Dysregulated gene expression predicts tumor aggressiveness in African-American prostate cancer patients.

Molecular mechanisms underlying the health disparity of prostate cancer (PCa) have not been fully determined. In this study, we applied bioinformatic approach to identify and validate dysregulated genes associated with tumor aggressiveness in African American (AA) compared to Caucasian American (CA) men with PCa. We retrieved and analyzed microarray data from 619 PCa patients, 412 AA and 207 CA, and we validated these genes in tumor tissues and cell lines by Real-Time PCR, Western blot, immunocytochemistry (ICC) and immunohistochemistry (IHC) analyses. We identified 362 differentially expressed genes in AA men and involved in regulating signaling pathways associated with tumor aggressiveness. In PCa tissues and cells, NKX3.1, APPL2, TPD52, LTC4S, ALDH1A3 and AMD1 transcripts were significantly upregulated (p < 0.05) compared to normal cells. IHC confirmed the overexpression of TPD52 (p = 0.0098) and LTC4S (p < 0.0005) in AA compared to CA men. ICC and Western blot analyses additionally corroborated this observation in PCa cells. These findings suggest that dysregulation of transcripts in PCa may drive the disparity of PCa outcomes and provide new insights into development of new therapeutic agents against aggressive tumors. More studies are warranted to investigate the clinical significance of these dysregulated genes in promoting the oncogenic pathways in AA men.

Drug-induced gynecomastia.

Drugs account for about 20% of gynecomastia cases in men. As a number of factors can alter the estrogen:androgen ratio, several pathophysiologic mechanisms are associated with drugs causing this disorder. Antiandrogens, protease inhibitors, and nucleoside reverse transcriptase inhibitors are the most common drug causes of gynecomastia, whereas first-generation antipsychotics, spironolactone, verapamil, and cimetidine are less common causes. Other drugs have been reported rarely as causes. Treatment may involve switching to an alternative agent or may require surgery or irradiation if the causative agent cannot be discontinued. We reviewed the literature on drug-induced gynecomastia and provided another perspective by reviewing data from the United States Food and Drug Administration's Adverse Event Reporting System. Epidemiologic studies are needed to provide a more accurate description of the frequency of drug-induced gynecomastia.

Bilateral cingulotomy and anterior capsulotomy applied to patients with aggressiveness.

OBJECTIVE: To perform a preliminary study on the effects and safety of bilateral cingulotomy and anterior capsulotomy in patients with aggressive behavior. PATIENTS AND METHODS: Twenty-three psychiatric patients showing aggressive behavior refractory to conventional treatment were initially evaluated. The subjects were clinically selected using the Overt Aggression Scale (OAS) and the Global Assessment of Functioning Scale (GAF). Each case was carefully reviewed by the Ethics Committee of Mexico's General Hospital. Once selection criteria were met, stereotactic lesions were made using radiofrequency on the anterior limb of the internal capsule and supragenual cingulum. Statistical differences were evaluated with a Wilcoxon test at 6 months and at 4 years. RESULTS: Ten patients underwent surgery. Their OAS and GAF scores decreased after the procedure at the 6-month (p < 0.05) and at the 4-year (p = 0.068) follow-up. Four patients showed mild and transitory postsurgical complications (hyperphagia and somnolence). CONCLUSIONS: Bilateral anterior capsulotomy in combination with cingulotomy may reduce aggressive behavior and improve clinical evaluations. Very strict clinical and ethical evaluations were applied prior to considering patients for this treatment.

Maternal hepatic growth response to pregnancy in the mouse.

Pregnancy is characterized by physiological adjustments in the maternal compartment. In this investigation, the influence of pregnancy on maternal liver was examined in CD-1 mice. Dramatic changes were observed in the size of the maternal liver during pregnancy. Livers doubled in weight from the non-pregnant state to day 18 of pregnancy. The pregnancy-induced hepatomegaly was a physiological event of liver growth confirmed by DNA content increase and detection of hepatocyte hyperplasia and hypertrophy. Growth of the liver was initiated following implantation and peaked at parturition. The expression and/or activities of key genes known to regulate liver regeneration, a phenomenon of liver growth compensatory to liver mass loss, were investigated. The results showed that pregnancy-dependent liver growth was associated with interleukin (IL)-6, tumor necrosis factor α, c-Jun and IL-1ß, but independent of hepatocyte growth factor, fibroblast growth factor 1, tumor necrosis factor receptor 1, constitutive androstane receptor and pregnane X receptor. Furthermore, maternal liver growth was associated with the activation of hepatic signal transducer and activator of transcription 3, ß-catenin and epidermal growth factor receptor, but pregnancy did not activate hepatic c-Met. The findings suggest that the molecular mechanisms regulating pregnancy-induced liver growth and injury-induced liver regeneration exhibit overlapping features but are not identical. In summary, the liver of the mouse adapts to the demands of pregnancy via a dramatic growth response driven by hepatocyte proliferation and size increase.

Human growth hormone: 45-kDa isoform with extraordinarily stable interchain disulfide links has attenuated receptor-binding and cell-proliferative activities.

BACKGROUND: Human growth hormone (hGH) is a complex mixture of molecular isoforms. Gaps in our knowledge exist regarding the structures and biological significances of the uncharacterized hGH molecular variants. Mercaptoethanol-resistant 45-kDa human growth hormone (MER-45 kDa hGH) is an extraordinarily stable disulfide-linked hGH homodimer whose biological significance is unknown. OBJECTIVES: To elucidate the pharmacokinetic abilities of dimeric MER-45-kDa hGH to bind to GH and prolactin (PRL) receptors and to elucidate its abilities to stimulate cell proliferation in lactogen-induced and somatogen-induced in vitro cell proliferation bioassays. DESIGN: The binding of MER-45-kDa hGH to GH and PRL receptors was tested in radioreceptor assays (RRAs). Competitive displacements of [(125)I]-bovine GH from bovine liver membranes, [(125)I]-ovine PRL from lactating rabbit mammary gland membranes and [(125)I]-hGH from human IM-9 lymphocytes by unlabelled GHs, PRLs or dimeric MER-45-kDa hGH were evaluated. The abilities of dimeric MER-45-kDa hGH to stimulate proliferation of lactogen-responsive Nb2 lymphoma cells and to stimulate proliferation of somatogen-responsive T47-D human breast cancer cells were assessed by incubation of cells with GHs or PRLs and subsequently measuring growth using the MTS cell proliferation assay. RESULTS: Dimeric MER-45-kDa hGH, compared to monomeric hGH, had reduced binding affinities to both GH and prolactin receptors. In a bovine liver GH radioreceptor assay its ED(50) (197.5 pM) was 40.8% that of monomeric hGH. In a human IM-9 lymphocyte hGH RRA its ED(50) (2.96 nM) was 26.2% that of monomeric hGH. In a lactating rabbit mammary gland prolactin RRA its ED(50) (3.56 nM) was 16.8% that of a monomeric hGH. Dimeric MER-45-kDa hGH, compared to monomeric hGH, had a diminished capacity to stimulate proliferation of cells in vitro. In a dose-response relationship assessing proliferation of Nb2 lymphoma cells its ED(50) (191 pM) was 18.0% that of monomeric hGH. While monomeric hGH stimulated a 2.2-fold proliferation of T47-D human breast cancer cells above vehicle control, dimeric MER-45-kDa hGH was unable to stimulate the cells to proliferate and slightly inhibited their proliferation to 77.6% that of control. CONCLUSIONS: The topological arrangement of monomeric hGHs to form an unusually stable disulfide-linked dimer markedly diminishes hGH's binding affinities to both GH and PRL receptors and also drastically attenuates its ability to stimulate proliferation of cells in vitro.

Gene profiling of maternal hepatic adaptations to pregnancy.

BACKGROUND: Maternal metabolic demands change dramatically during the course of gestation and must be co-ordinated with the needs of the developing placenta and fetus. The liver is critically involved in metabolism and other important functions. However, maternal hepatic adjustments to pregnancy are poorly understood. AIM: The aim of the study was to evaluate the influences of pregnancy on the maternal liver growth and gene expression profile. METHODS: Holtzman Sprague-Dawley rats were mated and sacrificed at various stages of gestation and post-partum. The maternal livers were analysed in gravimetric response, DNA content by PicoGreen dsDNA quantitation reagent, hepatocyte ploidy by flow cytometry and hepatocyte proliferation by ki-67 immunostaining. Gene expression profiling of non-pregnant and gestation d18.5 maternal hepatic tissue was analysed using a DNA microarray approach and partially verified by northern blot or quantitative real-time PCR analysis. RESULTS: During pregnancy, the liver exhibited approximately an 80% increase in size, proportional to the increase in body weight of the pregnant animals. The pregnancy-induced hepatomegaly was a physiological event of liver growth manifested by increases in maternal hepatic DNA content and hepatocyte proliferation. Pregnancy did not affect hepatocyte polyploidization. Pregnancy-dependent changes in hepatic expression were noted for a number of genes, including those associated with cell proliferation, cytokine signalling, liver regeneration and metabolism. CONCLUSIONS: The metabolic demands of pregnancy cause marked adjustments in maternal liver physiology. Central to these adjustments are an expansion in hepatic capacity and changes in hepatic gene expression. Our findings provide insights into pregnancy-dependent hepatic adaptations.

Hypoxia-inducible factor-dependent production of profibrotic mediators by hypoxic hepatocytes.

BACKGROUND/AIMS: During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B and plasminogen activator inhibitor-1 (PAI-1) in the liver, during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1alpha in liver cell types. Accordingly, the hypothesis was tested that HIF-1alpha is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells. METHODS: In this study, hepatocytes were isolated from the livers of control and HIF-1alpha- or HIF-1beta-deficient mice and exposed to hypoxia. RESULTS: Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1alpha and upregulated PAI-1, vascular endothelial cell growth factor and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, the levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1alpha-deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2alpha, may also regulate these genes. In support of this, HIF-2alpha was activated in hypoxic hepatocytes, and exposure of HIF-1beta-deficient hepatocytes to 1% oxygen completely prevented upregulation of PAI-1, vascular endothelial cell growth factor and ADM-1, suggesting that HIF-2alpha may also contribute to upregulation of these genes in hypoxic hepatocytes. CONCLUSIONS: Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.

MAP dendrimer elicits antibodies for detecting rat and mouse GH-binding proteins.

The membrane-bound rat GH-R and an alternatively spliced isoform, the soluble rat GH-BP, are comprised of identical N-terminal GH-binding domains; however, their C-terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent MAP dendrimer with four identical branches of a C-terminal peptide sequence of the rat GH-BP (GH-BP(263-279)) was synthesized and used as an immunogen in rabbits. Solid-phase peptide synthesis of four GH-BP(263-279) segments onto a tetravalent Lys(2)-Lys-beta-Ala-OH core peptide was carried out using Fmoc chemistry. The mass of the RP-HPLC-purified synthetic product, 8398 Da, determined by ESI-MS, was identical to expected mass. Three anti-rat GH-BP(263-279) MAP antisera, BETO-8039, BETO-8040, and BETO-8041, at dilutions of 10(-3), recognized both the rat GH-BP(263-279) MAP and recombinant mouse GH-BP with ED(50)s within a range of 5-10 fmol, but did not cross-react with BSA in dot blot analyses. BETO-8041 antisera (10(-3) dilution) recognized GH-BPs of rat serum and liver having M(r)s ranging from 35 to 130 kDa, but did not recognize full-length rat GH-Rs. The antisera also detected recombinant mouse GH-BPs. In summary, the tetravalent rat GH-BP(263-279) MAP dendrimer served as an effective immunogenic antigen in eliciting high titer antisera specific for the C-termini of both rat and mouse GH-BPs. The antisera will facilitate studies aimed at improving our understanding of the biology of GH-BPs.

Extraordinarily stable disulfide-linked homodimer of human growth hormone.

Although a 22-kDa human growth hormone (hGH) is the predicted protein product of the hGH-N gene, a pleiotropic collection of uncharacterized molecular weight and charge isoforms is also produced. Using chromatography and preparative SDS-PAGE under reducing conditions we isolated an unusually stable mercaptoethanol-resistant (MER) 45-kDa hGH. A 5-h incubation at 100 degrees C in the presence of 2-mercaptoethanol was required to convert approximately 90% of MER-45-kDa hGH into a 22-kDa hGH. Other reductants were not as effective in splitting MER-45-kDa hGH. After fracturing MER-45-kDa hGH, the 22-kDa hGH fragments would spontaneously reassociate if the reductant was removed; however, alkylation of cysteine residues prevented their reassociation. Identical amino acid sequences for the first six N-terminal residues were obtained for MER-45-kDa hGH and its 22-kDa hGH cleavage product. Structural identity of MER-45-kDa hGH and 22-kDa hGH was demonstrated by MALDI-TOF mass spectrometry of tryptic digests. MER-45-kDa hGH did not break up upon incubation with EDTA and EGTA. The significance of this work to our understanding of the structure of hGH isoforms is that it demonstrates that MER-45-kDa hGH is not a single chain polypeptide but is instead a homodimer of 22-kDa hGH monomers. The MER-45-kDa hGH dimer is held together by interchain disulfide bonds and not by divalent metal cation bridges. Additionally, MER-45-kDa hGH's interchain disulfide links are exceptionally resistant to reducing agents and thus confer extreme stability to the homodimer.