing and expansion of stem cells, progenitor cells, CD44v6 was involved in differentiation processes, particularly of lymphoid progenitor cells.

Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5′-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3′-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (α) and 40-kDa (β) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA.

Sphingolipid activator proteins (SAPs) are small, nonenzymic glycoproteins that stimulate lysosomal degradation of various sphingolipids. SAP-1, SAP-2, and two additional potential activator proteins are derived from a common precursor by proteolytic processing. A severe case of sphingolipid storage disease that led to death within 16 weeks was attributed to a possible total deficiency of the SAPs generated by this gene (Harzer, K., Paton, B. C., Poulos, A., Kustermann-Kuhn, B., Roggendorf, W., Grisar, T., and Popp, M. (1989) Eur. J. Pediatr. 149, 31-39). Analysis of the SAP precursor cDNA from the patient and his fetal sibling showed an A to T transversion in the initiation codon. Allele-specific oligonucleotide hybridization revealed that both parents are heterozygous carriers for this mutation. In pulse-chase experiments using antisera raised against SAP-1 or SAP-2, no cross-reacting material could be detected in the patients' fibroblasts.

Full-length cDNAs coding for the human GM2-activator protein has been isolated and characterized, and its genomic structure studied in two overlapping clones in λ-EMBL-4 isolated from a human brain genomic library. Two different cDNAs were found that were identical to the 5′-terminus to nt 1311 (counted from the A of the initiation codon, ATG) including the entire protein coding sequence. However, they were entirely dissimilar in the 3′-non-coding sequences. The genomic clones covered 94% of the full-length cDNA sequence. Three introns were found. The last exon spans contiguously the carboxyl terminus of the protein and the entire 3′-untranslated region of one of the two cDNAs with different 3′-ends. The origin of the 3′-portion of the other cDNA clone is not clear at this time.

We describe a novel mutation in exon 1 of the androgen receptor gene in a patient with complete androgen insensitivity (CAIS). Endocrine findings were typical for androgen insensitivity (testosterone serum levels in the upper limit of normal males and increased LH serum concentrations). Biochemical investigations in cultured genital skin fibroblasts of the patient showed a normal 5α-reductase activity but a complete absence of androgen binding. Western blot analysis revealed no detectable protein product. Sequence analysis of the entire coding region of the androgen receptor gene resulted in the identification of a 2-bp deletion in codon 472, causing frameshift and introduction of a premature stop codon 27 codons downstream of the mutation.

The action of androgen by way of the AR is required for the development of male gonads and external genitalia. The interplay between androgens and the somatotropic axis, in particular the IGFs in sexual development, is currently under thorough investigation. The IGF system is thought to mediate the androgen action in androgen-responsive cells. To investigate the interaction of androgens with the IGF system, we compared the expression of IGFs and IGF-binding proteins in cultured genital skin fibroblasts from nine patients with the syndrome of complete androgen insensitivity with that in genital skin fibroblasts from 10 normally virilized males. Mutations in the AR gene and/or abnormalities of the AR protein in the immunoblot were detected in all complete androgen insensitivity genital skin fibroblast strains. They caused a complete failure of DHT binding. RIA and RT-PCR demonstrated that the genital skin fibroblast strains expressed IGF-II, IGF-binding protein-2, and IGF-binding protein-3, but no IGF-I. Most strikingly, complete androgen insensitivity genital skin fibroblast strains produced significantly lower IGF-II (P < 0.001; 42.2 +/- 9.7 vs. 106.9 +/- 11.8 ng/mg protein) and IGF-II mRNA (P < 0.01, by RT-PCR) than control genital skin fibroblast strains. The production of IGF-binding protein-2 was also decreased (P < 0.03) in complete androgen insensitivity genital skin fibroblasts, whereas that of IGF-binding protein-3 did not differ. Furthermore, high levels of IGF-binding protein-5 mRNA were detected in all genital skin fibroblast strains, whereby the 28-kDa band in the ligand blot, probably representing IGF-binding protein-5, was more abundant in complete androgen insensitivity genital skin fibroblasts. Exposure of the genital skin fibroblasts to T (5 x 10(-8) M) had only weak effects on the expression of IGFs and IGF-binding proteins. In conclusion, although the mechanism underlying these differences requires further study, it is conceivable that in addition to the endocrine actions of IGF-I, IGF-II and IGF-binding protein-2, as local growth factors, are involved in the mediation of androgen action and growth of genital tissues.

The GM2 activator is a hexosaminidase A-specific glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. Genetic deficiency of GM2 activator leads to a neurological disorder, an atypical form of Tay-Sachs disease (GM2 gangliosidosis variant AB). Here, we describe a G506 to C transversion (Arg169 to Pro) in the mRNA of an infantile patient suffering from GM2-gangliosidosis variant AB. Using the polymerase chain reaction amplification and direct-sequencing technique, we found the patient to be homozygous for the mutation, whereas the parents were, as expected, heterozygous. BHK cells transfected with a construct of mutant cDNA gave no GM2 activator protein detectable by the Western blotting technique, whereas those transfected by a wild-type cDNA construct showed a significant level of human GM2 activator protein. The substitution of proline for the normal Arg169 therefore appears to result in premature degradation of the mutant GM2 activator, either during the post-translational processing steps or after reaching the lysosome. The basis for the phenotype of GM2 gangliosidosis variant AB may therefore be either inactivation of the physiological activator function by the point mutation or instability of the mutant protein.

GM2-gangliosidoses are neurological disorders caused by a genetic deficiency of either the β-hexosaminidase A or the GM2 activator, a glycolipid binding protein. In a patient with an immunologically proven GM2 activator protein deficiency, A T412 → C transition (counted from A of the initiation codon) was found in the coding sequence, which results in the substitution of Arg for the normal Cys107 in the mature GM2 activator protein. The remainder of the coding sequence remained entirely normal.

The organization of 14 exons covering 97% of the cDNA sequence of human cerebroside sulfate activator protein precursor has been determined from two overlapping EMBL-4 human genomic clones extending over 17kb. All exons and exon/intron splice junctions and five introns were sequenced. Exon 8 consists of only 9 bp and is involved in alternative splicing which generates three different mRNAs of cerebroside sulfate activator precursor.