Role of KCNAB2 expression in modulating hormone secretion in somatotroph pituitary adenoma.

OBJECTIVE: Prior profiling of the human pituitary adenoma (PA) DNA methylome showed the potassium channel subunit-encoding gene KCNAB2 to be highly differentially methylated between nonfunctional PAs (NFPAs) and growth hormone (GH)-secreting PAs, with greater KCNAB2 methylation detected in secretory PAs. KCNAB2 encodes an aldo-keto reductase that, among other things, negatively regulates members of the voltage-gated potassium channel (Kv) family. In this study, the authors aimed to determine whether modulation of Kcnab2 expression would alter GH secretion in the GH3 mammosomatotroph rat cell line. In addition, they examined whether dosing GH3 cells with the antiarrhythmic drug quinidine, a known inhibitor of Kv and voltage-gated sodium channels, would affect hormonal secretion. METHODS: Previously generated RNA-seq data were reanalyzed to compare KCNAB2 expression levels in human NFPAs and GH-secreting PAs. Kcnab2 was overexpressed in GH3 cells using plasmid transfection and knocked down using shRNA, with confirmation by quantitative polymerase chain reaction (qPCR). GH concentrations in cell culture supernatants collected 24 hours after cell seeding were measured using enzyme-linked immunosorbent assay (ELISA). Separately, quinidine was administered to GH3 cells at graduated doses. GH and prolactin concentrations in supernatants collected 48 hours after quinidine treatment were measured by fluorometric immunoassay. RESULTS: Modulation of expression at the transcript level in GH3 cells resulted in proportionate changes in the expression of GH mRNA and secretion of GH peptide, as confirmed by qPCR and ELISA. Specifically, partial knockdown of Kcnab2 was associated with fewer GH RNA transcripts and less GH secretion compared with controls, while augmentation of Kcnab2 expression was associated with more GH transcripts and secretion than the controls. Administration of quinidine (≥ 50 µM) reduced both GH and prolactin secretion in a dose-dependent fashion (p ≤ 0.05). CONCLUSIONS: GH secretion in a somatotroph cell line is partially dependent on KCNAB2 gene expression and may be mitigated in vitro by quinidine. These results collectively suggest a potential new target and pharmacological candidate to be considered in the development of clinical therapeutics for acromegaly.

Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens.

The study of pathophysiological mechanisms in human liver disease has been constrained by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. We and others have previously shown that mouse mature hepatocytes can be converted to liver progenitor-like cells in vitro with defined chemical factors. Here we describe a protocol achieving efficient conversion of human primary hepatocytes into liver progenitor-like cells (HepLPCs) through delivery of developmentally relevant cues, including NAD + -dependent deacetylase SIRT1 signaling. These HepLPCs could be expanded significantly during in vitro passage. The expanded cells can readily be converted back into metabolically functional hepatocytes in vitro and upon transplantation in vivo. Under three-dimensional culture conditions, differentiated cells generated from HepLPCs regained the ability to support infection or reactivation of hepatitis B virus (HBV). Our work demonstrates the utility of the conversion between hepatocyte and liver progenitor-like cells for studying HBV biology and antiviral therapies. These findings will facilitate the study of liver diseases and regenerative medicine.

Inhibition of SIRPα in dendritic cells potentiates potent antitumor immunity.

Despite their central function in tumor immunity, dendritic cells (DCs) can respond to inhibitory signals and become tolerogenic, curtailing T cell responses in vivo. Here, we provide the evidence for an inhibitory function of signal regulatory protein (SIRP) α in DC survival and activation. In tumors from human liver cancer patients, infiltrative DCs expressed elevated levels of SIRPα, which is correlated with the induction of immune tolerance within the tumors. Silencing of SIRPα resulted in a significant increase in the longevity of antigen-pulsed DCs in the draining lymph nodes. In addition, SIRPα controls the activation and output of DCs. Silencing of DC-expressed SIRPα induced spontaneous and enhanced production of IL12 and costimulatory molecules, resulting in more potent cytotoxic T lymphocyte responses, including the eradication of previously established solid tumors. SIRPα exerted such effects, at least in part, via the association and sequestration of p85 subunit of PI3K. Thus, SIRPα is a critical regulator of DC lifespan and activity, and its inhibition might improve the clinical efficacy of DC-based tumor vaccines.

Gene and protein expression in pituitary corticotroph adenomas: a systematic review of the literature.

OBJECT Functional corticotroph pituitary adenomas (PAs) secrete adrenocorticotropic hormone (ACTH) and are the cause of Cushing's disease, which accounts for 70% of all cases of Cushing's syndrome. Current classification systems for PAs rely primarily on laboratory hormone findings, tumor size and morphology, invasiveness, and immunohistochemical findings. Likewise, drug development for functional ACTH-secreting PAs (ACTH-PAs) is limited and has focused largely on blocking the production or downstream effects of excess cortisol. The authors aimed to summarize the findings from previous studies that explored gene and protein expression of ACTH-PAs to prioritize potential genetic and protein targets for improved molecular diagnosis and treatment of Cushing's disease. METHODS A systematic literature review was performed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A PubMed search of select medical subject heading (MeSH) terms was performed to identify all studies that reported gene- and protein-expression findings in ACTH-PAs from January 1, 1990, to August 24, 2014, the day the search was performed. The inclusion criteria were studies on functional ACTH-PAs compared with normal pituitary glands, on human PA tissue only, with any method of analysis, and published in the English language. Studies using anything other than resected PA tissue, those that compared other adenoma types, those without baseline expression data, or those in which any pretreatment was delivered before analysis were excluded. RESULTS The primary search returned 1371 abstracts, of which 307 were found to be relevant. Of those, 178 were selected for secondary full-text analysis. Of these, 64 articles met the inclusion criteria and an additional 4 studies were identified from outside the search for a total of 68 included studies. Compared with the normal pituitary gland, significant gene overexpression in 43 genes and 22 proteins was reported, and gene underexpression in 58 genes and 15 proteins was reported. Immunohistochemistry was used in 39 of the studies, and reverse transcriptase polymerase chain reaction was used in 26 of the studies, primarily, and as validation for 4 others. Thirteen studies used both immunohistochemistry and reverse transcriptase polymerase chain reaction. Other methods used included microarray, in situ hybridization, Northern blot analysis, and Western blot analysis. Expression of prioritized genes emphasized in multiple studies were often validated on both the gene and protein levels. Genes/proteins found to be overexpressed in ACTH-PAs relative to the normal pituitary gland included hPTTG1/securin, NEUROD1/NeuroD1 (Beta2), HSD11B2/11ß-hydroxysteroid dehydrogenase 2, AKT/Akt, protein kinase B, and CCND1/cyclin D1. Candidate genes/proteins found to be underexpressed in ACTH-PAs relative to the normal pituitary gland included CDKN1B/p27(Kip1), CDKN2A/p16, KISS1/kisspeptin, ACTHR/ACTH-R, and miR-493. CONCLUSIONS On the basis of the authors' systematic review, many significant gene and protein targets that may contribute to tumorigenesis, invasion, and hormone production/secretion of ACTH have been identified and validated in ACTH-PAs. Many of these potential targets have not been fully analyzed for their therapeutic and diagnostic potential but may represent candidate molecular targets for biomarker development and drug targeting. This review may help catalyze additional research efforts using modern profiling and sequencing techniques and alteration of gene expression.

p53 promotes inflammation-associated hepatocarcinogenesis by inducing HMGB1 release.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) develops in response to chronic hepatic injury. Although induced cell death is regarded as the major component of p53 tumor-suppressive activity, we recently found that sustained p53 activation subsequent to DNA damage promotes inflammation-associated hepatocarcinogenesis. Here we aim at exploring the mechanism linking p53 activation and hepatic inflammation during hepatocarcinogenesis. METHODS: p53(-/-) hepatocytes expressing inducible p53 and primary wild type hepatocytes were treated to induce p53 expression. The supernatants were collected and analyzed for the presence of released inflammatory cytokines. Ethyl pyruvate was used in a rat model of carcinogen-induced hepatocarcinogenesis to examine its effect on p53-dependent chronic hepatic injury, inflammation, and tumorigenesis. RESULTS: Here we show that cytoplasmic translocation and circulating levels of potent inflammatory molecule high-mobility group protein 1 (HMGB1) were greater in wild type rats than in p53(+/-) rats following carcinogen administration. Restoration of p53 expression in p53-null hepatocytes or induction of endogenous p53 in wild type hepatocytes gives rise to the release of HMGB1. Administration of the HMGB1 release inhibitor ethyl pyruvate, which does not affect p53-mediated hepatic apoptosis, substantially prevented carcinogen-induced cirrhosis and tumorigenesis in rat livers. CONCLUSIONS: These results suggest that although p53 is usually regarded as a tumor suppressor, its constant activation can promote pro-tumorigenic inflammation, at least in part, via inducing HMGB1 release. Application of HMGB1 inhibitors when restoring p53 in cancer therapy might protect against pro-tumorigenic effects while leaving p53-mediated clearance of malignant cells intact.

Rats deficient for p53 are susceptible to spontaneous and carcinogen-induced tumorigenesis.

The p53 tumor suppressor gene is highly mutated in human cancers. Individuals who inherit one p53 mutant allele are susceptible to a wide range of tumor types, including breast cancer and sarcoma. We recently generated p53 knockout rats through gene targeting in embryonic stem cells. Here we show that rats homozygous for the null allele are prone to early onset spontaneous sarcomas and lymphoma with high incidence of metastases. Heterozygous rats are also highly predisposed to cancer, but with a delayed onset and a wider spectrum of tumor types compared with homozygotes. Importantly, up to 20% of female heterozygotes developed breast cancer and about 70% of the tumors were positive for estrogen receptor. Exposing p53-deficient rats to a low dose of the carcinogen diethylnitrosamine dramatically decreased the latency for sarcoma development and survival time compared with equivalently treated wild-type rats. These unique features make this knockout line a valuable model for investigating human malignancy and in vivo carcinogenicity of chemicals and therapeutic compounds.

Rapid and cost-effective gene targeting in rat embryonic stem cells by TALENs.

The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stem (ES) cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease (TALEN)-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.

Generating gene knockout rats by homologous recombination in embryonic stem cells.

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell-based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ∼1 year to complete, from derivation of ES cells to generation of knockout rats.

Genetic manipulations in the rat: progress and prospects.

PURPOSE OF REVIEW: Several advances have been made to manipulate the rat genome in the last 2 years. This review aims to describe these advances in rat genetic manipulations, with an emphasis on their current status and their prospects and applications in the postgenomic era. RECENT FINDINGS: Authentic rat embryonic stem cells were derived in 2008 using the 2i/3i culture system. This led to the generation of the first gene knockout rats via embryonic stem cell-based gene targeting. The development of zinc-finger nucleases (ZFNs) provided an alternative approach that avoids the necessity of germline competent embryonic stem cells. Meanwhile, improvements have been made to the well established random mutagenesis mediated by transposons or N-ethyl-N-nitrosourea (ENU). The in-vitro rat spermatogonial stem cell (SSC) system has greatly optimized these phenotype-driven approaches for future applications. SUMMARY: The rat has long been a prime model organism in physiological, pharmacological and neurobehavioral studies. The recent advances of rat reverse genetic approaches, together with the classical ENU and transposon mutagenesis system, will contribute tremendously to the deciphering of gene functions and the creation of rat disease models.

Beyond knockout rats: new insights into finer genome manipulation in rats.

The ability to "knockout" specific genes in mice via embryonic stem (ES) cell-based gene-targeting technology has significantly enriched our understanding of gene function in normal and disease phenotypes. Improvements on this original strategy have been developed to enable the manipulation of genomes in a more sophisticated fashion with unprecedented precision. The rat is the model of choice in many areas of scientific investigation despite the lack of rat genetic toolboxes. Most Recent advances of zinc finger nucleases (ZFNs) and rat ES cells are diminishing the gap between rat and mouse with respect to reverse genetic approaches. Importantly, the establishment of rat ES cell-based gene targeting technology, in combination with the unique advantages of using rats, provides new, exciting opportunities to create animal models that mimic human diseases more faithfully. We hereby report our recent results concerning finer genetic modifications in the rat, and propose their potential applications in addressing biological questions.