Detection of GNAQ mutations and reduction of cell viability in uveal melanoma cells with functionalized gold nanoparticles.

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Early treatment may improve any chances of preventing metastatic disease, but diagnosis of small UM is challenging. Up to 95 % of all UMs carry somatic mutations in the G-coupled proteins GNAQ and GNA11 promoting anchorage-independent growth and proliferation. About 50 % of UMs are fatal. Once metastatic, patients have limited options for successful therapy. METHODS: We have developed functionalized gold nanoparticles (AuNPs) to visualize transcripts of mutant GNAQ mRNA in living cells. In addition to their suitability as a specific tool for GNAQ mutation detection, we have developed a novel linker that enables conjugation of siRNAs to AuNPs allowing for greater and more rapid intracellular release of siRNAs compared to previously described approaches. RESULTS: Binding of modified AuNPs to matching target mRNA leads to conformational changes, resulting in a detectable fluorescent signal that can be used for mutation detection in living cells. Knockdown of GNAQ with siRNA-AuNPs effectively reduced downstream signals and decreased cell viability in GNAQ mutant uveal melanoma cells. CONCLUSION: AuNPs may in future be developed to serve as sensors for mutations of vital importance. The new release system for siRNA-AuNP improves previous systems, which conceivably will be useful for future therapeutic gene regulatory approaches.

Mutations in GNA11 in uveal melanoma.

BACKGROUND: Uveal melanoma is the most common intraocular cancer. There are no effective therapies for metastatic disease. Mutations in GNAQ, the gene encoding an alpha subunit of heterotrimeric G proteins, are found in 40% of uveal melanomas. METHODS: We sequenced exon 5 of GNAQ and GNA11, a paralogue of GNAQ, in 713 melanocytic neoplasms of different types (186 uveal melanomas, 139 blue nevi, 106 other nevi, and 282 other melanomas). We sequenced exon 4 of GNAQ and GNA11 in 453 of these samples and in all coding exons of GNAQ and GNA11 in 97 uveal melanomas and 45 blue nevi. RESULTS: We found somatic mutations in exon 5 (affecting Q209) and in exon 4 (affecting R183) in both GNA11 and GNAQ, in a mutually exclusive pattern. Mutations affecting Q209 in GNA11 were present in 7% of blue nevi, 32% of primary uveal melanomas, and 57% of uveal melanoma metastases. In contrast, we observed Q209 mutations in GNAQ in 55% of blue nevi, 45% of uveal melanomas, and 22% of uveal melanoma metastases. Mutations affecting R183 in either GNAQ or GNA11 were less prevalent (2% of blue nevi and 6% of uveal melanomas) than the Q209 mutations. Mutations in GNA11 induced spontaneously metastasizing tumors in a mouse model and activated the mitogen-activated protein kinase pathway. CONCLUSIONS: Of the uveal melanomas we analyzed, 83% had somatic mutations in GNAQ or GNA11. Constitutive activation of the pathway involving these two genes appears to be a major contributor to the development of uveal melanoma. (Funded by the National Institutes of Health and others.).

A novel PPAR response element in the murine iNOS promoter.

The nuclear hormone receptor peroxisome proliferation activated receptor gamma (PPARgamma) is a modulator of inflammation including down-regulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. PPARgamma agonists reduce iNOS expression and NO production in a dose-dependent manner in macrophages, mesangial cells and other inflammatory cells. However, the mechanisms involved in the inhibition of iNOS expression by PPARgamma and its agonists are not fully understood. Here we show that the PPARgamma agonist ciglitazone dose-dependently inhibited a murine iNOS-luciferase reporter construct by up to 50% in transfected mesangial cells. Blocking de novo protein synthesis in mesangial cells had no effect on PPARgamma agonist activity, indicating that ciglitazone acts directly to inhibit iNOS transcription. We identified a novel PPAR response element (PPRE) in the murine iNOS promoter that is homologous to the PPRE consensus sequence. In binding assays PPARgamma directly binds to this response element in vitro and can function as a positive element in response to PPARgamma agonists when placed in front of a reporter gene. Site-directed mutagenesis of this PPRE in a murine iNOS promoter/reporter construct did not block the inhibitory activity of a synthetic PPARgamma agonist on the iNOS promoter/reporter construct in transfected mesangial cells. However, the mutated construct exhibited lower basal expression, and higher expression in response to inflammatory stimuli compared to the intact construct. These data suggest that the iNOS PPRE contributes to positive basal expression and negative expression of iNOS in response to inflammatory stimuli. The PPRE is not necessary, however, for synthetic PPARgamma agonists to inhibit iNOS expression.

Serum vascular endothelial growth factor (VEGF) levels correlate with number and location of micrometastases in a murine model of uveal melanoma.

BACKGROUND: A preliminary animal study was performed to determine if hepatic micrometastases from uveal melanoma secrete vascular endothelial growth factor (VEGF) that is measurable in serum. METHODS: We analysed the serum of a C57Bl/6 mouse model of uveal melanoma (n=10) at days 4, 7, 14 and 21 post-inoculation for VEGF levels. We compared the serum VEGF levels with the number and location of hepatic micrometastases and their respective expression of VEGF mRNA. RESULTS: Serum VEGF levels rose after inoculation of C57Bl/6 mice eyes with B16LS9 cutaneous melanoma cells. Beginning on day 14 there was a statistically significant (p<0.05) increase in VEGF levels, rising to an average peak level of 37.985 pg/ml at day 21. Peak serum VEGF levels correlated with the total number of hepatic micrometastases (R=0.444) and there was moderate correlation of peak VEGF serum levels with micrometastases in more hypoxic locations (R=0.572). VEGF mRNA expression by micrometastases was highest in the most hypoxic regions of the hepatic lobule. CONCLUSIONS: Hepatic micrometastastic melanoma arising in a mouse model of ocular melanoma secretes VEGF. The number and location of the micrometastases correlate with serum VEGF levels.

Anterior diffuse retinoblastoma: mutational analysis and immunofluorescence staining.

Retinoblastoma is the most common primary intraocular tumor of childhood and may be heritable or occur sporadically. Anterior diffuse retinoblastoma is an uncommon variant that is thought to be sporadic. We describe a child with anterior diffuse retinoblastoma who presented with a pseudohypopyon. Genetic analysis showed a germline mutation of the RB1 allele that is potentially heritable. Immunofluorescence staining was positive for transforming growth factor beta and for vascular endothelial growth factor and negative for inducible nitric oxide synthase and for hypoxia inducible factor alpha in the tumor seeds, indicating acquisition of nonischemia-mediated survival factors of the tumor seeds in the aqueous humor.

Conjunctival lichen planus in a patient with herpes simplex virus keratitis.

PURPOSE: To report a case of lichen planus in a patient with a history of herpes simplex virus keratitis. METHODS: Case report. RESULTS: A 60-year-old woman with chronic conjunctivitis and a history of herpes simplex virus keratitis was evaluated for irritation and a plaque on her right upper and lower eyelid palpebral conjunctivae. A surgical excision showed acanthosis and an underlying lichenoid infiltrate with a thickened basement membrane. CONCLUSION: Lichen planus of the conjunctiva can be present in the absence of cicatrization in a patient with chronic irritation of the conjunctiva.

Inflammatory modulation of PPAR gamma expression and activity.

Nitric oxide (NO) production increases with age in the lupus-prone MRL/lpr mouse, paralleling disease activity. One mechanism for excess NO production in MRL/lpr mice may be a defect in down-regulatory mechanisms of the iNOS pathway. A potential modulator of NO is the nuclear hormone receptor peroxisome proliferation activated receptor gamma (PPARgamma). We demonstrate that renal PPARgamma protein expression was altered as disease progressed in MRL/lpr mice, which paralleled increased iNOS protein expression. Additionally, MRL/lpr-derived primary mesangial cells expressed less PPARgamma than BALB/c mesangial cells and produced more NO in response to LPS and IFNgamma. Furthermore, PPARgamma activity was reduced in mesangial cells following exposure to inflammatory mediators. This activity was restored with the addition of a NOS enzyme inhibitor. These results indicate that the activation of inflammatory pathways may lead to reduced activity and expression of PPARgamma, further exacerbating the disease state.

Nephritogenic anti-DNA antibodies regulate gene expression in MRL/lpr mouse glomerular mesangial cells.

OBJECTIVE: Lupus-associated IgG anti-double-stranded DNA antibodies are thought to be pathogenic in the kidney due to cross-reaction with glomerular antigens, leading subsequently to immune complex formation in situ and complement activation. We undertook this study to determine if pathogenic anti-DNA antibodies may also contribute to renal damage by directly influencing mesangial gene expression. METHODS: Complementary DNA microarray gene profiling was performed in primary mesangial cells (derived from lupus-prone MRL/lpr mice) treated with pathogenic, noncomplexed anti-DNA antibodies. Significant gene up-regulation induced by anti-DNA antibodies as determined by microarray analysis was further investigated by real-time polymerase chain reaction and methods to detect the relevant proteins. Induction of proinflammatory genes by pathogenic antibodies was confirmed by comparing gene expression in glomeruli of old versus young MRL/lpr mice, and by antibody injection in vivo. RESULTS: Pathogenic, but not nonpathogenic, antibodies significantly induced a number of transcripts, including CXCL1/KC, LCN2, iNOS, CX3CL1/fractalkine, SERPINA3G, and IkappaBalpha ("marker genes"). Blocking of Fcgamma receptors or using Fcgamma chain-knockout mesangial cells had no effect on the gene regulation effect of the pathogenic antibody R4A, indicating a non-Fc-dependent mechanism. The glomerular expression of these marker genes increased over time with the development of glomerular antibody deposition and active nephritis in MRL/lpr mice. Moreover, injection of R4A into SCID mice in vivo significantly up-regulated glomerular marker gene expression. CONCLUSION: These findings indicate that the renal pathogenicity of anti-DNA antibodies may be attributed in part to their ability to directly modulate gene expression in kidney mesangial cells through both Fc-dependent and non-Fc-dependent mechanisms.

Peroxisome proliferation-activated receptor (PPAR)gamma is not necessary for synthetic PPARgamma agonist inhibition of inducible nitric-oxide synthase and nitric oxide.

Peroxisome proliferation-activated receptor (PPAR)gamma agonists inhibit inducible nitric-oxide synthase (iNOS), tumor necrosis factor-alpha, and interleukin-6. Because of these effects, synthetic PPARgamma agonists, including thiazolidinediones, are being studied for their impact on inflammatory disease. The anti-inflammatory concentrations of synthetic PPARgamma agonists range from 10 to 50 microM, whereas their binding affinity for PPARgamma is in the nanomolar range. The specificity of synthetic PPARgamma agonists for PPARgamma at the concentrations necessary for anti-inflammatory effects is thus in question. We report that PPARgamma is not necessary for the inhibition of iNOS by synthetic PPARgamma agonists. RAW 264.7 macrophages possess little PPARgamma, yet lipopolysaccharide (LPS)/interferon (IFN)gamma-induced iNOS was inhibited by synthetic PPARgamma agonists at 20 microM. Endogenous PPARgamma was inhibited by the transfection of a dominant-negative PPARgamma construct into murine mesangial cells. In the transfected cells, synthetic PPARgamma agonists inhibited iNOS production at 10 microM, similar to nontransfected cells. Using cells from PPARgamma Cre/lox conditional knockout mice, baseline and LPS/IFNgamma-induced nitric oxide levels were higher in macrophages lacking PPARgamma versus controls. However, synthetic PPARgamma agonists inhibited iNOS at 10 microM in the PPARgamma-deficient cells, similar to macrophages from wild-type mice. These results indicate that PPARgamma is not necessary for inhibition of iNOS expression by synthetic PPARgamma agonists at concentrations over 10 microM. Intrinsic PPARgamma function, in the absence of synthetic agonists, however, may play a role in inflammatory modulation.