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1.
Cancer Res ; 69(13): 5458-66, 2009 Jul 01.
Article in English | MEDLINE | ID: mdl-19549908

ABSTRACT

Cisplatin-derived anticancer therapy has been used for three decades despite its side effects. Other types of organometallic complexes, namely, some ruthenium-derived compounds (RDC), which would display cytotoxicity through different modes of action, might represent alternative therapeutic agents. We have studied both in vitro and in vivo the biological properties of RDC11, one of the most active compounds of a new class of RDCs that contain a covalent bond between the ruthenium atom and a carbon. We showed that RDC11 inhibited the growth of various tumors implanted in mice more efficiently than cisplatin. Importantly, in striking contrast with cisplatin, RDC11 did not cause severe side effects on the liver, kidneys, or the neuronal sensory system. We analyzed the mode of action of RDC11 and showed that RDC11 interacted poorly with DNA and induced only limited DNA damages compared with cisplatin, suggesting alternative transduction pathways. Indeed, we found that target genes of the endoplasmic reticulum stress pathway, such as Bip, XBP1, PDI, and CHOP, were activated in RDC11-treated cells. Induction of the transcription factor CHOP, a crucial mediator of endoplasmic reticulum stress apoptosis, was also confirmed in tumors treated with RDC11. Activation of CHOP led to the expression of several of its target genes, including proapoptotic genes. In addition, the silencing of CHOP by RNA interference significantly reduced the cytotoxicity of RDC11. Altogether, our results led us to conclude that RDC11 acts by an atypical pathway involving CHOP and endoplasmic reticulum stress, and thus might provide an interesting alternative for anticancer therapy.


Subject(s)
Cell Division/drug effects , Endoplasmic Reticulum/genetics , Melanoma, Experimental/pathology , Organometallic Compounds/therapeutic use , Ruthenium/therapeutic use , Transcription Factor CHOP/genetics , Animals , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Cisplatin/therapeutic use , Cisplatin/toxicity , Endoplasmic Reticulum/drug effects , Flow Cytometry , Glioblastoma/drug therapy , Glioblastoma/pathology , Humans , Luciferases/genetics , Melanoma, Experimental/drug therapy , Mice , Mice, Inbred C57BL , RNA/genetics , RNA/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction
2.
Dev Biol ; 305(2): 685-94, 2007 May 15.
Article in English | MEDLINE | ID: mdl-17400205

ABSTRACT

The Onecut (OC) transcription factor HNF-6 (OC-1) is required during embryogenesis for pancreatic specification, morphogenesis and endocrine differentiation. In mammals, HNF-6 has two paralogs, OC-2 and OC-3, which share DNA-binding and transcriptional activation properties and have expression patterns that overlap with that of HNF-6. This suggested that OC-2 and OC-3 play redundant roles with HNF-6 in pancreas development. Here, we have addressed this hypothesis by analyzing the phenotype of mice knockout for the Onecut factors. We found that neither OC-2 nor OC-3 is required for pancreas specification. However, OC-2 plays partially redundant roles with HNF-6 in pancreas morphogenesis and in the differentiation of endocrine precursors. As similar molecular events drive endocrine differentiation in the pancreas and gastrointestinal tract, we also investigated if Onecut factors are involved in enteroendocrine differentiation. OC-2 and OC-3 were found to delineate specific antero-posterior regions of the gut around embryonic day 12.5. Later on, OC2 was expressed in several gut cell types, whereas OC-3 behaved as a specific marker of the enteroendocrine lineage. However, OC-2 and OC-3, alone or in combination, were dispensable for gut development and enteroendocrine differentiation. In conclusion, our data reveal partially redundant roles for HNF-6 and OC-2 in developing pancreas and identify new markers for antero-posterior patterning of the gut and for enteroendocrine differentiation.


Subject(s)
Cell Differentiation/physiology , Enteroendocrine Cells/cytology , Insulin-Secreting Cells/cytology , Onecut Transcription Factors/physiology , Pancreas/cytology , Pancreas/embryology , Animals , Body Patterning/genetics , Cell Line , Enteroendocrine Cells/metabolism , Female , Gene Expression Regulation, Developmental , Hepatocyte Nuclear Factor 6/genetics , Homeodomain Proteins/genetics , Insulin-Secreting Cells/metabolism , Male , Mice , Mice, Knockout , Onecut Transcription Factors/deficiency , Onecut Transcription Factors/genetics , Pancreas/metabolism , Rats , Transcription Factors/deficiency , Transcription Factors/genetics
3.
Dev Biol ; 286(1): 217-24, 2005 Oct 01.
Article in English | MEDLINE | ID: mdl-16122727

ABSTRACT

Recently, the expression of the peptide hormone ghrelin was detected in alpha-cells of the islets of Langerhans as well as in epsilon-cells, a newly discovered endocrine cell type, but it remains unclear how the latter is related in lineage to the four classical islet cell types, alpha-, beta-, delta-, and PP-cells. Here, we provide further evidence that ghrelin is predominantly produced in the alpha-cells of mouse islets but also in single hormone ghrelin-secreting epsilon-cells. We additionally demonstrate that pancreatic epsilon-cells derive from Neurogenin3-expressing precursor cells and their genesis depends on Neurogenin3 activity. Furthermore, our data indicate that the number of ghrelin-producing cells is differentially regulated during pancreas morphogenesis by the homeodomain-containing transcription factors Arx, Pax4, and Pax6. Arx mutants lack ghrelin+ glucagon+ alpha-cells whereas Pax4 mutants develop an excess of these cells. Importantly, the ghrelin+ glucagon- epsilon-cell population is not affected following Arx or Pax4 disruption. In contrast, the loss of Pax6 provokes an unexpected increase of the ghrelin+ glucagon- epsilon-cell number which is not due to increased proliferation. Thus, we demonstrate that the development of ghrelin-producing cells is differentially dependent on Neurogenin3 in different domains of the gastrointestinal tract and that, in the endocrine pancreas, epsilon-cell genesis does not require Arx or Pax4 activities but is antagonized by Pax6.


Subject(s)
Glucagon/metabolism , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Peptide Hormones/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Eye Proteins/genetics , Eye Proteins/metabolism , Gene Expression Regulation, Developmental , Ghrelin , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Islets of Langerhans/growth & development , Mice , Mice, Mutant Strains , Mice, Transgenic , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , PAX6 Transcription Factor , Paired Box Transcription Factors/deficiency , Paired Box Transcription Factors/genetics , Paired Box Transcription Factors/metabolism , Peptide Hormones/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription Factors/metabolism
4.
Mol Endocrinol ; 18(11): 2765-76, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15297605

ABSTRACT

The basic helix-loop-helix transcription factor Neurogenin 3 (NGN3) controls endocrine cell fate specification in uncommitted pancreatic progenitor cells. Ngn3-deficient mice do not develop any islet cells and are diabetic. All the major islet cell types, including insulin-producing beta-cells, derive from Ngn3-positive endocrine progenitor cells. Therefore, the characterization of this population of immature cells is of particular interest for the development of novel strategies for cell replacement therapies in type 1 diabetes. To explore further the biology of islet progenitor cells we have generated a mouse in which Ngn3-expressing cells are labeled with the enhanced yellow fluorescent protein (EYFP) using a knock-add-on strategy. In this approach, the EYFP cDNA is introduced into the 3'-untranslated region of the proendocrine transcription factor, Neurogenin 3, without deleting any endogenous coding or regulatory sequences. In Ngn3(EYFP/+) and Ngn3(EYFP/EYFP) mice, the EYFP protein is targeted to Ngn3-expressing progenitors in the developing pancreas, and islets develop normally. Islet progenitors can be purified from whole embryonic pancreas by fluorescence-activated cell sorting from Ngn3(EYFP/+) mice and their development can be monitored in real time in pancreas explant cultures. These experiments showed that endocrine progenitors can form de novo and expand, in vitro, in the absence of signals from the surrounding mesenchyme, suggesting that endocrine commitment is a default pathway. The Ngn3(EYFP) mice represent a valuable tool to study islet cell development and neogenesis in normal and diabetic animals as well as for the determination of the conditions to generate beta-cells in vitro.


Subject(s)
Bacterial Proteins/genetics , Fluorescent Dyes , Islets of Langerhans/cytology , Islets of Langerhans/embryology , Luminescent Proteins/genetics , Nerve Tissue Proteins/genetics , Stem Cells/cytology , Animals , Bacterial Proteins/analysis , Bacterial Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors , Cell Differentiation , Flow Cytometry , Islets of Langerhans/metabolism , Luminescent Proteins/analysis , Luminescent Proteins/metabolism , Mesoderm/metabolism , Mice , Mice, Mutant Strains , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/metabolism , Pancreas/growth & development , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Stem Cells/chemistry , Stem Cells/metabolism , Tissue Distribution
5.
EMBO J ; 21(23): 6338-47, 2002 Dec 02.
Article in English | MEDLINE | ID: mdl-12456641

ABSTRACT

Endocrine cells of the pancreas and the gastrointestinal tract derive from multipotent endodermal stem cells. We have shown previously that the basic helix- loop-helix (bHLH) transcription factor neurogenin3 (ngn3) is required for the specification of the endocrine lineage in uncommitted progenitors in the developing pancreas. We investigate herein the expression and the function of ngn3 in the control of endocrine cell development in the intestinal and gastric epithelium. Our results indicate that as in the pancreas, gastrointestinal endocrine cells derive from ngn3-expressing progenitors. Mice homozygous for a null mutation in ngn3 fail to generate any intestinal endocrine cells, and endocrine progenitor cells are lacking. The other main intestinal epithelial cell types differentiate properly. In contrast, in the glandular stomach, the differentiation of the gastrin- (G cells) and somatostatin (D cells)-secreting cells is impaired whereas serotonin- (enterochromaffin EC cells), histamine- (enterochromaffin-like ECL cells) and ghrelin (X/A cells)-expressing cells are still present. Thus, ngn3 is strictly required for endocrine cell fate specification in multipotent intestinal progenitor cells, whereas gastric endocrine development is both ngn3 dependent and independent.


Subject(s)
Cell Differentiation/physiology , Enteroendocrine Cells/metabolism , Nerve Tissue Proteins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors , Epithelium/embryology , Epithelium/metabolism , Intestinal Mucosa/metabolism , Intestines/embryology , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Promoter Regions, Genetic
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