The Transcription Factor NURR1 Exerts Concentration-Dependent Effects on Target Genes Mediating Distinct Biological Processes

The transcription factor NURR1 plays a pivotal role in the development and maintenance of neurotransmitter phenotype in midbrain dopamine neurons. Conversely, decreased NURR1 expression is associated with a number of dopamine-related CNS disorders, including Parkinson’s disease and drug addiction. In order to better understand the nature of NURR1-responsive genes and their potential roles in dopamine neuron differentiation and survival, we used a human neural cellular background (SK-N-AS cells) in which to generate a number of stable clonal lines with graded NURR1 gene expression that approximated that seen in DA cell-rich human substantia nigra. Gene expression profiling data from these NURR1-expressing clonal lines were validated by quantitative RT-PCR and subjected to bioinformatic analyses. The present study identified a large number of NURR1-responsive genes and demonstrated the potential importance of concentration-dependent NURR1 effects in the differential regulation of distinct NURR1 target genes and biological pathways. These data support the promise of NURR1-based CNS therapeutics for the neuroprotection and/or functional restoration of DA neurons

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Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97166/1/jfo12080.pd

Ventral midbrain correlation between genetic variation and expression of the dopamine transporter gene in cocaine-abusing versus non-abusing subjects

Altered activity of the human dopamine transporter gene (hDAT) is asssociated with several common and severe brain disorders including cocaine abuse. However, there is little a priori information on whether such alteration was due to nature (genetic variantion) or nurture (human behaviors such as cocaine abuse). This study investigated the correlation between seven markers throughout hDAT and its mRNA levels in postmortem ventral midbrain tissues from 18 cocaine abusers and 18 strictly matched drug-free controls in the African American population. Here we show that one major haplotype with same frequency in cocaine abusers versus drug-free controls displays a 37.1%-reduction of expression levels in cocaine abusers, compared to matched controls (P = 0.0057). The most studied genetic marker, variable number tandem repeats (VNTR) located in Exon 15 (3′VNTR), is not correlated with hDAT mRNA levels. A 5′ upstream VNTR (rs70957367) has repeat numbers positively correlated with expression levels in controls (r2 0.9536, P = 0.0235) but this positive correlation disappears in cocaine abusers. The findings suggest that varying hDAT activity is attributed to both genetics and cocaine abuse

Inhibitors of histone deacetylase and MCL-1 synergistically reduce proliferation in malignant melanoma

Melanoma is a skin cancer that arises in melanocytes; it is the fifth most common cancer in the United States with approximately 100,000 new cases per year. Current treatments for malignant melanoma are surgical excision, radiation therapy and systemic therapy; however, the five-year survival rate for patients with stage IV is 29.8%. There is an urgent unmet clinical need to investigate novel treatments for these patients. Panobinostat is an orally available histone deacetylase inhibitor used in several hematologic malignancies, but it was ineffective as a single agent against melanoma in Phase 1. To address the insufficiency of options for melanoma patients, we treated a panel of cultured melanoma cell lines with panobinostat and the novel preclinical MCL-1 inhibitor AZD5991. We hypothesized the addition of AZD5991 (currently in phase 3) would enhance the antiproliferative effect of panobinostat in vitro. MTT and ATP-based proliferation assays demonstrated a significant reduction in proliferation when treated with either panobinostat or AZD5991. Isobologram analysis revealed that much lower concentrations of each drug was required to increase caspase 3/7 activity, induce a panel of Growth and DNA Damage (GADD) gene transcripts, and reduce proliferation when the drugs were added in combination. These in vitro studies revealed that panobinostat and AZD5991 synergistically inhibit melanoma growth. Increased caspase activation and the accumulation of GADD transcripts suggests apoptosis is a key feature of the antiproliferative mechanism. Ongoing studies are focused to further characterize panobinostat/AZD5991- induced cell death and to validate our cell culture observations in patient-derived xenograft models

Time-dependent alterations in calmodulin and its mRNAs after acute and repeated, intermittent amphetamine.

Regimens of repeated amphetamine dosing that elicit behavioral sensitization in rats also induce withdrawal-dependent alterations in calmodulin. This thesis examines if amphetamine-induced changes in calmodulin are time-dependent and if amphetamine also alters calmodulin gene transcription. To delineate time-dependent changes in calmodulin and assess whether amphetamine alters calmodulin gene transcription, calmodulin protein and mRNA of the three calmodulin genes were measured in dopaminergic cell body and terminal areas following acute or repeated amphetamine. Rats were injected twice weekly with 2.5 mg/kg amphetamine or saline i.p. and sacrificed three hours, three days or seven days after the tenth injection. Rats treated once were sacrificed three hours later. Acute amphetamine induced an increase in calmodulin in striatum and ventral mesencephalon but not in prefrontal cortex or nucleus accumbens. Elevations in calmodulin mRNAs were found which could contribute to increases in protein. The amphetamine-induced increase in calmodulin and calmodulin mRNAs in striatum and mesencephalon after the tenth dose was severely blunted as compared to the response to a single dose. Three days following the tenth amphetamine injection, elevations in calmodulin content and calmodulin mRNA were apparent in the cell body area (mesencephalon), but there was a decrease in both measurements in the dopaminergic terminal areas. However, calmodulin content in the striatum and prefrontal cortex was increased after seven days of withdrawal. These findings demonstrate that both calmodulin protein and calmodulin mRNA are altered by acute amphetamine and exhibit time-dependent changes during and after repeated amphetamine.Ph.D.Biological SciencesCellular biologyHealth and Environmental SciencesNeurosciencesPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131487/2/9909946.pd

Comparison of Amino Acid Positron Emission Tomographic Radiotracers for Molecular Imaging of Primary and Metastatic Brain Tumors

Positron emission tomography (PET) is an imaging technology that can detect and characterize tumors based on their molecular and biochemical properties, such as altered glucose, nucleoside, or amino acid metabolism. PET plays a significant role in the diagnosis, prognostication, and treatment of various cancers, including brain tumors. In this article, we compare uptake mechanisms and the clinical performance of the amino acid PET radiotracers (L-[methyl- 11 C]methionine [MET], 18 F-fluoroethyl-tyrosine [FET], 18 F-fluoro-L- dihydroxy-phenylalanine [FDOPA], and 11 C-alpha-methyl-L-tryptophan [AMT]) most commonly used for brain tumor imaging. First, we discuss and compare the mechanisms of tumoral transport and accumulation, the basis of differential performance of these radioligands in clinical studies. Then we summarize studies that provided direct comparisons among these amino acid tracers and to clinically used 2-deoxy-2[ 18 F]fluoro-D-glucose [FDG] PET imaging. We also discuss how tracer kinetic analysis can enhance the clinical information obtained from amino acid PET images. We discuss both similarities and differences in potential clinical value for each radioligand. This comparative review can guide which radiotracer to favor in future clinical trials aimed at defining the role of these molecular imaging modalities in the clinical management of brain tumor patients

Increased Heat Shock Protein 70 Gene Expression in the Brains of Cocaine‐Related Fatalities may be Reflective of Postdrug Survival and Intervention rather than Excited Delirium

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94232/1/jfo2212.pd

Preferential expression of functional IL-17R in glioma stem cells: potential role in self-renewal

Gliomas are the most common primary brain tumor and one of the most lethal solid tumors. Mechanistic studies into identification of novel biomarkers are needed to develop new therapeutic strategies for this deadly disease. The objective for this study was to explore the potential direct impact of IL-17-IL-17R interaction in gliomas. Immunohistochemistry and flow cytometry analysis of 12 tumor samples obtained from patients with high grade gliomas revealed that a considerable population (2-19%) of cells in all malignant gliomas expressed IL-17RA, with remarkable co-expression of the glioma stem cell (GSC) markers CD133, Nestin, and Sox2. IL-17 enhanced the self-renewal of GSCs as determined by proliferation and Matrigel® colony assays. IL-17 also induced cytokine/chemokine (IL-6, IL-8, interferon-γ-inducible protein [IP-10], and monocyte chemoattractant protein-1 [MCP-1]) secretion in GSCs, which were differentially blocked by antibodies against IL-17R and IL-6R. Western blot analysis showed that IL-17 modulated the activity of signal transducer and activator of transcription 3 (STAT3), nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), glycogen synthase kinase-3β (GSK-3β) and β-catenin in GSCs. While IL-17R-mediated secretion of IL-6 and IL-8 were significantly blocked by inhibitors of NF-κB and STAT3; NF-κB inhibitor was more potent than STAT3 inhibitor in blocking IL-17-induced MCP-1 secretion. Overall, our results suggest that IL-17-IL-17R interaction in GSCs induces an autocrine/paracrine cytokine feedback loop, which may provide an important signaling component for maintenance/self-renewal of GSCs via constitutive activation of both NF-κB and STAT3. The results also strongly implicate IL-17R as an important functional biomarker for therapeutic targeting of GSCs