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  • Inactivation of HNSCC cells by 90Y-labeled cetuximab strictly depends on the number of induced DNA double-strand breaks. 23345302

    Radioimmunotherapy is considered to have great potential for efficient and highly specific treatment of tumors. The aim of this study was to determine the efficacy of radioimmunotherapy when using (90)Y-labeled cetuximab and to determine to what degree induction and repair of DNA double-strand breaks (DSBs) are decisive for this approach.This study was performed with 9 cell lines of squamous cell carcinoma of the head and neck (HNSCC) differing strongly in epidermal growth factor receptor (EGFR) expression. The radionuclide (90)Y was coupled by the chelator trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-A″-DTPA)/linker construct to the EGFR-directed antibody cetuximab to yield (90)Y-Y-CHX-A″-DTPA-cetuximab with a specific activity of approximately 1.2 GBq/mg. EGFR expression was determined by immunofluorescence and Western blotting, cetuximab binding by fluorescence-activated cell sorter analysis, the number of DSBs by immunofluorescence staining γH2AX/53BP1-positive repair foci, and cell survival by colony formation.For the 9 HNSCC cell lines, cetuximab binding correlated with the amount of EGFR present in the cell membrane (r(2) = 0.967, P less than 0.001). When cells were exposed to (90)Y-Y-CHX-A″-DTPA-cetuximab, the number of induced DSBs increased linearly with time (r(2) = 0.968, P = 0.016). This number was found to correlate with the amount of membranous EGFR (r(2) = 0.877, P = 0.006). Most DSBs were repaired during incubation at 37°C, but the small number of remaining DSBs still correlated with the amount of membranous EGFR (24 h: r(2) = 0.977, P less than 0.001; 48 h: r(2) = 0.947, P less than 0.001). Exposure to (90)Y-Y-CHX-A″-DTPA-cetuximab also resulted in efficient cell killing, whereby the extent of cell killing correlated strongly with the respective number of remaining DSBs (r(2) = 0.989, P less than 0.001) and with the amount of membranous EGFR (r(2) = 0.967, P less than 0.001). No cell killing was observed for UTSCC15 cells with low EGFR expression, in contrast to the strong reduction of 86% measured for UTSCC14 cells showing a strong overexpression of EGFR.(90)Y-Y-CHX-A″-DTPA-cetuximab affected cell survival through the induction of DSBs. This treatment was especially efficient for HNSCC cells strongly overexpressing EGFR, whereas no effect was seen for cells with low levels of EGFR expression. Therefore, EGFR-directed radioimmunotherapy using (90)Y-Y-CHX-A″-DTPA-cetuximab appears to be a powerful tool that can be used to inactivate tumors with strong EGFR overexpression, which are often characterized by a pronounced radioresistance.
    Document Type:
    Reference
    Product Catalog Number:
    05-636
    Product Catalog Name:
    Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 - (Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301)

  • Manganese ethylene-bis-dithiocarbamate and selective dopaminergic neurodegeneration in rat: a link through mitochondrial dysfunction. 12558996

    Manganese ethylene-bis-dithiocarbamate (Mn-EBDC) is the major active element of maneb, a pesticide linked to parkinsonism in certain individuals upon chronic exposure. Additionally, it has been shown to produce dopaminergic neurodegeneration in mice systemically coexposed to another pesticide, 1,1'-dimethyl-4,4'-bipyridinium (paraquat). Here, we described a rat model in which selective dopaminergic neurodegeneration was produced by delivering Mn-EBDC directly to the lateral ventricles. After establishing this model, we tested whether Mn-EBDC provoked dopamine efflux in the striatum, a well-known phenomenon produced by the mitochondrial inhibitor 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that causes parkinsonism in humans, as well as in some animals. Finally, we investigated whether Mn-EBDC directly inhibited mitochondrial function in vitro using isolated brain mitochondria. Our data demonstrated that Mn-EBDC induced extensive striatal dopamine efflux that was comparable with that induced by MPP+, and that Mn-EBDC preferentially inhibited mitochondrial complex III. As mitochondrial dysfunction is pivotal in the pathogenesis of Parkinson's disease (PD), our results support the proposal that exposure to pesticides such as maneb, or other naturally occurring compounds that inhibit mitochondrial function, may contribute to PD development.
    Document Type:
    Reference
    Product Catalog Number:
    AB1511

  • Proteasomal inhibition induced by manganese ethylene-bis-dithiocarbamate: relevance to Parkinson's disease. 15350641

    Maneb, a widely used fungicide, has been associated with Parkinsonism in humans. In experimental models, maneb and its major active element, manganese ethylene-bis-dithiocarbamate (Mn-EBDC) cause selective nigrostriatal neurodegeneration in mice and in rats, respectively. To investigate the mechanisms underlying this neurodegeneration, we studied the effects of Mn-EBDC on proteasomal function, which is decreased in patients with Parkinson's disease (PD), in a dopaminergic neuronal cell line (MES 23.5 or MES). The results demonstrated that exposure of MES cells to 6 microM Mn-EBDC for 7 days produced not only significant neurotoxicity but also inhibition of proteasomal chymotrypsin-like and postglutamyl peptidase activities. Proteasomal dysfunction was accompanied by formation of cytoplasmic inclusions that were positive for alpha-synuclein immunostaining and significantly increased sodium dodecyl sulfate-insoluble alpha-synuclein aggregation seen by Western blot analysis. In addition, there was a significant increase in oxidative stress, evidenced by elevated total protein carbonyl content, in cells treated with Mn-EBDC. Manipulation of intracellular reduced glutathione levels with N-acetyl-L-cysteine or L-buthionine sulfoximine pretreatment to modulate Mn-EBDC-mediated oxidative stress altered Mn-EBDC-mediated neurotoxicity, proteasomal dysfunction, and alpha-synuclein aggregation in these cells. These data suggest that neurotoxicity-induced by Mn-EBDC is at least partially attributable to Mn-EBDC-mediated proteasomal inhibition, and that the proteasome may be an important target by which environmental exposure modifies the risk for developing PD in vulnerable populations.
    Document Type:
    Reference
    Product Catalog Number:
    AB5038
    Product Catalog Name:
    Anti-Synuclein α Antibody - (Anti-Synuclein α Antibody)

  • Two cell circuits of oriented adult hippocampal neurons on self-assembled monolayers for use in the study of neuronal communication in a defined system. 23611164

    In this study, we demonstrate the directed formation of small circuits of electrically active, synaptically connected neurons derived from the hippocampus of adult rats through the use of engineered chemically modified culture surfaces that orient the polarity of the neuronal processes. Although synaptogenesis, synaptic communication, synaptic plasticity, and brain disease pathophysiology can be studied using brain slice or dissociated embryonic neuronal culture systems, the complex elements found in neuronal synapses makes specific studies difficult in these random cultures. The study of synaptic transmission in mature adult neurons and factors affecting synaptic transmission are generally studied in organotypic cultures, in brain slices, or in vivo. However, engineered neuronal networks would allow these studies to be performed instead on simple functional neuronal circuits derived from adult brain tissue. Photolithographic patterned self-assembled monolayers (SAMs) were used to create the two-cell "bidirectional polarity" circuit patterns. This pattern consisted of a cell permissive SAM, N-1[3-(trimethoxysilyl)propyl] diethylenetriamine (DETA), and was composed of two 25 μm somal adhesion sites connected with 5 μm lines acting as surface cues for guided axonal and dendritic regeneration. Surrounding the DETA pattern was a background of a non-cell-permissive poly(ethylene glycol) (PEG) SAM. Adult hippocampal neurons were first cultured on coverslips coated with DETA monolayers and were later passaged onto the PEG-DETA bidirectional polarity patterns in serum-free medium. These neurons followed surface cues, attaching and regenerating only along the DETA substrate to form small engineered neuronal circuits. These circuits were stable for more than 21 days in vitro (DIV), during which synaptic connectivity was evaluated using basic electrophysiological methods.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple
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