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1.
Semin Nephrol ; 40(1): 14-27, 2020 01.
Article in English | MEDLINE | ID: mdl-32130962

ABSTRACT

Clear cell renal cell carcinoma (ccRCC) is a major cancer yet has long evaded extensive efforts to target it chemotherapeutically. Recent efforts to characterize its proteome and metabolome in a grade-defined manner has resulted in a global proteometabolomic reprogramming model yielding a number of potential drug targets, many of which are under the control of transcription factor and MYC proto-oncogene, bHLH transcription factor. Furthermore, through the use of conventional technologies such as immunohistochemistry, protein moonlighting, a phenomenon wherein a single protein performs more than one distinct biochemical or biophysical functions, is emerging as a second mode of operation for ccRCC metabolo-proteomic reprogramming. This renders the subcellular localization of the grade-defining biomarkers an additional layer of grade-defining ccRCC molecular signature, although its functional significance in ccRCC etiology is only beginning to emerge.


Subject(s)
Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Basic Helix-Loop-Helix Transcription Factors , Biomarkers, Tumor/metabolism , Cellular Reprogramming , Citric Acid Cycle , Gluconeogenesis , Glutathione/metabolism , Glycolysis , Humans , Metabolomics , Neoplasm Grading , Proteomics , Proto-Oncogene Mas , Proto-Oncogene Proteins c-myc , Urea/metabolism
2.
Methods Mol Biol ; 2047: 421-437, 2020.
Article in English | MEDLINE | ID: mdl-31552669

ABSTRACT

One of the first steps in studies of gene function is the spatiotemporal analysis of patterns of gene expression. Indirect immunohistochemistry is a method that allows the detection of a protein of interest by incubating a histological section with an antibody or antiserum raised against the protein, and then localizing this primary antibody with a tagged secondary antibody. To determine the cellular source of a protein of interest, or if a specific antibody is not available, specific transcripts can be localized using in situ hybridization. A histological section is incubated with a labeled RNA probe that is complementary to the target transcript; after hybridization with the target transcript the labeled RNA probe can be identified with an antibody. Here we describe materials and methods used to perform basic indirect immunohistochemistry and in situ hybridization on frozen sections through the developing chicken brain, emphasizing controls and potential problems that may be encountered.


Subject(s)
Brain/growth & development , Brain/metabolism , RNA/analysis , Animals , Chick Embryo , Cryoultramicrotomy , Immunohistochemistry , In Situ Hybridization , Tissue Fixation
3.
Am J Physiol Renal Physiol ; 315(6): F1855-F1868, 2018 12 01.
Article in English | MEDLINE | ID: mdl-30280600

ABSTRACT

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.


Subject(s)
Arginine/metabolism , Cell Proliferation , Energy Metabolism , Kidney/metabolism , Polycystic Kidney, Autosomal Dominant/metabolism , Animals , Arginine/deficiency , Arginine/pharmacology , Argininosuccinate Synthase/genetics , Argininosuccinate Synthase/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Disease Models, Animal , Energy Metabolism/drug effects , Female , Genetic Predisposition to Disease , Humans , Kidney/drug effects , Kidney/pathology , Male , Metabolomics/methods , Mice, Knockout , Phenotype , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/pathology , Receptors, Cell Surface/deficiency , Receptors, Cell Surface/genetics , Signal Transduction , TRPP Cation Channels/deficiency , TRPP Cation Channels/genetics
4.
J Urol ; 184(5): 2143-9, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20850841

ABSTRACT

PURPOSE: We evaluated the effect of roscovitine (Sigma-Aldrich®), a pharmacological inhibitor of cyclin dependent kinase, on renal cell carcinoma cell lines in vitro. MATERIALS AND METHODS: We exposed several renal cell carcinoma cell lines to roscovitine and examined apoptotic signaling pathways using immunoblotting and immunohistochemistry. RESULTS: As expected, roscovitine caused dose and time dependent inhibition of cyclin dependent kinase 2 autophosphorylation, and of cyclin dependent kinase mediated Pol II phosphorylation in the ACHN (p53-wt) and 786-O (p53 inactive) renal cell carcinoma cell lines (ATCC®). Roscovitine also induced apoptosis in each cell line within a narrow concentration range (about 10 µg/ml). Apoptosis induction was more efficient in ACHN than in 786-O cells and at least partly due to p53 activity. In ACHN cells roscovitine induced apoptosis was associated with p21 induction, and decreased Akt1, XIAP and phospho-Rb expression. These changes also depended on p53 and were not present (p21) or showed a different dose pattern (Akt1, XIAP and phospho-Rb) in 786-O cells. Partial restoration of roscovitine induced apoptosis in 786-O cells by the Mdm-2 inhibitor nutlin-3 (Sigma-Aldrich) suggests that the inactivating mutation of VHL in these cells and its destabilizing effect on p53 are responsible for the decreased sensitivity to apoptosis. CONCLUSIONS: Our data extend previous studies documenting the pro-apoptotic effect of roscovitine and to our knowledge show for the first time that this activity is restricted to a narrow dose range in renal cell carcinoma cells and partly depends on p53. Thus, roscovitine is a novel potential chemotherapy in a subset of patients with renal cell carcinoma if a narrow therapeutic window is used. These data also provide insight into the role of VHL mutation and p53 in the renal cell carcinoma response to therapeutic cyclin dependent kinase manipulation.


Subject(s)
Apoptosis/drug effects , Carcinoma, Renal Cell/enzymology , Carcinoma, Renal Cell/pathology , Cyclin-Dependent Kinases/antagonists & inhibitors , Kidney Neoplasms/enzymology , Kidney Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Tumor Suppressor Protein p53/drug effects , Tumor Suppressor Protein p53/physiology , Roscovitine , Tumor Cells, Cultured
5.
Neuron ; 65(1): 80-93, 2010 Jan 14.
Article in English | MEDLINE | ID: mdl-20152115

ABSTRACT

During development of the central nervous system, precise synaptic connections between presynaptic and postsynaptic neurons are formed. While significant progress has been made in our understanding of AMPA receptor trafficking during synaptic plasticity, less is known about the molecules that recruit AMPA receptors to nascent synapses during synaptogenesis. Here we identify a type II transmembrane protein (SynDIG1) that regulates AMPA receptor content at developing synapses in dissociated rat hippocampal neurons. SynDIG1 colocalizes with AMPA receptors at synapses and at extrasynaptic sites and associates with AMPA receptors in heterologous cells and brain. Altered levels of SynDIG1 in cultured neurons result in striking changes in excitatory synapse number and function. SynDIG1-mediated synapse development is dependent on association with AMPA receptors via its extracellular C terminus. Intriguingly, SynDIG1 content in dendritic spines is regulated by neuronal activity. Altogether, we define SynDIG1 as an activity-regulated transmembrane protein that regulates excitatory synapse development.


Subject(s)
Excitatory Postsynaptic Potentials/physiology , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Receptors, AMPA/metabolism , Synapses/physiology , Amino Acid Sequence , Animals , Cell Line , Female , Gene Expression Regulation , Hippocampus/cytology , Humans , Membrane Proteins/genetics , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/physiology , Patch-Clamp Techniques , Pregnancy , Rats , Receptors, AMPA/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Synapses/ultrastructure
6.
Mol Cell Biol ; 27(23): 8178-89, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17893326

ABSTRACT

During development, Sonic hedgehog (Shh) regulates the proliferation of cerebellar granule neuron precursors (GNPs) in part via expression of Nmyc. We present evidence supporting a novel role for the Mad family member Mad3 in the Shh pathway to regulate Nmyc expression and GNP proliferation. Mad3 mRNA is transiently expressed in GNPs during proliferation. Cultured GNPs express Mad3 in response to Shh stimulation in a cyclopamine-dependent manner. Mad3 is necessary for Shh-dependent GNP proliferation as measured by bromodeoxyuridine incorporation and Nmyc expression. Furthermore, Mad3 overexpression, but not that of other Mad proteins, is sufficient to induce GNP proliferation in the absence of Shh. Structure-function analysis revealed that Max dimerization and recruitment of the mSin3 corepressor are required for Mad3-mediated GNP proliferation. Surprisingly, basic-domain-dependent DNA binding of Mad3 is not required, suggesting that Mad3 interacts with other DNA binding proteins to repress transcription. Interestingly, cerebellar tumors and pretumor cells derived from patched heterozygous mice express high levels of Mad3 compared with adjacent normal cerebellar tissue. Our studies support a novel role for Mad3 in cerebellar GNP proliferation and possibly tumorigenesis, and they challenge the current paradigm that Mad3 should antagonize Nmyc by competition for direct DNA binding via Max dimerization.


Subject(s)
Cerebellum/cytology , Neurons/cytology , Neurons/metabolism , Repressor Proteins/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Animals , Cell Line , Cell Proliferation , Cerebellum/metabolism , Gene Expression , Hedgehog Proteins/metabolism , Humans , Medulloblastoma/metabolism , Medulloblastoma/pathology , Mice , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , Proto-Oncogene Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/genetics , Up-Regulation/genetics
7.
J Neurosci ; 26(12): 3087-101, 2006 Mar 22.
Article in English | MEDLINE | ID: mdl-16554460

ABSTRACT

Endogenous mechanisms underlying the remodeling of neuronal circuitry after mammalian CNS injury or disease remain primarily unknown. Here, we investigated axonal plasticity after optic nerve injury and found that macrophages recruited into the injury site and adult retinal ganglion cell (RGC) axons, which undergo injury-induced sprouting and terminal remodeling, were linked by their respective expression of a ligand and receptor pair active in axon guidance. Recruited macrophages specifically upregulated mRNA encoding the guidance molecule EphB3 and expressed EphB proteins capable of binding Ephrin B molecules in vivo and in vitro. Injured adult RGC axons in turn expressed EphrinB3, a known receptor for EphB3, and RGC axons bound recombinant EphB3 protein injected into the optic nerve. In vitro, EphB3 supported adult RGC axon outgrowth, and axons turned toward a source of this guidance molecule. In vivo, both reduction of EphB3 function in adult heterozygous animals and loss of function in homozygous animals greatly decreased RGC axon re-extension or sprouting after optic nerve injury. Comparisons of axon re-extension in EphB3 null and wild-type littermates showed that this loss of axonal plasticity was not attributable to a difference in intrinsic axon growth potential. Rather, the results indicated an essential role for local optic nerve-derived EphB3 in regulating adult RGC axon plasticity after optic nerve injury. Of note, the loss of EphB3 did not affect the ability of injured RGC axons to elaborate complex terminal branching, suggesting that additional EphB3-independent mechanisms governed adult axon branching triggered by CNS damage.


Subject(s)
Growth Cones/metabolism , Nerve Regeneration/physiology , Neuronal Plasticity/physiology , Optic Nerve Injuries/metabolism , Receptor, EphB3/genetics , Retinal Ganglion Cells/metabolism , Animals , Cell Communication/physiology , Disease Models, Animal , Down-Regulation/physiology , Ephrin-B3/metabolism , Female , Growth Cones/ultrastructure , Macrophages/metabolism , Male , Mice , Mice, Knockout , Mice, Mutant Strains , Mice, Transgenic , Nerve Growth Factors/metabolism , Optic Nerve/cytology , Optic Nerve/metabolism , Optic Nerve Injuries/physiopathology , Protein Binding/physiology , RNA, Messenger/genetics , Receptor, EphB3/metabolism , Retinal Ganglion Cells/cytology , Up-Regulation/genetics
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