Haloperidol-associated stealth liposomes: a potent carrier for delivering genes to human breast cancer cells

Sigma receptors are membrane-bound proteins that are overexpressed in certain human malignancies including breast cancer. These receptors show very high affinity for various sigma ligands including neuroleptics like haloperidol. We hypothesized that in associating haloperidol-linked lipid into the cationic lipid-DNA complex, we can specifically target and deliver genes to breast cancer cells that overexpress sigma receptors. In the present study, haloperidol was chemically modified to conjugate at the distal end of the polyethylene glycollinked phospholipid, which was then incorporated into the cationic liposome known to condense and deliver genes inside cells. The resulting haloperidol-conjugated targeted lipoplex showed at least 10-fold higher (p < 0.001) reporter gene expression in MCF-7 cells than control lipoplex. The reporter gene expression of the targeted lipoplex was significantly blocked by haloperidol (p < 0.001) and by another sigma ligand, 1,3-ditolylguanidine (p < 0.001) in the majority of cationic lipid to DNA charge ratios (+/-). Spironolactone-mediated sigma receptor down-regulation enabled MCF-7 to show 10-fold lower transgene expression with targeted lipoplex compared with that obtained in spironolactone-untreated cells. The targeted lipoplex generated nonspecific gene expression in sigma receptor-nonexpressing human cancer cells such as Hela, KB, HepG2, and Chinese hamster ovary cells. Moreover, the transgene expression remained unabated in physiologically relevant serum concentrations. This is the first study to demonstrate that haloperidol-targeted gene delivery systems can mediate efficient targeting of genes to sigma receptor-overexpressing breast cancer cells, thereby becoming a novel class of therapeutics for the treatment of human cancers.     

A transfection compound series based on a versatile Tris linkage

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.     

Microbubble‐mediated sonoporation for highly efficient transfection of recalcitrant human B‐ cell lines

Sonoporation has not been widely explored as a strategy for the transfection of heterologous genes into notoriously difficult‐to‐transfect mammalian cell lines such as B cells. This technology utilizes ultrasound to create transient pores in the cell membrane, thus allowing the uptake of extraneous DNA into eukaryotic and prokaryotic cells, which is further enhanced by cationic microbubbles. This study investigates the use of sonoporation to deliver a plasmid encoding green fluorescent protein (GFP) into three human B‐cell lines (Ramos, Raji, Daudi). A higher transfection efficiency (TE) of >42% was achieved using sonoporation compared with <3% TE using the conventional lipofectamine method for Ramos cells. Upon further antibiotic selection of the transfected population for two weeks, we successfully enriched a stable population of GFP‐positive Ramos cells (>70%). Using the same strategy, Raji and Daudi B cells were also successfully transfected and enriched to 67 and 99% GFP‐positive cells, respectively. Here, we present sonoporation as a feasible non‐viral strategy for stable and highly efficient heterologous transfection of recalcitrant B‐cell lines. This is the first demonstration of a non‐viral method yielding transfection efficiencies significantly higher (42%) than the best reported values of electroporation (30%) for Ramos B‐cell lines.     

Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.     

On the Mechanism of Cationic Amphiphile-mediated Transfection. To Fuse or not to Fuse: Is that the Question?

Due to charge interaction, cationic lipids spontaneously associate with nucleic acids, resulting in the formation of so-called lipoplexes. Lipoplexes are membranous structures that are capable of transducing genes into cells, eventually leading to expression of the genes (transfection). The mechanism involved in the cellular uptake of lipoplexes is most likely endocytosis, which occurs after nonspecific charge-mediated binding to cellular receptors. An important step in the transfection process following the actual internalization of lipoplexes is the release of the lipoplex and/or its DNA into the cytoplasm in order to evade lysosomal degradation. Here, the membranous nature of the lipoplex seems to be crucial in that it allows the exchange of lipids between the endosomal membrane and the lipoplex, which results in membrane perturbations that are a prerequisite in the endosomal escape of DNA. Interestingly, a hexagonal phase of the lipoplexes has been correlated with efficient transfection and it can be envisaged that such a phase could be instrumental in the creation of membrane perturbations. Subsequent to its release into the cytoplasm, the DNA has to be transferred into the nucleus. The nuclear import of DNA is most likely a protein-mediated process. In addition, the nuclear uptake of DNA may be facilitated at the time of nuclear envelope disassembly during mitosis. Currently, cationic liposomes are widely used as gene carrier system to deliver nucleic acids into cells in culture to study the cell-biological functioning of genes plus accompanying proteins. Ultimately, cationic lipids may be used in gene therapeutic protocols.     

How are Nucleic Acids Released in Cells from Cationic Lipid-Nucleic Acid Complexes?

Abstract

Cationic lipid-nucleic acid complexes are widely used to deliver oligonucleotides, RNA and DNA into cells. Although much has been learned about the structure and forces that hold the complex together, an understanding of the mechanism of release of the nucleic acids from the complex into cells has been lacking. Recent studies have shown that anionic liposomes with compositions similar to the cytoplasmic face of the endosomal membrane are potent agents for inducing the rapid release of oligonucleotides and DNA from cationic lipid-nucleic acid complexes. Based upon these results, we propose that after the cationic lipid/nucleic complex is internalized by endocytosis it destabilizes the endosomal membrane. This destabilization induces flip-flop of anionic lipids from the cytoplasmic facing monolayer, which laterally diffuse into the complex and form a charge neutral ion-pair with the cationic lipids. This results in displacement of the nucleic acid from the cationic lipid and subsequent release of the nucleic acid into cytoplasm of the cell. We review the data that show the proposed mechanism accounts for a variety of observations on cationic lipid/nucleic acid complex-cell interactions.     

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.     

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).     

Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine™ RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.     

Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST–Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST–Z2 is able to condense 130–150 kb bacterial artificial chromosomes (BACs) into protein–DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein–BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR^– cells by GST–Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST–Z2-mediated gene transfer. Because DNA condensation by GST–Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.     

Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures

A critical issue in the management of head and neck tumors is radioprotection of the salivary glands. We have investigated whether siRNA-mediated gene knock down of pro-apoptotic mediators can reduce radiation-induced cellular apoptosis in salivary gland cells in vitro. We used novel, pH-responsive nanoparticles to deliver functionally active siRNAs into cultures of salivary gland cells. The nanoparticle molecules are comprised of cationic micelles that electrostatically interact with the siRNA, protecting it from nuclease attack, and also include pH-responsive endosomolytic constituents that promote release of the siRNA into the target cell cytoplasm. Transfection controls with Cy3-tagged siRNA/nanoparticle complexes showed efficiently internalized siRNAs in more than 70% of the submandibular gland cells. We found that introduction of siRNAs specifically targeting the Pkcδ or Bax genes significantly blocked the induction of these pro-apoptotic proteins that normally occurs after radiation in cultured salivary gland cells. Furthermore, the level of cell death from subsequent radiation, as measured by caspase-3, TUNEL, and mitochondrial disruption assays, was significantly decreased. Thus, we have successfully demonstrated that the siRNA/nanoparticle-mediated knock down of pro-apoptotic genes can prevent radiation-induced damage in submandibular gland primary cell cultures.     

Chaperonin CCT-Mediated AIB1 Folding Promotes the Growth of ERα-Positive Breast Cancer Cells on Hard Substrates

Clinical observations have revealed a strong association between estrogen receptor alpha (ERα)-positive tumors and the development of bone metastases, however, the mechanism underlying this association remains unknown. We cultured MCF-7 (ERα-positive) on different rigidity substrates. Compared with cells grown on more rigid substrates (100 kPa), cells grown on soft substrates (10 kPa) exhibited reduced spreading ability, a lower ratio of cells in the S and G2/M cell cycle phases, and a decreased proliferation rate. Using stable isotope labeling by amino acids (SILAC), we further compared the whole proteome of MCF-7 cells grown on substrates of different rigidity (10 and 100 kPa), and found that the expression of eight members of chaperonin CCT increased by at least 2-fold in the harder substrate. CCT folding activity was increased in the hard substrate compared with the soft substrates. Amplified in breast cancer 1 (AIB1), was identified in CCT immunoprecipitates. CCT folding ability of AIB1 increased on 100-kPa substrate compared with 10- and 30-kPa substrates. Moreover, using mammalian two-hybrid protein-protein interaction assays, we found that the polyglutamine repeat sequence of the AIB1 protein was essential for interaction between CCTζ and AIB1. CCTζ-mediated AIB1 folding affects the cell area spreading, growth rate, and cell cycle. The expressions of the c-myc, cyclin D1, and PgR genes were higher on hard substrates than on soft substrate in both MCF-7 and T47D cells. ERα and AIB1 could up-regulate the mRNA and protein expression levels of the c-myc, cyclin D1, and PgR genes, and that 17 β-estradiol could enhance this effects. Conversely, 4-hydroxytamoxifen, could inhibit these effects. Taken together, our studies demonstrate that some ERα-positive breast cancer cells preferentially grow on more rigid substrates. CCT-mediated AIB1 folding appears to be involved in the rigidity response of breast cancer cells, which provides novel insight into the mechanisms of bone metastasis.     

The Genome Landscape of the African Green Monkey Kidney-Derived Vero Cell Line

Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes were identified in the 2.97-Gb genome sequence. A homozygous ∼9-Mb deletion on chromosome 12 caused the loss of the type I interferon gene cluster and cyclin-dependent kinase inhibitor genes in Vero cells. In addition, an ∼59-Mb loss of heterozygosity around this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. Moreover, a genomic analysis of Vero cells revealed a female Chlorocebus sabaeus origin and proviral variations of the endogenous simian type D retrovirus. These results revealed the genomic basis for the non-tumourigenic permanent Vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines and developing new tools in the quality control of Vero cells.     

Cationic liposome-mediated delivery of siRNAs in adult mice

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for dissecting gene function and drug target validation. siRNAs can be synthesized in large quantities and thus can be used to analyze a large number of sequences emerging from genome projects in a cost-effective manner. However, the major obstacle to the use of siRNAs as therapeutics is the difficulty involved in effective in vivo delivery. We used a fluorescein-labeled siRNA to investigate cationic liposome-mediated intravenous and intraperitoneal delivery in adult mice. We show that this simple approach can deliver siRNAs into various cell types. In addition, we show that in contrast to mouse cells, siRNAs can activate the non-specific pathway in human freshly isolated monocytes, resulting in TNF-alpha and IL-6 production. Taken together, the data provide a basis for lipid-mediated systemic delivery of siRNAs and indicate that certain siRNA sequences can activate the innate immunity response genes that can be beneficial for the treatment of cancer.     

Efficient transfer of chromosome-based DNA constructs into mammalian cells

Artificial chromosomes, engineered minichromosomes and other chromosome-based DNA constructs are promising new vectors for use in gene therapy, protein production and transgenics. However, a major drawback in the application of chromosome-based DNA is the lack of a suitable and convenient procedure for large-scale cellular introduction, which is particularly frustrated by their size (1 by 2 microm). Here we present a method to transfer Artificial Chromosome Expression systems (ACEs) into mammalian cells, which relies on a combined approach of using cationic amphiphiles and high frequency ultrasound. Thus, when cells were preincubated with liposomes consisting of the cationic lipid SAINT-2 and the phospholipid dioleoylphosphatidylethanolamine (molar ratio 1:1), followed by ultrasound, ACEs could be introduced into mammalian cells, which resulted in the expression of ACEs-harbored reporter genes, such as Green Fluorescent Protein. Depending on cell type, transfection efficiencies ranged from 12% to 53%. Interestingly, no detectable delivery occurred when cells were treated alone with either ultrasound or liposomes. Evidence is provided, based on cellular entry of differently sized beads and trypan-blue permeation, which supports a mechanism in which integration of the lipids creates unstable membrane domains, which are particularly prone to ultrasound-induced pore formation. Time- and temperature-dependent experiments indicate that these pores display a transient stability. Hence, following ultrasound, the pores disappear as a function of time as suggested by a time-window for ACEs entry, and trypan blue exclusion, 80% of the cells becoming stained immediately following ultrasound, dropping to approximately 20% after 30 min. Co-expression of different genes in conjunction with fluorescence in situ hybridization (FISH) analysis indicates that the current procedure provides a means to introduce functionally active artificial chromosomes into eukaryotic cells.     

Whole-genome expression profiling defines the HrpL regulon of Pseudomonas syringae pv. tomato DC3000, allows de novo reconstruction of the Hrp cis clement, and identifies novel coregulated genes

Pseudomonas syringae pv. tomato DC3000 is a model pathogen of tomato and Arabidopsis that uses a hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to deliver virulence effector proteins into host cells. Expression of the Hrp system and many effector genes is activated by the HrpL alternative sigma factor. Here, an open reading frame-specific whole-genome microarray was constructed for DC3000 and used to comprehensively identify genes that are differentially expressed in wild-type and deltahrpL strains. Among the genes whose differential regulation was statistically significant, 119 were upregulated and 76 were downregulated in the wild-type compared with the deltahrpL strain. Hierarchical clustering revealed a subset of eight genes that were upregulated particularly rapidly. Gibbs sampling of regions upstream of HrpL-activated operons revealed the Hrp promoter as the only identifiable regulatory motif and supported an iterative refinement involving real-time polymerase chain reaction testing of additional HrpL-activated genes and refinements in a hidden Markov model that can be used to predict Hrp promoters in P. syringae strains. This iterative bioinformatic-experimental approach to a comprehensive analysis of the HrpL regulon revealed a mix of genes controlled by HrpL, including those encoding most type III effectors, twin-arginine transport (TAT) substrates, other regulatory proteins, and proteins involved in the synthesis or metabolism of phytohormones, phytotoxins, and myo-inositol. This analysis provides an extensively verified, robust method for predicting Hrp promoters in P. syringae genomes, and it supports subsequent identification of effectors and other factors that likely are important to the host-specific virulence of P. syringae.     

Cationic phosphoramidate alpha-oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis C virus IRES-mediated translation

A potential means to improve the efficacy of steric-blocking antisense oligonucleotides (ON) is to increase their affinity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electrostatic repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucleoside linkages into ON with a non-natural α-anomeric configuration. Cationic α-ON bound with high affinity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modifications, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic α-ON and their RNA targets were not substrates for RNase H. The penalty in T_m inflicted by a single mismatch, however, was high; suggesting that they are well suited as sequence-specific, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have confirmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic α-ON in cell culture.     

A platform of high-efficiency nonviral gene transfer in mouse osteoblast cells in vitro

We have previously established mouse genetic models and identified the genetic components of quantitative trait loci (QTL) on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and bone's anabolic response to mechanical loading. However, these regions contain dozens of unknown genes that are needed for functional testing. In this study, we provided a protocol of nucleoporation with high efficiency by using a commercial nucleofection buffer and Gene Pulser to deliver a test gene into bone cells for functional studies. We cloned an osteoblast differentiation-specific geneosterix (Osx) from a mouse bone cDNA library into a pHGCX expression vector and used nucleoporation to deliver pHGCX/Flag-Osx into the nuclei of MC3T3-E1 cells. We then examined the transfection efficiency transgene expression, and function. Our results have demonstrated that nucleoporation can deliver a transgene into MC3T3-E1 osteoblast cells with approx 94% transfection efficiency, and express a functional Flag-Osx fusion protein capable of inducing cell differentiation as measured by an incease in alkaline phosphatase (ALP) activity. Therefore, this experimental system provides a rapid, safe, and efficient cell-based model of high-throughput phenotypic screening to identify candidate genes from physically mapped regions that are important for osteoblast differentiation.