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1.  The Basic Helix-Loop-Helix Transcription Factor E47 Reprograms Human Pancreatic Cancer Cells to a Quiescent Acinar State With Reduced Tumorigenic Potential 
Pancreas  2015;44(5):718-727.
Supplemental digital content is available in the text.
Pancreatic ductal adenocarcinoma (PDA) initiates from quiescent acinar cells that attain a Kras mutation, lose signaling from basic helix-loop-helix (bHLH) transcription factors, undergo acinar-ductal metaplasia, and rapidly acquire increased growth potential. We queried whether PDA cells can be reprogrammed to revert to their original quiescent acinar cell state by shifting key transcription programs.
Human PDA cell lines were engineered to express an inducible form of the bHLH protein E47. Gene expression, growth, and functional studies were investigated using microarray, quantitative polymerase chain reaction, immunoblots, immunohistochemistry, small interfering RNA, chromatin immunoprecipitation analyses, and cell transplantation into mice.
In human PDA cells, E47 activity triggers stable G0/G1 arrest, which requires the cyclin-dependent kinase inhibitor p21 and the stress response protein TP53INP1. Concurrently, E47 induces high level expression of acinar digestive enzymes and feed forward activation of the acinar maturation network regulated by the bHLH factor MIST1. Moreover, induction of E47 in human PDA cells in vitro is sufficient to inhibit tumorigenesis.
Human PDA cells retain a high degree of plasticity, which can be exploited to induce a quiescent acinar cell state with reduced tumorigenic potential. Moreover, bHLH activity is a critical node coordinately regulating human PDA cell growth versus cell fate.
PMCID: PMC4464938  PMID: 25894862
pancreatic ductal adenocarcinoma; ID3; bHLH signaling; E47; reprogramming
3.  Insulin Biosynthetic Interaction Network Component, TMEM24, Facilitates Insulin Reserve Pool Release 
Cell reports  2013;4(5):10.1016/j.celrep.2013.07.050.
Insulin homeostasis in pancreatic β-cell is now recognized as a critical element in the progression of obesity and type II diabetes (T2D). Proteins that interact with insulin to direct its sequential synthesis, folding, trafficking and packaging into reserve granules to manage release in response to elevated glucose remains largely unknown. Using a conformation based approach combined with mass spectrometry we have now generated the insulin biosynthetic interaction network (insulin BIN), a proteomic roadmap in the β-cell that describes the sequential interacting partners of insulin along the secretory axis. The insulin BIN revealed an abundant C2 domain-containing transmembrane protein 24 (TMEM24) that manages glucose-stimulated insulin secretion from reserve pool of granules, a critical event impaired in patients with T2D. Identification of TMEM24 in the context of a comprehensive set of sequential insulin binding partners provides a molecular description of the insulin secretory pathway in β-cells.
PMCID: PMC3839581  PMID: 24012759
4.  Id3 upregulates BrdU incorporation associated with a DNA damage response, not replication, in human pancreatic β-cells 
Islets  2011;3(6):358-366.
Elucidating mechanisms of cell cycle control in normally quiescent human pancreatic β-cells has the potential to impact regeneration strategies for diabetes. Previously we demonstrated that Id3, a repressor of basic Helix-Loop-Helix (bHLH) proteins, was sufficient to induce cell cycle entry in pancreatic duct cells, which are closely related to β-cells developmentally. We hypothesized that Id3 might similarly induce cell cycle entry in primary human β-cells. To test this directly, adult human β-cells were transduced with adenovirus expressing Id3. Consistent with a replicative response, β-cells exhibited BrdU incorporation. Further, Id3 potently repressed expression of the cyclin dependent kinase inhibitor p57Kip2, a gene which is also silenced in a rare β-cell hyperproliferative disorder in infants. Surprisingly, however, BrdU positive β-cells did not express the proliferation markers Ki67 and pHH3. Instead, BrdU uptake reflected a DNA damage response, as manifested by hydroxyurea incorporation, γH2AX expression and 53BP1 subcellular relocalization. The uncoupling of BrdU uptake from replication raises a cautionary note about interpreting studies relying solely upon BrdU incorporation as evidence of β-cell proliferation. The data also establish that loss of p57Kip2 is not sufficient to induce cell cycle entry in adult β-cells. Moreover, the differential responses to Id3 between duct and β-cells reveal that β-cells possess intrinsic resistance to cell cycle entry not common to all quiescent epithelial cells in the adult human pancreas. The data provide a much needed comparative model for investigating the molecular basis for this resistance in order to develop a strategy for improving replication competence in β-cells.
PMCID: PMC3329516  PMID: 21964314
DNA damage; regeneration; replication
5.  HNF4α Antagonists Discovered by a High-Throughput Screen for Modulators of the Human Insulin Promoter 
Chemistry & biology  2012;19(7):806-818.
Hepatocyte Nuclear Factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic β-cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.
PMCID: PMC3447631  PMID: 22840769
6.  Phenothiazine Neuroleptics Signal to the Human Insulin Promoter as Revealed by a Novel High-Throughput Screen 
Journal of Biomolecular Screening  2010;15(6):663-670.
A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic β-cell. A cell line from human islets in which the expression of insulin and other β-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of β-cell differentiated function.
PMCID: PMC3374493  PMID: 20547533
diabetes; chlorpromazine; ethopropazine
7.  Derivation of a Retinoid X Receptor Scaffold from Peroxisome Proliferator-Activated Receptor γ Ligand 1-Di(1H-indol-3-yl)methyl-4-trifluoromethylbenzene 
ChemMedChem  2009;4(7):1106-1119.
1-Di(1H-indol-3-yl)methyl-4-trifluoromethylbenzene (DIM-Ph-4-CF3) is reported to inhibit cancer cell growth and to act as a transcriptional agonist of peroxisome proliferator-activated receptor γ (PPARγ) and nuclear receptor 4A subfamily member 1 (NR4A1). In addition, DIM-Ph-4-CF3 exerts anticancer effects independent of these receptors because PPARγ antagonists do not block its inhibition of cell growth, and the small pocket in the NR4A1 crystal structure suggests no ligand can bind. Because PPARγ and NR4A1 heterodimerize with retinoid X receptor (RXR), and several PPARγ ligands transcriptionally activate RXR, DIM-Ph-4-CF3 was investigated as an RXR ligand. DIM-Ph-4-CF3 displaces 9-cis-retinoic acid from RXRα but does not transactivate RXRα. Structure-based design using DIM-Ph-4-CF3 as a template led to the RXRα transcriptional agonist (E)-3-[5-di(1-methyl-1H-indol-3-yl)methyl-2-thienyl]acrylic acid. Its docked pose in the RXRα ligand binding domain suggests that binding is stabilized by interactions of its carboxylate group with arginine 316, its indoles with cysteines 269 and 432, and its 1-methyl groups with hydrophobic residues lining the binding pocket. As is expected of a selective activator of RXRα, but not of RARs and PPARγ, this RXRα agonist, unlike DIM-Ph-4-CF3, does not appreciably decrease cancer cell growth or induce apoptosis at pharmacologically relevant concentrations.
PMCID: PMC3031428  PMID: 19378296
antitumor agents; dimethylarenes; receptors; retinoids; RXR; TR3
8.  CENP-A, a protein required for chromosome segregation in mitosis, declines with age in islet but not exocrine cells 
Aging (Albany NY)  2010;2(11):785-790.
Beta-cell replication dramatically declines with age. Here, we report that the level of CENP-A, a protein required for cell division, declines precipitously with age in an islet-specific manner. CENP-A is essentially undetectable after age 29 in humans. However, exocrine cells retain CENP-A expression. The decline in islet-cell CENP-A expression is more striking in humans than in mice, where CENP-A expression continues to be detectable at low levels even in elderly mice. The mechanism by which CENP-A declines appears to be post-transcriptional, as there was no correlation between CENP-A mRNA levels and age or islet purity. This finding has implications for efforts to induce beta-cell replication as a treatment for diabetes.
PMCID: PMC3006021  PMID: 21068465
β-cell; replication; pancreas; diabetes
9.  Real-time Bioluminescence Imaging of Macroencapsulated Fibroblasts Reveals Allograft Protection in Rhesus Monkeys (Macaca mulatta) 
Transplantation  2009;88(1):38-41.
Encapsulation of cells has the potential to eliminate the need for immunosuppression for cellular transplantation. Recently, the TheraCyte® device was shown to provide long-term immunoprotection of murine islets in the NOD/SCID mouse model of diabetes. In this report, translational studies were undertaken using skin fibroblasts from an unrelated rhesus monkey donor that were transduced with an HIV-1-derived lentiviral vector expressing firefly luciferase permitting the use of bioluminescence imaging (BLI) to monitor cell survival over time and in a noninvasive manner.
Encapsulated cells were transplanted subcutaneously (N=2) or cells were injected without encapsulation (N=1) and outcomes compared. BLI was performed to monitor cell survival.
The BLI signal from the encapsulated cells remained robust post-insertion, and in one animal persisted for up to 1 year. In contrast, the control animal that received unencapsulated cells exhibited a complete loss of cell signal within 14 days.
These data demonstrate that TheraCyte® encapsulation of allogeneic cells provides robust immune protection in transplanted rhesus monkeys.
PMCID: PMC2744215  PMID: 19584678
Encapsulation; Transplantation; Optical Imaging; Rhesus Monkeys
10.  Human β-cell Precursors Mature Into Functional Insulin-producing Cells in an Immunoisolation Device: Implications for Diabetes Cell Therapies 
Transplantation  2009;87(7):983-991.
Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human β-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human β-cells and their progenitors and (2) the engraftment of encapsulated murine β-cells in allo- and autoimmune settings.
Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets.
Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary β-cells ameliorated diabetes without stimulating a detectable T-cell response.
We demonstrate for the first time that human β-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of β-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.
PMCID: PMC2715156  PMID: 19352116
Encapsulation; Diabetes; Islet transplantation; Immunoisolation
11.  Islet Specific Wnt Activation in Human Type II Diabetes 
Experimental Diabetes Research  2009;2008:728763.
The Wnt pathway effector gene TCF7L2 has been linked to type II diabetes, making it important to study the role of Wnt signaling in diabetes pathogenesis. We examined the expression of multiple Wnt pathway components in pancreases from normal individuals and type II diabetic individuals. Multiple members of the Wnt signaling pathway, including TCF7L2, Wnt2b, β-catenin, pGSK3β, TCF3, cyclinD1, and c-myc, were undetectable or expressed at low levels in islets from nondiabetic individuals, but were also upregulated specifically in islets of type II diabetic patients. Culture of pancreatic tissue and islet isolation led to Wnt activation that was reversed by the Wnt antagonist sFRP, demonstrating that Wnt activation in that setting was due to soluble Wnt factors. These data support a model in which the Wnt pathway plays a dynamic role in the pathogenesis of type II diabetes and suggest manipulation of Wnt signaling as a new approach to β-cell-directed diabetes therapy.
PMCID: PMC2628766  PMID: 19165345
To examine the mechanism by which growth-stimulated pancreatic β-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and βlox5, a cell line derived from human β-cells which progressively dedifferentiated in culture. MIN6/βlox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that βlox5 expresses a dominant trans-acting factor(s) that represses β-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary β-cells as well as MIN6, but was repressed in hybrids. Hes6 overexpression using a retroviral vector led to a decrease in Hes1 levels, an increase in β-cell transcription factors and partial restoration of insulin expression. We conclude that the balance of Notch activators and inhibitors may play an important role in maintaining the β-cell differentiated state.
PMCID: PMC1852427  PMID: 17300753
β-cell; insulin; cell fusion; differentiation; islet; somatic cell hybrids

Results 1-12 (12)