The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway is frequently hyper-activated upon vemurafenib treatment of melanoma. We have here investigated the relationship between SRY-box 10 (SOX10), forkhead box 3 (FOXD3) and microphthalmia-associated transcription factor (MITF) in the regulation of the receptor tyrosine-protein kinase ERBB3, and its cognate ligand neuregulin 1-beta (NRG1-beta). We found that both NRG1-beta and ERBB3 mRNA levels were elevated as a consequence of MITF depletion, induced by either vemurafenib or MITF small interfering RNA (siRNA) treatment. Elevation of ERBB3 receptor expression after MITF depletion caused increased activation of the PI3K pathway in the presence of NRG1-beta ligand. Together, our results suggest that MITF may play a role in the development of acquired drug resistance through hyper-activation of the PI3K pathway.
Photochemical internalization (PCI) is a technology based on a photosensitizer that photochemically destabilizes endosomal membranes after illumination, resulting in the release of endocytosed material into the cytosol. In this study, we investigated the potential of using polyethylenimine (PEI) for light-controlled delivery of small interfering RNA (siRNA) molecules via the endocytic pathway. PEI formulations with different molecular weights (MW) and chemical forms (linear [L]/branched [B]) were investigated for their capacity to deliver siRNA molecules with or without PCI at variable nitrogen/phosphorus (N/P) ratios and illumination doses. By targeting the S100A4 gene in an osteosarcoma cell model system, potent gene silencing was observed in samples treated with PCI compared with samples not treated with PCI. The effect of light-controlled gene silencing was dependent on several factors, including light-doses and MW, chemical form, as well as on the N/P ratio of the PEI formulations. This study demonstrates the first success in using PEI formulations as siRNA carriers for light-controlled gene silencing with the objective of future use in in vivo applications.
Analysis of DNA variation in biological samples most frequently utilizes the polymerase chain reaction (PCR) performed on extracted genomic DNA, followed by visualization of alleles using various methodologies. Few reports have demonstrated that amplification of DNA from plasma and serum samples is possible. We have performed DNA amplification on a large set of serum samples (n = 2955). Here, we report that known hereditary mutations in the BRCA gene can efficiently be analyzed in serum samples collected and stored over several decades. Fragments were PCR-amplified following a short initial denaturation of the serum sample in a standard microwave oven. Fragment analysis was subsequently performed using a DNA capillary-sequencing instrument. The PCR success rates were fragment- and size-dependent ranging from 83.2% to 97.9%. Of the 11,820 polymerase chain reactions performed, the overall PCR success rate was 91.3% (10,796/11,820), which is comparable to PCR performed on genomic DNA. The advantage of the method described herein is its ability to utilize archival material stored in serum biobanks for long periods of time.
Novel strategies for efficient delivery of small interfering RNA (siRNA) molecules with a potential for targeting are required for development of RNA interference (RNAi) therapeutics. Here, we present a strategy that is based on delivery of siRNA molecules through the endocytic pathway, in order to develop a method for site-specific gene silencing. To achieve this, we combined the use of cationic lipids and photochemical internalization (PCI). Using the human S100A4 gene as a model system, we obtained potent gene silencing in four tested human cancer cell lines following PCI induction when using the cationic lipid jetSI-ENDO. Gene silencing was shown at both the RNA and protein levels, with no observed PCI toxicity when using the jetSI reagent and an optimized PCI protocol. This novel induction method opens for in vivo site-specific delivery of siRNA molecules toward a sequence of interest.
In this study, we have investigated the possibility of combining a cyclodextrin-containing polymer (CDP) with siRNA molecules to modulate gene expression in a light-directed manner through photochemical internalization (PCI) technology. We utilized S100A4 as a model gene to evaluate the efficacy of gene silencing. After optimization of carrier/cargo ratio and illumination dose, real-time reverse transcriptase-polymerase chain reaction data showed between 80% and 90% silencing in the siRNA samples treated with PCI compared with untreated control. In contrast, only a 0%-10% silencing effect was detected in the siRNA samples without PCI treatment, demonstrating the potency of light-specific delivery of siRNA molecules. Light-directed siRNA delivery was shown in 2 different cell lines with corresponding potency. Further, time-lapse results demonstrated maximum gene silencing only at 5 hours after endosomal release, implying, for example, rapid carrier decondensation when using the CDP. This work represents a first success in using a CDP delivery agent, without endosomolytic properties for siRNA gene silencing in a light-directed manner, opening the opportunity to use CDPs for light-directed siRNA gene silencing in vivo.
A promising method that offers both time- and site-specific delivery of macromolecules is photochemical internalization technology (PCI). Here, we have characterized various polyamidoamine (PAMAM) carriers [generation (G) 0-7], for light-directed delivery of nucleic acids in vitro by the use of PCI technology. A number of parameters for optimal delivery of nucleic acids into human cancer cells, that is, various light-doses, carrier-doses, and small interfering RNA (siRNA)/messenger RNA (mRNA) doses were investigated for either up- or down-regulation of enhanced green fluorescent protein (EGFP) gene expression. In summary, our results showed in an osteosarcoma cell line (OHS) [EGFP] model system the possibility for efficient light-directed siRNA silencing (>80% silencing) when using PAMAM G3 to G7 as carriers. Surprisingly, no EGFP mRNA up-regulation was detected either with or without PCI after EGFP mRNA/PAMAM (G0-G7) transfection in standard OHS cells. We have here identified properties for PAMAM formulations enabling light-directed siRNA delivery with the aim of developing a site-specific strategy for delivery of nucleic acids in vivo.
A promising strategy for increased intracellular delivery of nucleic acids with the benefit for targeting is photochemical internalization (PCI). PCI relies on the use of a photosensitizing compound that photochemically destroys membranes in the endocytic pathway after illumination, resulting in cytosolic transfer of endosomal content. PCI technology combined with biodegradable polyamino acid carriers and nucleic acids delivers effective targeting and improved biosafety. In an in vitro model system, we have evaluated various poly-l-lysine (PLL), poly-l-histidine (PLH), and poly-l-arginine (PLA) formulations for light-directed small interference RNA (siRNA) gene silencing and messenger RNA (mRNA) delivery. We find that PLA formulations are suitable as siRNA and mRNA carriers in a strictly light-directed manner.
We here demonstrate for the first time that 5-carboxytetramethylrhodamine (TAMRA) covalently linked to nuclear localization signal (NLS)-conjugated peptide nucleic acids (PNAs) are photosensitizers (PSs) with the capacity to initiate photochemical damage to endocytic membranes, resulting in release of endocytosed material into cytosol. Our results show that TAMRA/PNA/NLS conjugates work as multifunctional molecules by offering cellular uptake, PNA-directed gene silencing, and the possibility for targeting in a light-controlled manner. In addition to PNA-directed gene silencing, we demonstrate that TAMRA/PNA/NLS molecules may function as a PS for light-controlled release of small interfering RNA molecules from the endocytic pathway when combined with an appropriate carrier. Using these strategies, we could silence the S100A4 gene at both protein and mRNA levels in a light-controlled manner, without any detectable reduction in cell viability. Our data demonstrate the possibility for light-controlled delivery of macromolecules entrapped within endocytic vesicles using multifunctional TAMRA/PNA/NLS molecules as PSs.
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