Multipronged approach to identify and validate a novel upstream regulator of <i>Sncg</i> in mouse retinal ganglion cells

Chintalapudi, Sumana R; Morales-Tirado, Vanessa M.; Williams, Robert W.; Jablonski, Monica M.

doi:10.1111/febs.13620

Cited by 16 publications

(16 citation statements)

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“…A final volume of 10 μL was loaded into each well after combination of TaqMan® Universal Master Mix, cDNA, primers and Nuclease Free water. Plates were run using Roche® LightCycler 480 and data were analyzed using the Comparative Ct Method as in [24, 25]. …”

Section: Qpcr Analysesmentioning

confidence: 99%

Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

et al. 2017

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BackgroundRetinoblastoma (Rb) is the most common primary intraocular tumor in children. Local treatment of the intraocular disease is usually effective if diagnosed early; however advanced Rb can metastasize through routes that involve invasion of the choroid, sclera and optic nerve or more broadly via the ocular vasculature. Metastatic Rb patients have very high mortality rates. While current therapy for Rb is directed toward blocking tumor cell division and tumor growth, there are no specific treatments targeted to block Rb metastasis. Two such targets are matrix metalloproteinases-2 and -9 (MMP-2, −9), which degrade extracellular matrix as a prerequisite for cellular invasion and have been shown to be involved in other types of cancer metastasis. Cancer Clinical Trials with an anti-MMP-9 therapeutic antibody were recently initiated, prompting us to investigate the role of MMP-2, −9 in Rb metastasis.MethodsWe compare MMP-2, −9 activity in two well-studied Rb cell lines: Y79, which exhibits high metastatic potential and Weri-1, which has low metastatic potential. The effects of inhibitors of MMP-2 (ARP100) and MMP-9 (AG-L-66085) on migration, angiogenesis, and production of immunomodulatory cytokines were determined in both cell lines using qPCR, and ELISA. Cellular migration and potential for invasion were evaluated by the classic wound-healing assay and a Boyden Chamber assay.ResultsOur results showed that both inhibitors had differential effects on the two cell lines, significantly reducing migration in the metastatic Y79 cell line and greatly affecting the viability of Weri-1 cells. The MMP-9 inhibitor (MMP9I) AG-L-66085, diminished the Y79 angiogenic response. In Weri-1 cells, VEGF was significantly reduced and cell viability was decreased by both MMP-2 and MMP-9 inhibitors. Furthermore, inhibition of MMP-2 significantly reduced secretion of TGF-β1 in both Rb models.ConclusionsCollectively, our data indicates MMP-2 and MMP-9 drive metastatic pathways, including migration, viability and secretion of angiogenic factors in Rb cells. These two subtypes of matrix metalloproteinases represent new potential candidates for targeted anti-metastatic therapy for Rb.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3418-y) contains supplementary material, which is available to authorized users.

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Section: Qpcr Analysesmentioning

confidence: 99%

Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

et al. 2017

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“…RNA was extracted from cells or tissue using RNeasy ® Mini Kit (Qiagen Inc., Valencia, CA, USA) following manufacturer's conditions and published protocols. [14][15][16] We used 100 ng of RNA material for complementary DNA (cDNA) synthesis. Resulting cDNA material was pre-amplified prior to assay set up.…”

Section: Qpcr Analysesmentioning

confidence: 99%

Sirtuin Inhibitor as a Novel Cell Cycle Checkpoint and Regulator of the TP53-MDM2 Pathway in Uveal Melanoma

Goldsmith¹,

McEwen²,

Kibe³

et al. 2019

Ophthalmol Open J.

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Cite this articleGoldsmith ZK, McEwen MW, Kibe MW, et al. Sirtuin inhibitor as a novel cell cycle checkpoint and regulator of the TP53-MDM2 pathway in uveal melanoma. ABSTRACT PurposeThe liver is the most common site of uveal melanoma (UM) metastasis with approximately 50% of UM patients being affected. With no proven therapies that mitigate metastases the mortality rate is 85% within the first year after detection of the liver disease. In this study, we provide a mechanistic understanding of the de-regulation of the TP53-MDM2 pathway in UM, which plays a central role in tumor biology. MethodsWe investigated the TP53-MDM2 signaling pathway in the microenvironment of liver metastases taken from both a murine orthotopic xenograft and post-mortem metastatic UM human liver. These findings were studied in-depth using both primary and metastatic UM cell lines treated with the MDM2 antagonist Nutlin-3a and the sirtuin inhibitor and transcriptional activator of TP53, Tenovin-6. ResultsDe-regulation of the TP53-MDM2 signaling pathway is specific to the liver microenvironment, providing a survival mechanism for UM metastases. Tenovin-6, not Nutlin-3a, reduced UM cell survival by increasing the percentage of cell death and reducing the percentage of proliferating cells. Tenovin-6 increased acetylation of p53, reduced ubiquitination of the protein, and acted as a cell cycle regulator. ConclusionOur findings suggest that in patients with metastatic UM de-regulation of TP53-MDM2 signaling pathway promotes growth of the liver metastases and provides pre-clinical information on the potential of targeting of the TP53-MDM2 signaling pathway via Tenovin-6.

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“…Five to six million variants segregate among the BXD family (Wang et al, 2016), giving them a genetic complexity approaching that of human populations, but with the major advantage of being able to replicate any and all individuals and thereby explore gene-byenvironment, gene-by-age, and gene-by-therapy effects (Chintalapudi et al, 2017). Genome and phenome data sets for this family have been assembled in what is essentially an open source electronic health care database (gn2.genenetwork.org) that is now widely used as an experimental platform for personalized and probabilistic medicine (Koutnikova et al, 2009;Jablonski et al, 2011;Lu et al, 2011a;Lu et al, 2011b;Swaminathan et al, 2013;Chintalapudi et al, 2015;Lu et al, 2016;Chintalapudi et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Systems Genetics of Optic Nerve Axon Necrosis During Glaucoma

et al. 2020

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In this study, we identify genomic regions that modulate the number of necrotic axons in optic nerves of a family of mice, some of which have severe glaucoma, and define a set of high priority positional candidate genes that modulate retinal ganglion cell (RGC) axonal degeneration. A large cohort of the BXD family were aged to greater than 13 months of age. Optic nerves from 74 strains and the DBA/2J (D2) parent were harvested, sectioned, and stained with p-phenylenediamine. Numbers of necrotic axons per optic nerve crosssection were counted from 1 to 10 replicates per genotype. Strain means and standard errors were uploaded into GeneNetwork 2 for mapping and systems genetics analyses (Trait 18614). The number of necrotic axons per nerve ranged from only a few hundred to more than 4,000. Using conventional interval mapping as well as linear mixed model mapping, we identified a single locus on chromosome 12 between 109 and 112.5 Mb with a likelihood ratio statistic (LRS) of~18.5 (p genome-wide~0.1). Axon necrosis is not linked to locations of major known glaucoma genes in this family, including Gpnmb, Tyrp1, Cdh11, Pou6f2, and Cacna2d1. This indicates that although these genes contribute to pigmentary dispersion or elevated IOP, none directly modulates axon necrosis. Of 156 positional candidates, eight genes-CDC42 binding protein kinase beta (Cdc42bpb); eukaryotic translation initiation factor 5 (Eif5); BCL2-associated athanogene 5 (Bag5); apoptogenic 1, mitochondrial (Apopt1); kinesin light chain 1 (Klc1); X-ray repair cross complementing 3 (Xrcc3); protein phosphatase 1, regulatory subunit 13B (Ppp1r13b); and transmembrane protein 179 (Tmem179)-passed stringent criteria and are high priority candidates. Several candidates are linked to mitochondria and/or axons, strengthening their plausible role as modulators of ON necrosis. Additional studies are required to validate and/or eliminate plausible candidates. Surprisingly, IOP and ON necrosis are inversely correlated across the BXD family in mice >13 months of age and these two traits share few genes among their top ocular and retinal correlates. These data suggest that the two traits are independently modulated or that a more complex and multifaceted approach is required to reveal their association.

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Multipronged approach to identify and validate a novel upstream regulator of Sncg in mouse retinal ganglion cells

Cited by 16 publications

References 68 publications

Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma

Sirtuin Inhibitor as a Novel Cell Cycle Checkpoint and Regulator of the TP53-MDM2 Pathway in Uveal Melanoma

Systems Genetics of Optic Nerve Axon Necrosis During Glaucoma

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