Changes in Retinoblastoma Cell Adhesion Associated with Optic Nerve Invasion▿ †

In the 1970s, several human retinoblastoma cell lines were developed from cultures of primary tumors. As the human retinoblastoma cell lines were established in culture, growth properties and changes in cell adhesion were described. Those changes correlated with the ability of the human retinoblastoma cell lines to invade the optic nerve and metastasize in orthotopic xenograft studies. However, the mechanisms that underlie these changes were not determined. We used the recently developed knockout mouse models of retinoblastoma to begin to characterize the molecular, cellular, and genetic changes associated with retinoblastoma tumor progression and optic nerve invasion. Here we report the isolation and characterization of the first mouse retinoblastoma cell lines with targeted deletions of the Rb family. Our detailed analysis of these cells as they were propagated in culture from the primary tumor shows that changes in cadherin-mediated cell adhesion are associated with retinoblastoma invasion of the optic nerve prior to metastasis. In addition, the same changes in cadherin-mediated cell adhesion correlate with the invasive properties of the human retinoblastoma cell lines isolated decades ago, providing a molecular mechanism for these earlier observations. Most importantly, our studies are in agreement with genetic studies on human retinoblastomas, suggesting that changes in this pathway are involved in tumor progression

Immunomodulation by gastrointestinal carbon black nanoparticle exposure in ovalbumin T cell receptor transgenic mice

<p>Humans could become exposed to carbon black nanoparticles (CBNPs) in consumer products or an occupational setting. In rodent models, acute respiratory, subcutaneous, and direct immune cell exposure to CBNPs has been shown to enhance allergic sensitization to co-administered ovalbumin (OVA) protein from chicken egg. However, little is known about the effects of ingested CBNPs on immunological responses and oral tolerance to food antigens. We hypothesized that ingestion of CBNPs would enhance the development of food allergy to OVA. Allergy prone DO11.10 mice were orally exposed to CBNPs every second day for 2 weeks (total dose 10.8 (LOW) or 108 μg (HI)), with and without (±) co-administered OVA. Systemic immune parameters were measured at necropsy. Exposure to OVA resulted in significant increases in serum anti-OVA IgG1, anti-OVA IgM, and anti-OVA IgA antibodies relative to vehicle control. Immunophenotyping revealed a reduction in the number of OVA-specific CD4⁺T helper cells upon OVA ± CBNP^HI treatment in the spleen. Yet, secretion of the allergy-associated Th2 cytokines IL-4, IL-9, and IL-13 was greater in OVA_323-339 peptide-pulsed splenocytes from OVA + CBNP^HI-treated mice compared with control. Transcriptome analysis at necropsy of splenocytes from OVA + CBNP^HI dose mice compared with OVA mice revealed increases in the allergy associated genes <i>Il4</i> and <i>Stat6</i> and decreases in <i>Csf3r</i> and <i>Retnlg</i>. Although oral exposure to high-dose CBNPs did not impact OVA-specific antibody production relative to OVA, we did observe increased expression of genes and cytokines associated with allergy in peripheral splenocytes. This work suggests that CBNP gastrointestinal exposure may potentiate allergy pathways.</p

Novel miRNA-31 and miRNA-200a-Mediated Regulation of Retinoblastoma Proliferation

<div><p>Retinoblastoma is the most common intraocular tumor in children. Current management includes broad-based treatments such as chemotherapy, enucleation, laser therapy, or cryotherapy. However, therapies that target specific pathways important for retinoblastoma progression could provide valuable alternatives for treatment. MicroRNAs are short, noncoding RNA transcripts that can regulate the expression of target genes, and their aberrant expression often facilitates disease. The identification of post-transcriptional events that occur after the initiating genetic lesions could further define the rapidly aggressive growth displayed by retinoblastoma tumors. In this study, we used two phenotypically different retinoblastoma cell lines to elucidate the roles of miRNA-31 and miRNA-200a in tumor proliferation. Our approach confirmed that miRNAs-31 and -200a expression is significantly reduced in human retinoblastomas. Moreover, overexpression of these two miRNAs restricts the expansion of a highly proliferative cell line (Y79), but does not restrict the growth rate of a less aggressive cell line (Weri1). Gene expression profiling of miRNA-31 and/or miRNA-200a-overexpressing cells identified differentially expressed mRNAs associated with the divergent response of the two cell lines. This work has the potential to enhance the development of targeted therapeutic approaches for retinoblastoma and improve the efficacy of treatment.</p></div

MicroRNAs- 31 and -200a are significantly downregulated in human retinoblastoma tumors and cell lines.

<p>(A) The expression level of miR-31 was measured in retinoblastomas as compared to normal pediatric retinas. (B) The expression level of miR-200a was measured in human retinoblastomas as compared to normal pediatric retinas. (C) The expression level of miR-31 and miR-200a was measured in human retinoblastoma cell lines (Y79, Weri1) as compared to normal pediatric retinas. Data represents mean and standard deviation from two independent experiments in triplicate. *denotes p< 0.05, ** denotes p< 0.003, *** denotes p<0.0005.</p

Increased expression of miRNAs-31 and -200a negatively regulates genes associated with proliferation and survival.

<p>(A- C) Expression of <i>ACOT7</i>, <i>DLL3</i>, <i>PPP6C</i>, and <i>STK40</i> mRNAs as measured by TaqMan qRT-PCR in human retinoblastoma cells (Y79 and Weri1) after transient overexpression of miR-31 (A), miR-200a (B), and co-transfected miRs-31 and -200a, “Mix” (C) as compared to negative control miRNA overexpressing cells. Data represents mean and standard deviation from two independent experiments. * denotes p< 0.05, ** denotes p< 0.01, *** denotes p< 0.0005.</p

<i>ACOT7</i> is a direct target of miRNA-200a.

<p>(A) The nucleotide sequence of the putative miR-200a binding site within the 3’UTR of <i>ACOT7</i>. As a negative control, a mutated version of the binding site (‘ACOT7-<i>mut</i>’) contains three different nucleotides (underlined). (B) Y79 retinoblastoma cells, transfected with a miR-200a mimic or a negative (control miR) mimic, were co-transfected with plasmid vectors containing a firefly luciferase gene coding region upstream of either the ACOT7-3’UTR (pMir-ACOT7) or the mutated ACOT7- 3’UTR (pMir-mut), in addition to a renilla luciferase plasmid for normalization purposes. Data represents mean and standard deviation from independent triplicates from two experiments. *** denotes p< 0.0001.</p

DLL3 and ACOT7 expression in Y79 cells following overexpression of miRNAs-31, -200a, or when overexpressed together (Mix).

<p>Immunofluorescence staining of DLL3 (A) and ACOT7 (B) in Y79 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). Quantification of immunofluorescence intensity of DLL3 (C) and ACOT7 (E); bar represents mean and standard deviation from two independent experiments with quadruplicate measurements. Western blot analysis of DLL3 (D) and ACOT7 (F) in Y79 cells transfected with a negative miRNA (control), miRNA-31, miRNA-200a, and miR-31/-200a (Mix). * denotes p< 0.05. Scale bar 20 μm.</p

Human retinoblastomas express ACOT7 and DLL3.

<p>Immunohistochemistry was performed on four retinoblastoma samples obtained from four individual pediatric patients. Hematoxylin and eosin staining of each retinoblastoma (retinoblastoma 1–4) shows identified tumor (Tumor) and adjacent retina (denoted with an asterisk). Control staining served as a negative control (no primary; secondary antibody only; hematoxylin counterstain). In four of four cases, retinoblastomas display expression of ACOT7 and DLL3.</p