The immunotoxicity of natural and depleted uranium: From cells to people.

Uranium is a naturally occurring element found in the environment as a mixture of isotopes with differing radioactive properties. Enrichment of mined material results in depleted uranium waste with substantially reduced radioactivity but retains the capacity for chemical toxicity. Uranium mine and milling waste are dispersed by wind and rain leading to environmental exposures through soil, air, and water contamination. Uranium exposure is associated with numerous adverse health outcomes in humans, yet there is limited understanding of the effects of depleted uranium on the immune system. The purpose of this review is to summarize findings on uranium immunotoxicity obtained from cell, rodent and human population studies. We also highlight how each model contributes to an understanding of mechanisms that lead to immunotoxicity and limitations inherent within each system. Information from population, animal, and laboratory studies will be needed to significantly expand our knowledge of the contributions of depleted uranium to immune dysregulation, which may then inform prevention or intervention measures for exposed communities.

The curcumin analog ca27 down-regulates androgen receptor through an oxidative stress mediated mechanism in human prostate cancer cells.

BACKGROUND: The androgen receptor (AR) plays a critical role in prostate cancer development and progression. Therefore, the inhibition of AR function is an established therapeutic intervention. Since the expression of the AR is retained and often increased in progressive disease, AR protein down-regulation is a promising therapeutic approach against prostate cancer. We show here that the curcumin analog 27 (ca27) down-regulates AR expression in several prostate cancer cell lines. METHODS: ca27 at low micromolar concentrations was tested for its effect on AR expression, AR activation, and induction of oxidative stress in human LNCaP, C4-2, and LAPC-4 prostate cancer cells. RESULTS: ca27 induced the down-regulation of AR protein expression in LNCaP, C4-2, and LAPC-4 cells within 12 hr. Further, ca27 led to the rapid induction of reactive oxygen species (ROS). To further support this finding, ca27 treatment led to the activation of the cellular redox sensor NF-E2-related factor 2 (Nrf2) and the induction of the Nrf2-regulated genes NAD(P)H quinone oxidoreductase 1 and aldoketoreductase 1C1. We show that ROS production preceded AR protein loss and that ca27-mediated down-regulation of the AR was attenuated by the antioxidant, N-acetyl cysteine. CONCLUSIONS: ca27 induces ROS and mediates AR protein down-regulation through an oxidative stress mechanism of action. Our results suggest that ca27 represents a novel agent for the elucidation of mechanisms of AR down-regulation, which could lead to effective new anti-androgenic strategies for the treatment of advanced prostate cancer.

Drug Repurposing from an Academic Perspective.

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the "valley of death" by bridging basic to clinical sciences.

Co-exposure of sodium arsenite and uranyl acetate differentially alters gene expression in CD3/CD28 activated CD4+ T-cells.

Communities in the western region of the United States experience environmental exposure to metal mixtures from living in proximity to numerous unremediated abandoned uranium mines. Metals including arsenic and uranium co-occur in and around these sites at levels higher than the United States Environmental Protection Agency maximum contaminant levels. To address the potential effect of these metals on the activation of CD4+ T-cells, we used RNA sequencing methods to determine the effect of exposure to sodium arsenite (1 µM and 10 µM), uranyl acetate (3 µM and 30 µM) or a mixture of sodium arsenite and uranyl acetate (1 µM sodium arsenite + 3 µM uranyl acetate). Sodium arsenite induced a dose dependent effect on activation associated gene expression; targeting immune response genes at the lower dose. Increases in oxidative stress gene expression were observed with both sodium arsenite doses. While uranyl acetate alone did not significantly alter activation associated gene expression, the mixture of uranyl acetate with sodium arsenite demonstrated a combined effect relative to sodium arsenite alone. The results demonstrate the need to investigate metal and metalloid mixtures at environmentally relevant concentrations to better understand the toxicological impact of these mixtures on T-cell activation, function and immune dysregulation.

Sustained expression of GRAIL during hematopoiesis results in dysregulated differentiation.

BACKGROUND/AIMS: Despite a sophisticated understanding of the hematopoietic developmental program at the transcriptional level, our understanding of the role of E3 ubiquitin ligases remains underdeveloped. The E3 ubiquitin ligase, GRAIL (RNF128), is expressed in the bone marrow, but its role is as yet undefined. In this study, we evaluate the effect of GRAIL expression during hematopoietic differentiation in vitro and in vivo. METHODS: Retroviral transduction of hematopoietic multipotent progenitor cells was used for methylcellulose colony assays and bone marrow reconstitution. RESULTS: Enforced expression of GRAIL in colony assays demonstrated skewing of hematopoietic lineage development toward granulocytic/monocytic cells. Bone marrow reconstitution experiments with progenitor cells expressing biologically varied levels of GRAIL demonstrated diminished erythropoiesis and megakaryopoiesis if GRAIL (endogenous or forced) is maintained throughout hematopoiesis. CONCLUSION: These data highlight a role for GRAIL during hematopoiesis and emphasize the importance of studying posttranslational effects to complement our current understanding of the transcriptional regulation of hematopoiesis.

A multifunctional androgen receptor screening assay using the high-throughput Hypercyt flow cytometry system.

The androgen receptor (AR) is a steroid hormone receptor which regulates transcription of androgen-sensitive genes and is responsible for the development and maintenance of male secondary sexual characteristics. Chemicals that interfere with AR activity may lead to pathological conditions in androgen-sensitive tissues. A variety of reporter systems have been developed, driven by androgen-sensitive promoters, which screen for chemicals that modulate androgenic activity. We have developed a flexible, high-throughput AR transcriptional activation assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, to facilitate the identification of novel modulators of AR transcriptional activity using flow cytometry. Androgen-independent human prostate cancer-derived PC3 cells were transiently cotransfected with an expression vector for the wild-type human AR and an androgen-sensitive promoter regulating the expression of destabilized enhanced GFP (dsEGFP). The transfected cells were stimulated with established androgenic and antiandrogenic compounds and assessed for increased or decreased dsEGFP expression. To screen for antagonists of AR transcription, the AR agonist R1881 was coadministered at submaximal concentrations with potential AR antagonists. The assay was formatted for high-throughput screening using the HyperCyt flow cytometry system. Agents with established androgenic and antiandrogenic activity were used for validation of the MARS assay. AR agonists were found to potently induce dsEGFP. Furthermore, AR agonists induced dsEGFP expression in a dose-dependent manner. Alternatively, AR antagonists blocked dsEGFP expression when coadministered with low-dose R1881, which also occurred in a dose-dependent manner. Modulators of AR transcriptional activity can be successfully identified by the MARS assay, utilizing a rapid, flexible, sensitive, and high-throughput format. Dose-response curves can be successfully generated for these compounds, allowing for an assessment of potency. Because of its simplicity and high-throughput compatibility, the MARS assay and HyperCyt system combined with flow cytometric analysis represents a valuable and novel addition to the current repertoire of AR transcriptional activation screening assays.

Antioxidants Abrogate Alpha-Tocopherylquinone-Mediated Down-Regulation of the Androgen Receptor in Androgen-Responsive Prostate Cancer Cells.

Tocopherylquinone (TQ), the oxidation product of alpha-tocopherol (AT), is a bioactive molecule with distinct properties from AT. In this study, AT and TQ are investigated for their comparative effects on growth and androgenic activity in prostate cancer cells. TQ potently inhibited the growth of androgen-responsive prostate cancer cell lines (e.g., LAPC4 and LNCaP cells), whereas the growth of androgen-independent prostate cancer cells (e.g., DU145 cells) was not affected by TQ. Due to the growth inhibitory effects induced by TQ on androgen-responsive cells, the anti-androgenic properties of TQ were examined. TQ inhibited the androgen-induced activation of an androgen-responsive reporter and inhibited the release of prostate specific antigen from LNCaP cells. TQ pretreatment was also found to inhibit AR activation as measured using the Multifunctional Androgen Receptor Screening assay. Furthermore, TQ decreased androgen-responsive gene expression, including TM4SF1, KLK2, and PSA over 5-fold, whereas AT did not affect the expression of androgen-responsive genes. Of importance, the antiandrogenic effects of TQ on prostate cancer cells were found to result from androgen receptor protein down-regulation produced by TQ that was not observed with AT treatment. Moreover, none of the androgenic endpoints assessed were affected by AT. The down-regulation of androgen receptor protein by TQ was abrogated by co-treatment with antioxidants. Overall, the biological actions of TQ were found to be distinct from AT, where TQ was found to be a potent inhibitor of cell growth and androgenic activity in androgen-responsive prostate cancer cells.

Longitudinal Assessment of Lung Cancer Progression in Mice Using the Sodium Iodide Symporter Reporter Gene and SPECT/CT Imaging.

Lung cancer has the highest mortality rate of any tissue-specific cancer in both men and women. Research continues to investigate novel drugs and therapies to mitigate poor treatment efficacy, but the lack of a good descriptive lung cancer animal model for preclinical drug evaluation remains an obstacle. Here we describe the development of an orthotopic lung cancer animal model which utilizes the human sodium iodide symporter gene (hNIS; SLC5A5) as an imaging reporter gene for the purpose of non-invasive, longitudinal tumor quantification. hNIS is a glycoprotein that naturally transports iodide (I-) into thyroid cells and has the ability to symport the radiotracer 99mTc-pertechnetate (99mTcO4-). A549 lung adenocarcinoma cells were genetically modified with plasmid or lentiviral vectors to express hNIS. Modified cells were implanted into athymic nude mice to develop two tumor models: a subcutaneous and an orthotopic xenograft tumor model. Tumor progression was longitudinally imaged using SPECT/CT and quantified by SPECT voxel analysis. hNIS expression in lung tumors was analyzed by quantitative real-time PCR. Additionally, hematoxylin and eosin staining and visual inspection of pulmonary tumors was performed. We observed that lentiviral transduction provided enhanced and stable hNIS expression in A549 cells. Furthermore, 99mTcO4- uptake and accumulation was observed within lung tumors allowing for imaging and quantification of tumor mass at two-time points. This study illustrates the development of an orthotopic lung cancer model that can be longitudinally imaged throughout the experimental timeline thus avoiding inter-animal variability and leading to a reduction in total animal numbers. Furthermore, our orthotopic lung cancer animal model is clinically relevant and the genetic modification of cells for SPECT/CT imaging can be translated to other tissue-specific tumor animal models.

Xenogeneic transplantation of porcine islets: an overview.

The extreme demand for human organs or tissues for transplantation has driven the search for viable alternatives. Pigs are considered a possible source of tissue for a number of reasons including shared physiology, plentiful supply, short gestation, and, more recently, the generation of transgenic animals. Porcine islets show promise as a source of islets for the treatment of type 1 diabetes mellitus. Porcine islets regulate glucose levels in the same physiologic range as humans, and porcine insulin has been used for years as an exogenous source of insulin for glucose control. In this review, we discuss the advantages and disadvantages of the use of adult or neonatal porcine islets, the immunologic challenges facing transplantation of xenogeneic islets, and the concerns regarding transmission of infectious agents between species. Porcine islets isolated from both adult and neonatal pigs are capable of restoring euglycemia in experimental animal models of diabetes. Adult islets are more difficult to isolate, whereas neonatal islets have great proliferation potential but require several weeks to function posttransplantation. Xenogeneic islets are susceptible to complement-mediated lysis after the binding of preformed natural antibodies and cellular immunity involving both macrophages and CD4+ T cells. In addition, the potential for transmission of porcine endogenous retroviruses, porcine cytomegalovirus, and porcine lymphotropic herpesvirus type 1 are all concerns that must be addressed. Despite the challenges facing xenotransplantation, the extreme need for donor organs and tissues continues to drive progress toward overcoming the unique issues associated with transplantation between species.

Microarray analysis demonstrates a role for Slug in epidermal homeostasis.

Slug (Snail2) is a member of the Snail family of zinc-finger transcription factors with regulatory functions in development, tissue morphogenesis, and tumor progression. Little is known about Slug in normal adult tissue; however, a role for Slug in the skin was suggested by our previous observations of Slug expression in normal murine keratinocytes and Slug induction at wound margins. To study the impact of Slug in the skin, we compared patterns of gene expression in epidermis from Slug-null and wild-type mice. A total of 139 genes had significantly increased, and 109 genes had significantly decreased expression in Slug knockout epidermis. Altered expression of selected genes in Slug knockout epidermis was validated by real-time PCR and immunohistochemistry. Previously reported Slug targets were identified, in addition to novel genes, including cytokeratins, adhesion molecules, and extracellular matrix components. Functional classification of altered gene expression was consistent with a role for Slug in keratinocyte development and differentiation, proliferation, apoptosis, adhesion, motility, as well as angiogenesis and response to environmental stimuli. These results highlight the utility of genetic models to study the in vivo impact of regulatory factors in unperturbed skin and suggest that Slug has significant activities in the adult epidermis.

GRAIL is up-regulated in CD4+ CD25+ T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype.

GRAIL (gene related to anergy in lymphocytes) is an ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase necessary for the induction of CD4(+) T cell anergy in vivo. We have extended our previous studies to characterize the expression pattern of GRAIL in other murine CD4(+) T cell types with a described anergic phenotype. These studies revealed that GRAIL expression is increased in naturally occurring (thymically derived) CD4(+) CD25(+) T regulatory cells (mRNA levels 10-fold higher than naive CD25(-) T cells). Further investigation demonstrated that CD25(+) Foxp3(+) antigen-specific T cells were induced after a "tolerizing-administration" of antigen and that GRAIL expression correlated with the CD25(+) Foxp3(+) antigen-specific subset. Lastly, using retroviral transduction, we demonstrated that forced expression of GRAIL in a T cell line was sufficient for conversion of these cells to a regulatory phenotype in the absence of detectable Foxp3. These data demonstrate that GRAIL is differentially expressed in naturally occurring and peripherally induced CD25(+) T regulatory cells and that the expression of GRAIL is linked to their functional regulatory activity.