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1.
Proc Natl Acad Sci U S A ; 109(15): 5729-34, 2012 Apr 10.
Article in English | MEDLINE | ID: mdl-22451918

ABSTRACT

Mutations in Drosophila merry-go-round (mgr) have been known for over two decades to lead to circular mitotic figures and loss of meiotic spindle integrity. However, the identity of its gene product has remained undiscovered. We now show that mgr encodes the Prefoldin subunit counterpart of human von Hippel Lindau binding-protein 1. Depletion of Mgr from cultured cells also leads to formation of monopolar and abnormal spindles and centrosome loss. These phenotypes are associated with reductions of tubulin levels in both mgr flies and mgr RNAi-treated cultured cells. Moreover, mgr spindle defects can be phenocopied by depleting ß-tubulin, suggesting Mgr function is required for tubulin stability. Instability of ß-tubulin in the mgr larval brain is less pronounced than in either mgr testes or in cultured cells. However, expression of transgenic ß-tubulin in the larval brain leads to increased tubulin instability, indicating that Prefoldin might only be required when tubulins are synthesized at high levels. Mgr interacts with Drosophila von Hippel Lindau protein (Vhl). Both proteins interact with unpolymerized tubulins, suggesting they cooperate in regulating tubulin functions. Accordingly, codepletion of Vhl with Mgr gives partial rescue of tubulin instability, monopolar spindle formation, and loss of centrosomes, leading us to propose a requirement for Vhl to promote degradation of incorrectly folded tubulin in the absence of functional Prefoldin. Thus, Vhl may play a pivotal role: promoting microtubule stabilization when tubulins are correctly folded by Prefoldin and tubulin destruction when they are not.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Molecular Chaperones/metabolism , Protein Subunits/metabolism , Tubulin/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Animals , Conserved Sequence , Drosophila melanogaster/cytology , Humans , Microtubules/metabolism , Mutation/genetics , Protein Binding , Protein Stability , Proteolysis , Spindle Apparatus/metabolism
2.
Stem Cells ; 26(4): 894-902, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18238852

ABSTRACT

The potential to differentiate human embryonic stem cells (hESCs) in vitro to provide an unlimited source of human hepatocytes for use in biomedical research, drug discovery, and the treatment of liver diseases holds great promise. Here we describe a three-stage process for the efficient and reproducible differentiation of hESCs to hepatocytes by priming hESCs towards definitive endoderm with activin A and sodium butyrate prior to further differentiation to hepatocytes with dimethyl sulfoxide, followed by maturation with hepatocyte growth factor and oncostatin M. We have demonstrated that differentiation of hESCs in this process recapitulates liver development in vivo: following initial differentiation, hESCs transiently express characteristic markers of the primitive streak mesendoderm before turning to the markers of the definitive endoderm; with further differentiation, expression of hepatocyte progenitor cell markers and mature hepatocyte markers emerged sequentially. Furthermore, we have provided evidence that the hESC-derived hepatocytes are able to carry out a range of hepatocyte functions: storage of glycogen, and generation and secretion of plasma proteins. More importantly, the hESC-derived hepatocytes express several members of cytochrome P450 isozymes, and these P450 isozymes are capable of converting the substrates to metabolites and respond to the chemical stimulation. Our results have provided evidence that hESCs can be differentiated efficiently in vitro to functional hepatocytes, which may be useful as an in vitro system for toxicity screening in drug discovery.


Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Hepatocytes/cytology , Liver/cytology , Liver/growth & development , Animals , Biomarkers/metabolism , Cell Culture Techniques/methods , Cells, Cultured , Embryonic Stem Cells/physiology , Hepatocytes/physiology , Humans , Mice , Organogenesis/physiology
3.
J Cell Biol ; 156(3): 437-51, 2002 Feb 04.
Article in English | MEDLINE | ID: mdl-11827981

ABSTRACT

Disruption of the function of the A-type Aurora kinase of Drosophila by mutation or RNAi leads to a reduction in the length of astral microtubules in syncytial embryos, larval neuroblasts, and cultured S2 cells. In neuroblasts, it can also lead to loss of an organized centrosome and its associated aster from one of the spindle poles, whereas the centrosome at the other pole has multiple centrioles. When centrosomes are present at the poles of aurA mutants or aurA RNAi spindles, they retain many antigens but are missing the Drosophila counterpart of mammalian transforming acidic coiled coil (TACC) proteins, D-TACC. We show that a subpopulation of the total Aurora A is present in a complex with D-TACC, which is a substrate for the kinase. We propose that one of the functions of Aurora A kinase is to direct centrosomal organization such that D-TACC complexed to the MSPS/XMAP215 microtubule-associated protein may be recruited, and thus modulate the behavior of astral microtubules.


Subject(s)
Centrosome/enzymology , Drosophila/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/enzymology , Mitosis/genetics , Protein Kinases/metabolism , Spindle Apparatus/enzymology , Xenopus Proteins , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Animals , Aurora Kinases , Binding Sites/genetics , Cell Compartmentation/physiology , Cell Cycle Proteins , Drosophila/embryology , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Female , Male , Microtubule-Associated Proteins/genetics , Microtubules/genetics , Mutation/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Kinases/genetics , Protein Serine-Threonine Kinases , Protein Structure, Tertiary/genetics , RNA/genetics , RNA/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spindle Apparatus/genetics
4.
Cancer Res ; 64(14): 4875-86, 2004 Jul 15.
Article in English | MEDLINE | ID: mdl-15256458

ABSTRACT

The acquisition of resistance to apoptosis, the cell's intrinsic suicide program, is essential for cancers to arise and progress and is a major reason behind treatment failures. We show in this article that small molecule antagonists of the sigma-1 receptor inhibit tumor cell survival to reveal caspase-dependent apoptosis. sigma antagonist-mediated caspase activation and cell death are substantially attenuated by the prototypic sigma-1 agonists (+)-SKF10,047 and (+)-pentazocine. Although several normal cell types such as fibroblasts, epithelial cells, and even sigma receptor-rich neurons are resistant to the apoptotic effects of sigma antagonists, cells that can promote autocrine survival such as lens epithelial and microvascular endothelial cells are as susceptible as tumor cells. Cellular susceptibility appears to correlate with differences in sigma receptor coupling rather than levels of expression. In susceptible cells only, sigma antagonists evoke a rapid rise in cytosolic calcium that is inhibited by sigma-1 agonists. In at least some tumor cells, sigma antagonists cause calcium-dependent activation of phospholipase C and concomitant calcium-independent inhibition of phosphatidylinositol 3'-kinase pathway signaling. Systemic administration of sigma antagonists significantly inhibits the growth of evolving and established hormone-sensitive and hormone-insensitive mammary carcinoma xenografts, orthotopic prostate tumors, and p53-null lung carcinoma xenografts in immunocompromised mice in the absence of side effects. Release of a sigma receptor-mediated brake on apoptosis may offer a new approach to cancer treatment.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Receptors, sigma/antagonists & inhibitors , Animals , Apoptosis/physiology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Calcium Signaling/drug effects , Carbazoles/pharmacology , Caspases/metabolism , Cattle , Cell Division/drug effects , Cell Division/physiology , Cell Line, Tumor , Enzyme Activation , Ethylenediamines/pharmacology , Haloperidol/pharmacology , Humans , Isoenzymes/metabolism , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Male , Mice , Mice, Nude , Phospholipase C delta , Piperazines/pharmacology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-akt , Type C Phospholipases/metabolism , Xenograft Model Antitumor Assays , Sigma-1 Receptor
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