A novel yeast expression system to study androgen action.
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Animals↗
Biomedical subjects
C Y Young
Publications and source records attributed to C Y Young.
Tumor-promoting phorbol ester-induced cell death and gene expression in a human prostate adenocarcinoma cell line.
12-O-tetradecanoyl phorbol ester (TPA) has profound cytotoxic effects on a human prostate cancer cell line, LNCaP. The TPA effect may be mediated via a protein kinase C (PKC) pathway, since staurosporine, a potent PKC inhibitor, could reverse the cell-killing effect. Our studies, based on cellular fragmentation, chromatin condensation, and nuclear fragmentation, suggest that the cell-killing effect is due to apoptosis. Moreover, we also examined expression of early growth response genes and androgen-induced genes in association with TPA-induced apoptosis. Northern blot analysis demonstrated that androgen induction of human glandular kallikrein-1 (hKLK2) mRNA was repressed by TPA in a concentration-dependent manner. A time course study showed that both hKLK2 and c-myc mRNAs were repressed by TPA as early as four hours. In contrast, the steady state mRNA levels for c-fos, c-jun, nerve growth factor induced gene A, and the orphan steroid receptor nur77 were rapidly induced within the first two hours of the treatment. Furthermore, transient co-transfection experiments demonstrated that c-fos and c-jun could repress androgen receptor-mediated gene induction. The above studies suggest that (1) the repression of androgen induction of gene expression by TPA-activated PKC is at least in part due to overexpression of c-jun and c-fos and (2) PKC may be a negative growth regulator in prostate cells.
Adenocarcinoma↗
Implementing a Physician's Workstation using client/server technology and the distributed computing environment.
PWS is a physician's workstation research prototype developed to explore the use of information management tools by physicians in the context of patient care. The original prototype was implemented in a client/server architecture using a broadcast message server. As we expanded the scope of the prototyping activities, we identified the limitations of the broadcast message server in the areas of scalability, security, and interoperability. To address these issues, we reimplemented PWS using the Open Software Foundation's Distributed Computing Environment (DCE). We describe the rationale for using DCE, the migration process, and the benefits achieved. Future work and recommendations are discussed.
Ambulatory Care Information Systems↗
Androgen induction of a human prostate-specific kallikrein, hKLK2: characterization of an androgen response element in the 5' promoter region of the gene.
The human prostate-specific kallikreins, human glandular kallikrein-1 (hKLK2) and prostate-specific antigen (hKLK3), have been shown to be regulated by androgens. To determine whether the androgen induction of these genes is transcriptionally regulated via an androgen response element, an hKLK2 promoter DNA fragment was linked to a promoterless chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with an androgen receptor expression vector in an androgen receptor-less human prostate cell line, PC-3. Dose response and steroid specificity experiments showed that the hKLK2 promoter confers androgen receptor-mediated gene induction in a ligand-specific manner. Moreover, 5' deletion constructs of the hKLK2 promoter DNA and internal deletion constructs devoid of the 5' half-site of the putative androgen responsive element (ARE) were used to show that the putative ARE is indeed acting as a functional ARE in prostate cells. In addition, multiple AREs from both hKLK2 and hKLK3 were able to reconstitute androgenic induction, further strengthening the argument that the AREs are functional. Although previous studies have shown that hKLK3 mRNA is expressed at a higher level than that of hKLK2, our results suggest that the hKLK2 ARE may have higher androgenic inducibility than the hKLK3 ARE. These results suggest that other cis-acting elements may be involved in coordinating in vivo androgenic induction of hKLK2 and hKLK3 genes.
Androgens↗
Automated identification of relevant patient information in a physician's workstation.
The introduction of computer-based patient records offers an opportunity to improve the ability of physicians to browse the medical record and to monitor patient events. We describe a methodology to identify relevant information in the patient record. This methodology combines a patient-specific physiological model with functions to determine relevance of patient information. The model consists of a qualitative representation of physiological parameters and influences, custom-tailored to a particular patient's problems, medications, and test results. We describe two applications of this model in the context of an integrated physician's workstation: automatic linking of relevant patient information for configuration of user-interface displays, and monitoring of patient events to prevent oversight of noteworthy information.
Computer Systems↗
Tumor-promoting phorbol ester down-regulates the androgen induction of prostate-specific antigen in a human prostatic adenocarcinoma cell line.
Prostate-specific antigen (PSA) is the most sensitive marker available for monitoring the progression of prostate cancer and response to therapy. In a previous study, we demonstrated tissue-specific expression of PSA glycoprotein and mRNA and its regulation through the androgen receptor. In this study, we examine the effects of protein kinase A (PKA) and protein kinase C (PKC) on the androgen regulation of PSA in a human adenocarcinoma cell line, LNCaP. Northern blot analysis demonstrated that forskolin, an activator of PKA, had no effect on the androgen regulation of PSA. However, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a direct activator of PKC, showed a time- and dose-dependent repression of the androgen regulation of PSA glycoprotein and mRNA. The biologically inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, had no effect. Staurosporine, a PKC inhibitor, blocked the TPA-mediated repression of the androgenic stimulation of PSA glycoprotein. In addition, the calcium ionophore, A23187, was able to simulate the actions of TPA, presumably through activation of PKC via calcium mobilization. In summary, the androgenic regulation of PSA protein and mRNA is repressed by tumor-promoting phorbol esters through the PKC pathway. This indicates that the effects of TPA may be secondary to repressed gene transcription or altered mRNA stability. In addition, this study emphasizes that the androgenic regulation of PSA is complex and may involve other extracellular transduction signals.
Alkaloids↗
Tissue-specific and hormonal regulation of human prostate-specific glandular kallikrein.
Kallikreins are involved in the posttranslational processing of a number of specific polypeptide precursors. Previously, human glandular kallikrein (hGK-1) mRNA has been identified in the prostate; however, the hGK-1 protein has not been identified and characterized. Therefore, its physiologic function in the prostate is not known. In this study, we have isolated a full-length hGK-1 cDNA from a human adenocarcinoma cell line, LNCaP. In vitro translation experiments demonstrated that the molecular size of the hGK-1 protein generated from this cDNA is similar to that of prostate-specific antigen (PSA), a protein which is produced exclusively in the prostate. In situ hybridization with a hGK-1-specific oligonucleotide probe (77 bases), which can differentiate hGK-1 mRNA from PSA mRNA, demonstrated the hGK-1 mRNA to be located in the prostate epithelium. The hGK-1 mRNA was colocalized with PSA mRNA in prostatic epithelia. Moreover, in situ hybridization studies revealed that the level of hGK-1 mRNA in human benign prostatic hyperplasia tissues is approximately half that of PSA mRNA. Furthermore, we have demonstrated that hGK-1 mRNA is under androgenic regulation in LNCaP cells. Time course analysis revealed that hGK-1 mRNA levels increased significantly at 5 h of mibolerone treatment and reached maximal levels by 9 h. In addition, hGK-1 mRNA levels were increased by dihydrotestosterone, but not by dexamethasone or diethylstilbestrol treatments. Flutamide, a nonmetabolized anti-androgen, repressed the androgenic effects. These studies suggest that expression of hGK-1 mRNA is regulated by androgen via the androgen receptor.
Adenocarcinoma↗
Hormonal regulation of prostate-specific antigen (PSA) glycoprotein in the human prostatic adenocarcinoma cell line, LNCaP.
Prostate-specific antigen (PSA) has emerged as the most useful marker for management of patients with prostate cancer. The regulation of this glycoprotein in vivo has important clinical implications. Indirect evidence indicates that the PSA glycoprotein might be regulated by androgens, and previous studies in this laboratory have demonstrated that PSA mRNA is upregulated by androgens. The current work reports a detailed study of PSA glycoprotein expression as influenced by steroid hormones in a human prostatic adenocarcinoma cell line, LNCaP. First, we have examined the steroid binding specificity of the androgen receptor in this cell line. In comparison with wild-type rat androgen receptor in prostate, the receptor in LNCaP cells has altered affinity for a number of steroids or analogs such as progesterone (R5020), antiprogesterone (RU486), two antiandrogens (cyperoterone acetate and hydroxyflutamide), and an androgen metabolite (epitestosterone). However, its affinity for androgens (mibolerone, dihydrotestosterone, and testosterone) is not changed. The receptor does not bind to the synthetic glucocorticoids (triaminolone acetonide and dexamethasone) nor to a synthetic estrogen DES (diethylstilbestrol). The change of the steroid binding specificity of the receptor is correlated with a single mutation (A----G at nucleotide #876 relative to the initiation codon) of the steroid binding domain of the receptor. The mutation and alteration of steroid-binding specificity of the androgen receptor is also correlated with PSA glycoprotein expression affected by different ligands tested. We have demonstrated that the PSA glycoprotein is upregulated by androgens and is affected by neither epidermal growth factor nor basic fibroblast growth factor. Moreover, PSA glycoprotein could be induced by R5020, estradiol, and epitestosterone; but neither glucocorticoids nor DES had any effect on PSA induction. Interestingly, although the antiandrogen, cyperotone acetate, had the ability to induce PSA, both RU486 and hydroxyflutamide could block androgen and progesterone induction of PSA glycoprotein. Therefore, we conclude that the PSA glycoprotein expression is influenced predominantly by androgens via its receptor, and the mutation of the receptor can affect the expression of this cellular gene by the steroids other than androgens.
Adenocarcinoma↗
Sequence and structure of the galactose adherence lectin of Entamoeba histolytica.
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Amino Acid Sequence↗
Hormonal regulation of prostate-specific antigen messenger RNA in human prostatic adenocarcinoma cell line LNCaP.
Prostate-specific antigen (PSA) is a member of the kallikrein gene family and is expressed exclusively in human prostatic epithelial cells. PSA protein has been an important biological marker for prostate cancers. Until now, very little was known about the regulation of PSA expression in prostatic cells. In this study, we have developed a specific oligonucleotide probe which recognizes PSA but not the human glandular kallikrein. This is crucial because both PSA and human glandular kallikrein are expressed in the prostate at relatively high levels and have high nucleotide sequence homology (greater than 82%). Utilizing a S-labeled PSA-specific probe, PSA mRNA was localized within the glandular epithelium of the prostate. Northern blot analysis detected a single 1.6-kilobase transcript in LNCaP cells, a cell line derived from a human prostate adenocarcinoma metastasis. Therefore, LNCaP cells were used to study the androgenic effects on PSA mRNA expression. A time course study demonstrated that PSA mRNA was induced by mibolerone (a nonmetabolizable synthetic androgen) and reached maximal levels after 9 h. The induction of PSA mRNA required as little as 0.3 nM mibolerone. In addition to mibolerone, PSA mRNA could be induced by the natural androgen, dihydrotestosterone, but not by the synthetic glucocorticoid, dexamethasone, or the synthetic estrogen, diethylstilbestrol. Moreover, in the presence of dihydrotestosterone, PSA mRNA was depressed by hydroxyflutamide (an antiandrogen). These results suggest strongly that the androgenic effects on PSA mRNA in LNCaP cells may be via the function of the androgen receptor.
Adenocarcinoma↗
Heterogenous database integration in a physician workstation.
We discuss the integration of a variety of data and information sources in a Physician Workstation (PWS), focusing on the integration of data from DHCP, the Veteran Administration's Distributed Hospital Computer Program. We designed a logically centralized, object-oriented data-schema, used by end users and applications to explore the data accessible through an object-oriented database using a declarative query language. We emphasize the use of procedural abstraction to transparently integrate a variety of information sources into the data schema.
Computer Communication Networks↗
An open systems architecture for development of a physician's workstation.
We are developing a physician's workstation consisting of highly integrated information management tools for use by physicians in patient care. We have designed and implemented an open systems, client/server architecture as a development platform which allows new applications to be easily added to the system. Applications cooperate by exchanging messages via a broadcast message server.
Ambulatory Care Information Systems↗
Physicians' workstations: integrated information management for clinicians.
This paper describes a project to specify, design, develop, and evaluate a physician's workstation for use in patient care. We conducted an ethnographic study of physicians' information needs in an outpatient setting, from which we derived a set of functional specifications for a physician's workstation. We have implemented an experimental prototype using an open systems, client/server architecture, and are exploring research issues in heterogeneous database integration, object-oriented database technology, model-based reasoning, and semantic integration. We plan to evaluate our workstation prototype in a clinical setting to assess its impact on quality of care and health care costs.
Artificial Intelligence↗
Modelling work practices: input to the design of a physician's workstation.
To ensure tight coupling between users' work practices and the system's model of these practices, designers need methods to generate accurate descriptions of what users actually do. This paper illustrates how we model physicians' information needs and translate knowledge about these needs into design specifications.
Artificial Intelligence↗
Molecular cloning of androgen receptors from divergent species with a polymerase chain reaction technique: complete cDNA sequence of the mouse androgen receptor and isolation of androgen receptor cDNA probes from dog, guinea pig and clawed frog.
We have cloned and sequenced 2.8 kilobases of cDNA encoding the mouse androgen receptor by RNA amplification with transcript sequencing. Sequence analysis predicts that this cDNA contains an open reading frame of 2697 nucleotides encoding a polypeptide of 899 amino acids. Androgen receptor cDNA probes of dog, guinea pig, and frog were also isolated and sequenced using consensus primers derived from human and rat androgen receptor cDNAs. Northern blot analysis with the species-specific probes revealed similarities in size between amphibian and mammalian mRNAs. These results demonstrate the utility of this technique in obtaining nucleic acid probes and sequence information of steroid receptors from different species. The sequence data and the Northern blot analysis of the receptors in different species demonstrate that the androgen receptor has been well-conserved during evolution.
Amino Acid Sequence↗
In situ hybridization of prostate-specific antigen mRNA in human prostate.
Prostate-specific antigen (PSA) mRNA was detected by in situ hybridization utilizing a 428 base pair [35S]-labelled cDNA probe from the 3' noncoding region of the PSA gene. Thirty six fresh surgical specimens were collected from patients undergoing radical retropubic prostatectomy for carcinoma of the prostate. Quantitative analysis of the levels of PSA mRNA in both the benign and malignant tissues was performed using an IBAS 2000 Image Analysis System. The results of this study demonstrated that there is a significant decrease in the expression of PSA mRNA in the carcinoma tissue when compared to the benign epithelium. The average binding (number of silver grains/1 x 10(4) microns. 2) for 20 specimens of malignant epithelium was 475 +/- 161 and 586 +/- 140 for 16 specimens of benign epithelium (p less than 0.05). Eleven patients had both benign and malignant tissue from the same surgical specimen available for study. From these paired specimens, the PSA mRNA expression was also significantly reduced in the malignant epithelium when compared to the benign epithelium, 445 +/- 162 and 588 +/- 135 respectively (p less than 0.005). The PSA protein was detected using a monoclonal antibody to PSA with an immunohistochemical staining technique. The PSA protein expression paralleled the expression of the PSA mRNA in the majority of the tissue sections. Many of the tumor specimens showed a heterogeneous expression of PSA, whereas all of the benign epithelium had a uniform high level of PSA expression. In conclusion, PSA mRNA and protein are located only within the glandular epithelial tissue, the expression of PSA protein parallels that of the PSA mRNA, and both the PSA protein and PSA mRNA are significantly decreased in the malignant epithelium when compared to benign prostatic epithelium.
Adenocarcinoma↗
Overexpression of a partial human androgen receptor in E. coli: characterization of steroid binding, DNA binding, and immunological properties.
The recent cloning of human androgen receptor (AR) cDNAs in this and other laboratories has provided valuable probes for investigating the structure and function of the AR at the molecular level. We now report the overexpression of a region of the human AR containing both the DNA- and hormone-binding domains in E. coli, which provides a means to produce large amounts of AR for analysis and use in functional studies. Under isopropyl-beta-D-thiogalactopyranoside induction, a tripartite protein, consisting of beta-galactosidase, a collagenase recognition site, and AR polypeptide, was produced in E. coli JM109 using pSS20 a as a vector. About 1 mg of the fused AR could be recovered per liter bacterial culture. The induced protein could readily be detected in a sodium dodecyl sulfate-polyacrylamide gel by Coomassie blue staining. Its identity was confirmed by Western blot analysis using antibodies to both beta-galactosidase and the AR. Scatchard analysis of the androgen-binding activity of the hybrid AR revealed high affinity binding to the synthetic androgen, Mibolerone (Kd, approximately 1.2 nM). Competition studies demonstrated the fusion protein's specificity for androgens. The hybrid receptor formed immune complexes with human anti-AR serum that sedimented at about 19S in 10-50% linear sucrose gradients containing 0.4 M KCl. Gel band shift assays revealed that the hybrid receptor protein forms specific complexes with a synthetic steroid response element derived from the mouse mammary tumor virus long terminal repeat region. These results demonstrate that the recombinant AR expressed in E. coli possesses many of the functional properties characteristic of DNA- and steroid-binding domains of the native AR.
Androgens↗
The androgen receptor of the testicular-feminized (Tfm) mutant mouse is smaller than the wild-type receptor.
The physicochemical and immunological properties of androgen receptors from kidney and brain of testicular-feminized (Tfm) mutant mice and wild-type mice were compared. Analysis by gel filtration and sucrose density gradients revealed that the mol wt of the mutant receptor was 66K (38A; 3.8S) which was significantly smaller than the 110K (53A; 4.6S) size of the wild-type androgen receptor (P less than 0.05). Mixing experiments failed to demonstrate any role for differential proteolysis in the size differences between these receptors. Interaction of the mutant androgen receptor with specific polyclonal antiandrogen receptor antibodies produced significantly smaller immune complexes than that formed with wild-type receptor (12S vs. 17S; P less than 0.01). This confirmed the smaller size of the Tfm mutant androgen receptor and suggested that it contained fewer epitopes. The Tfm kidney cytosols also demonstrated a decreased concentration of androgen receptor-binding activity relative to that of the wild type. Together, these results suggest that the androgen insensitivity associated with the Tfm phenotype is due to a deficiency of androgen receptor in target tissues and a qualitative defect in the androgen receptor protein itself.
Androgen-Insensitivity Syndrome↗